essentials of genetics - indian institute of technology...

Essentials of Genetics

Why does a commercial dairy cow produce four times as much milk as most other mammals? Why do we look like our cousins? Why do roses come in so many different colors? The answers to these and other questions about the diversity of living things involve processes that occur at the level of genes.

Essentials of Genetics is a brief guide through the core concepts of how genes are structured and how they drive biological diversity. This course can be used as a guide for introductory biology students, as a reference for advanced students, or as a self-guided exploration for general science enthusiasts. Topics covered include the nature of DNA and its relationship to the physical characteristics of organisms; the passage of DNA from organism to organism; and the variation of DNA within and across populations of organisms. Essentials of Genetics also connects these core concepts to the scientific process by discussing the key tools used to study DNA in the laboratory. Alongside each concept are links to biographies of scientists who made major contributions to the field, as well as to a broad set of detailed readings on advanced topics in modern genetics. Finally, Essentials of Genetics combines its descriptions of various core concepts with high-quality video animations of molecular processes to stimulate an intuitive physical understanding of genetics.

About the Authors

Lead Editors: Ilona Miko, Ph.D. and Lorrie LeJeune

Writers: Heidi Chial, Ph.D., Carrie Drovdlic, Maggie Koopman, Ph.D., Sarah Catherine Nelson, Ph. D., Angela Spivey, Robin Smith, Ph. D., WilliamsTown Communications.

Animations and Illustrations: Arkitek

Citation

Please cite this book as:

Miko, I. & LeJeune, L., eds. Essentials of Genetics. Cambridge, MA: NPG Education, 2009.

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Contents

Unit 1 What Is DNA? What Does DNA Do?

Unit 2 How Does DNA Move from Cell to Cell? Unit 3 How Is Genetic Information Passed between Organisms? Unit 4 How Do Scientists Study and Manipulate the DNA inside Cells? Unit 5 How Does Inheritance Operate at the Level of Whole Populations?

Essentials of Genetics

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PROGRESS

1.1 Introduction: What Is DNA? Prev Page

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Deoxyribonucleic acid, more commonly known as DNA, is a complex molecule that contains all of the information necessary to build and maintain an organism. All living things have DNA within their cells. In fact, nearly every cell in a multicellular organism possesses the full set of DNA required for that organism. However, DNA does more than specify the structure and function of living things — it also serves as the primary unit of heredity in organisms of all types. In other words, whenever organisms reproduce, a portion of their DNA is passed along to their offspring. This transmission of all or part of an organism's DNA helps ensure a certain level of continuity from one generation to the next, while still allowing for slight changes that contribute to the diversity of life.But what, exactly, is DNA? What smaller elements make up this complex molecule, how are these elements arranged, and how is information extracted from them? This unit answers each of these questions, and it also provides a basic overview of the process of DNA discovery.

Unit 1

Introduction: What Is DNA?

DNA Is a Structure That Encodes Biological Information

Discovery of the Function of DNA Resulted from the Work of Multiple Scientists

Cells Can Replicate Their DNA Precisely

The Information in DNA Is Decoded by Transcription

The Information in DNA Determines Cellular Function via Translation

Key Questions Where can I learn more about DNA?

Key Concepts DNA

chromosomes

Essentials of Genetics Contents Unit 1: What Is DNA? What Does DNA Do?

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PROGRESS

1.2 DNA Is a Structure That Encodes Biological Information Prev Page

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What do a human, a rose, and a bacterium have in common? Each of these things — along with every other organism on Earth — contains the molecular instructions for life, called deoxyribonucleic acid or DNA. Encoded within this DNA are the directions for traits as diverse as the color of a person's eyes, the scent of a rose, and the way in which bacteria infect a lung cell. DNA is found in nearly all living cells. However, its exact location within a cell depends on whether that cell possesses a special membrane-bound organelle called a nucleus. Organisms composed of cells that contain nuclei are classified as eukaryotes, whereas organisms composed of cells that lack nuclei are classified as prokaryotes. In eukaryotes, DNA is housed within the nucleus, but in prokaryotes, DNA is located directly within the cellular cytoplasm, as there is no nucleus available.But what, exactly, is DNA? In short, DNA is a complex molecule that consists of many components, a portion of which are passed from parent organisms to their offspring during the process of reproduction. Although each organism's DNA is unique, all DNA is composed of the same nitrogen-based molecules. So how does DNA differ from organism to organism? It is simply the order in which these smaller molecules are arranged that differs among individuals. In turn, this pattern of arrangement ultimately determines each organism's unique characteristics, thanks to another set of molecules that "read" the pattern and stimulate the chemical and physical processes it calls for.

What components make up DNA?

Figure 1: A single nucleotide contains a nitrogenous base (red), a deoxyribose sugar molecule (gray), and a phosphate group attached to the 5' side of the sugar (indicated by light gray). Opposite to the 5' side of the sugar molecule is the 3' side (dark gray), which has a free hydroxyl group attached (not shown).

At the most basic level, all DNA is composed of a series of smaller molecules called nucleotides. In turn, each nucleotide is itself made up of three primary components: a nitrogen-containing region known as a nitrogenous base, a carbon-based sugar molecule called deoxyribose, and a phosphorus-containing region known as a phosphate group attached to the sugar molecule (Figure 1). There are four different DNA nucleotides, each defined by a specific nitrogenous base: adenine (often abbreviated "A" in science writing), thymine (abbreviated "T"), guanine (abbreviated "G"), and cytosine (abbreviated "C") (Figure 2).


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Figure 2: The four nitrogenous bases that compose DNA nucleotides are shown in bright colors: adenine (A, green), thymine (T, red), cytosine (C, orange), and guanine (G, blue).

Although nucleotides derive their names from the nitrogenous bases they contain, they owe much of their structure and bonding capabilities to their deoxyribose molecule. The central portion of this molecule contains five carbon atoms arranged in the shape of a ring, and each carbon in the ring is referred to by a number followed by the prime symbol ('). Of these carbons, the 5' carbon atom is particularly notable, because it is the site at which the phosphate group is attached to the nucleotide. Appropriately, the area surrounding this carbon atom is known as the 5' end of the nucleotide. Opposite the 5' carbon, on the other side of the deoxyribose ring, is the 3' carbon, which is not attached to a phosphate group. This portion of the nucleotide is typically referred to as the 3' end (Figure 1). When nucleotides join together in a series, they form a structure known as a polynucleotide. At each point of juncture within a polynucleotide, the 5' end of one nucleotide attaches to the 3' end of the adjacent nucleotide through a connection called a phosphodiester bond (Figure 3). It is this alternating sugar-phosphate arrangement that forms the "backbone" of a DNA molecule.

Figure 3: All polynucleotides contain an alternating sugar-phosphate backbone. This backbone is formed when the 3' end (dark gray) of one nucleotide attaches to the 5' phosphate end (light gray) of an adjacent nucleotide by way of a phosphodiester bond.

How is the DNA strand organized? Although DNA is often found as a single-stranded polynucleotide, it assumes its most stable form when double stranded. Double-stranded DNA consists of two polynucleotides that are arranged such that the nitrogenous bases within one polynucleotide are attached to the nitrogenous bases within another polynucleotide by way of special chemical bonds called hydrogen bonds. This base-to-base bonding is not random; rather, each A in one strand always pairs with a T in the other strand, and each C always pairs with a G. The double-stranded DNA that results from this pattern of bonding looks much like a ladder with sugar-phosphate side supports and base-pair rungs.Note that because the two polynucleotides that make up double-stranded DNA are "upside down" relative to each other, their sugar-phosphate ends are anti-parallel, or arranged in opposite orientations. This means that one strand's sugar-phosphate chain runs in the 5' to 3' direction, whereas the other's runs in the 3' to 5' direction (Figure 4). It's also critical to understand that the specific sequence of A, T, C, and G nucleotides within an organism's DNA is unique to that individual, and it is this sequence that controls not only the operations within a particular cell, but within the organism as a whole.

Figure 4: Double-stranded DNA consists of two polynucleotide chains whose nitrogenous bases are connected by hydrogen bonds. Within this arrangement, each strand mirrors the other as a result of the anti-parallel orientation of the sugar-phosphate backbones, as well as the complementary nature of the A-T and C-G base pairing.



Figure 5: Rosalind Franklin used X-ray diffraction to obtain this image of DNA. Images like this one enabled the precise calculation of molecular distances within the double helix.

Beyond the ladder-like structure described above, another key characteristic of double-stranded DNA is its unique three-dimensional shape. The first photographic evidence of this shape was obtained in 1952, when scientist Rosalind Franklin used a process called X-ray diffraction to capture images of DNA molecules (Figure 5). Although the black lines in these photos look relatively sparse, Dr. Franklin interpreted them as representing distances between the nucleotides that were arranged in a spiral shape called a helix. Around the same time, researchers James Watson and Francis Crick were pursuing a definitive model for the stable structure of DNA inside cell nuclei. Watson and Crick ultimately used Franklin's images, along with their own evidence for the double-stranded nature of DNA, to argue that DNA actually takes the form of a double helix, a ladder-like structure that is twisted along its entire length (Figure 6). Franklin, Watson, and Crick all published articles describing their related findings in the same issue of Nature in 1953.

Figure 6: The double helix looks like a twisted ladder.

How is DNA packaged inside cells?

Figure 7: To better fit within the cell, long pieces of double-stranded DNA are tightly packed into structures called chromosomes.

Most cells are incredibly small. For instance, one human alone consists of approximately 100 trillion cells. Yet, if all of the DNA within just one of these cells were arranged into a single straight piece, that DNA would be nearly two meters long! So, how can this much DNA be made to fit within a cell? The answer to this question lies in the process known as DNA packaging, which is the phenomenon of fitting DNA into dense compact forms (Figure 7).

Chromatin

What does real chromatin look like?•Compare the relative sizes of the double helix, histones, and chromosomes•

During DNA packaging, long pieces of double-stranded DNA are tightly looped, coiled, and folded so that they fit easily within the cell. Eukaryotes accomplish this feat by wrapping their DNA around special proteins called histones, thereby compacting it enough to fit inside the nucleus (Figure 8). Together, eukaryotic DNA and the histone proteins that hold it together in a coiled form is called chromatin.



Figure 8: In eukaryotic chromatin, double-stranded DNA (gray) is wrapped around histone proteins (red).

© 2009 Nature Education.

DNA can be further compressed through a twisting process called supercoiling (Figure 9). Most prokaryotes lack histones, but they do have supercoiled forms of their DNA held together by special proteins. In both eukaryotes and prokaryotes, this highly compacted DNA is then arranged into structures called chromosomes. Chromosomes take different shapes in different types of organisms. For instance, most prokaryotes have a single circular chromosome, whereas most eukaryotes have one or more linear chromosomes, which often appear as X-shaped structures . At different times during the life cycle of a cell, the DNA that makes up the cell's chromosomes can be tightly compacted into a structure that is visible under a microscope, or it can be more loosely distributed and resemble a pile of string.

Figure 9: Supercoiled eukaryotic DNA.

How do scientists visualize DNA?

Figure 10: This karyotype depicts all 23 pairs of chromosomes in a human cell, including the sex-determining X and Y chromosomes that together make up the twenty-third set (lower right).

It is impossible for researchers to see double-stranded DNA with the naked eye — unless, that is, they have a large amount of it. Modern laboratory techniques allow scientists to extract DNA from tissue samples, thereby pooling together miniscule amounts of DNA from thousands of individual cells. When this DNA is collected and purified, the result is a whitish, sticky substance that is somewhat translucent.To actually visualize the double-helical structure of DNA, researchers require special imaging technology, such as the X-ray diffraction used by Rosalind Franklin. However, it is possible to see chromosomes with a standard light microscope, as long as the chromosomes are in their most condensed form. To see chromosomes in this way, scientists must first use a chemical process that attaches the chromosomes to a glass slide and stains or "paints" them. Staining makes the chromosomes easier to see under the microscope. In addition, the banding patterns that appear on individual chromosomes as a result of the staining process are unique to each pair of chromosomes, so they allow researchers to distinguish different chromosomes from one another. Then, after a scientist has visualized all of the chromosomes within a cell and captured images of them, he or she can arrange these images to make a composite picture called a karyotype (Figure 10).

Watch this video for a closer look at the relationship between chromosomes and the DNA double helix



What components make up DNA?

How is the DNA strand organized?

How is DNA packaged inside cells?

How do scientists visualize DNA?

Watch this video for a closer look at the relationship between chromosomes and the DNA double helix

Unit 1







Key Questions How can so much DNA be packed inside a chromosome?

What are karyotypes used for?

Who is James Watson?

Key Concepts DNA

nucleic acid

Chromosome

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PROGRESS

1.3 Discovery of the Function of DNA Resulted from the Work of Multiple Scientists

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The chemical nature and structure of DNA were not elucidated until the middle of the twentieth century. Prior to that point, scientists had spent years speculating about which of the many types of molecules within cells contained the hereditary information. Was it protein? Was it something else — perhaps even a molecule they had yet to discover? Eventually, researchers zeroed in on DNA as the substance responsible for the transfer of traits from one generation to the next. From there, the race was on to learn more about this remarkable molecule.

Who first identified DNA? More on the discovery of DNA

Where is the proof that DNA is the hereditary material?: The experiments of Griffith, Avery, Hershey, and Chase

•

Intrigued by the structure of DNA: The contribution of Maurice Wilkins•

Although James Watson and Francis Crick determined the double-helical structure of DNA, DNA itself was identified nearly 90 years earlier by Swiss chemist Friedrich Miescher. While studying white blood cells, Miescher isolated a previously unknown type of molecule that was slightly acidic and contained a high percentage of phosphorus. Miescher named this molecule "nuclein," which was later changed to "nucleic acid" and eventually to "deoxyribonucleic acid," or DNA. Interestingly, Miescher did not believe that nuclein was the carrier of hereditary information, because he thought it lacked the variability necessary to account for the incredible diversity among organisms. Rather, like most scientists of his time, Miescher believed that proteins were responsible for heredity, because they existed in such a wide variety of forms.

Who linked DNA to heredity? For multiple decades following Miescher's discovery, most scientists continued to believe that protein, not DNA, was the carrier of hereditary information. This changed in 1944, when biologist Oswald Avery performed a series of groundbreaking experiments with the bacteria that cause pneumonia. At the time, scientists knew that some types of these bacteria (called "S type") had an outer layer called a capsule, but other types (called "R type") did not. Through a series of experiments, Avery and his colleagues found that only DNA could change R type bacteria into S type. This meant that something about DNA allowed it to carry instructions from one cell to another. This was not true of any other substances within the bacteria, including protein. This result highlighted DNA as the "transforming factor," thereby making it the best candidate for the hereditary material.

Who confirmed Avery's findings?


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Figure 1: A bacteriophage infects a bacterial cell by inserting its own DNA into that cell.

As is often the case with such discoveries, Avery's findings were largely unaccepted as evidence for DNA as the hereditary material until separate experiments were performed by other scientists. Thus, eight years later, Alfred Hershey and Martha Chase further confirmed that protein was not the hereditary material through their work with bacteriophages, which are viruses that infect bacteria (Figure 1). Bacteriophages are composed of only two substances: protein and DNA. By using radioactive labels that would integrate specifically into either DNA or protein, but not both, Hershey and Chase were able to show that DNA is the only material transferred directly from bacteriophages into bacteria when the bacteria are infected by these viruses. This observation was important, because Hershey and Chase knew that the end result of bacteriophage infection was the production of more viruses in multiple copies.

Figure 2: Viral DNA (in purple) hijacks the bacterial cell, forcing it to make copies of this DNA and manufacture new viruses.

But just how did the injection of viral DNA into a bacterium create new viruses? Hershey and Chase admitted that they were unsure of the answer to this question; however, they knew it didn't have anything to do with protein, but did have something to do with DNA. Thanks to additional research, scientists now know that the DNA in a virus can take over a bacterial cell, causing it to replicate only the viral DNA and to create new viruses (Figure 2). This process is a form of hijacking, wherein the viral life-form takes over the regular machinery inside another life-form (in this case, a single bacterial cell). Under normal circumstances, a bacterial cell will reproduce by a form of cell division called binary fission. When Avery, MacLeod, McCarty, Hershey, and Chase performed their experiments, scientists knew that binary fission involved the copying of the hereditary substance and the redistribution of this substance into two new cells. So, when DNA was proven to be the material responsible for controlling the operations inside a single cell, it became easier to understand how the process of cell division and the transfer of the DNA could control the characteristics of newly born cells. Therefore, although they did not state it explicitly, Hershey and Chase had presented experiments that clearly suggested that DNA controls the production of more DNA, and that DNA itself was the substance that directed the construction and function of living things.Only one year after Hershey and Chase performed these experiments, James Watson and Francis Crick determined the three-dimensional structure of DNA. This discovery enabled investigators to put together the story of how DNA carries hereditary information from cell to cell. Indeed, the experiments connecting heredity and the structure of DNA were happening in parallel, so the next few years would be an exciting time for the discovery of DNA function.

Who first identified DNA?

Who linked DNA to heredity?

Who confirmed Avery's findings?

Unit 1







Key Questions How do scientists explore chromosomes?

How does DNA replicate?

What crucial contribution did Rosalind Franklin provide for the discovery of DNA?

What role did Linus Pauling play in DNA research?

Key Concepts DNA

meiosis

chromosome

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PROGRESS

1.4 Cells Can Replicate Their DNA Precisely Prev Page

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Replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules. DNA replication is one of the most basic processes that occurs within a cell. Each time a cell divides, the two resulting daughter cells must contain exactly the same genetic information, or DNA, as the parent cell. To accomplish this, each strand of existing DNA acts as a template for replication.

How is DNA replicated? Replication occurs in three major steps: the opening of the double helix and separation of the DNA strands, the priming of the template strand, and the assembly of the new DNA segment. During separation, the two strands of the DNA double helix uncoil at a specific location called the origin. Several enzymes and proteins then work together to prepare, or prime, the strands for duplication. Finally, a special enzyme called DNA polymerase organizes the assembly of the new DNA strands. The following description of this three-stage process applies generally to all cells, but specific variations within the process may occur depending on organism and cell type.

What triggers replication?

Figure 1: Helicase (yellow) unwinds the double helix.

The initiation of DNA replication occurs in two steps. First, a so-called initiator protein unwinds a short stretch of the DNA double helix. Then, a protein known as helicase attaches to and breaks apart the hydrogen bonds between the bases on the DNA strands, thereby pulling apart the two strands. As the helicase moves along the DNA molecule, it continues breaking these hydrogen bonds and separating the two polynucleotide chains (Figure 1).

Figure 2: While helicase and the initiator protein (not shown) separate the two polynucleotide chains, primase (red) assembles a primer. This primer permits the next step in the replication process.


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Meanwhile, as the helicase separates the strands, another enzyme called primase briefly attaches to each strand and assembles a foundation at which replication can begin. This foundation is a short stretch of nucleotides called a primer (Figure 2).

How are DNA strands replicated?

Figure 3: Beginning at the primer sequence, DNA polymerase (shown in blue) attaches to the original DNA strand and begins assembling a new, complementary strand.

After the primer is in place on a single, unwound polynucleotide strand, DNA polymerase wraps itself around that strand, and it attaches new nucleotides to the exposed nitrogenous bases. In this way, the polymerase assembles a new DNA strand on top of the existing one (Figure 3).

Figure 4: Each nucleotide has an affinity for its partner. A pairs with T, and C pairs with G.

As DNA polymerase makes its way down the unwound DNA strand, it relies upon the pool of free-floating nucleotides surrounding the existing strand to build the new strand. The nucleotides that make up the new strand are paired with partner nucleotides in the template strand; because of their molecular structures, A and T nucleotides always pair with one another, and C and G nucleotides always pair with one another. This phenomenon is known as complementary base pairing (Figure 4), and it results in the production of two complementary strands of DNA.

Figure 5: A new DNA strand is synthesized. This strand contains nucleotides that are complementary to those in the template sequence.

Base pairing ensures that the sequence of nucleotides in the existing template strand is exactly matched to a complementary sequence in the new strand, also known as the anti-sequence of the template strand. Later, when the new strand is itself copied, its complementary strand will contain the same sequence as the original template strand. Thus, as a result of complementary base pairing, the replication process proceeds as a series of sequence and anti-sequence copying that preserves the coding of the original DNA.

How long does replication take? More on replication

How does DNA polymerase work?•What does the molecular structure of a nucleotide look like?•What does the lagging strand look like?•

In the prokaryotic bacterium E. coli, replication can occur at a rate of 1,000 nucleotides per second. In comparison, eukaryotic human DNA replicates at a rate of 50 nucleotides per second. In both cases, replication occurs so quickly because multiple polymerases can synthesize two new strands at the same time by using each unwound strand from the original DNA double helix as a template. One of these original strands is called the leading strand, whereas the other is called the lagging strand. The leading strand is synthesized continuously, as shown in Figure 5. In contrast, the lagging strand is synthesized in small, separate fragments that are eventually joined together to form a complete, newly copied strand.

Watch this video for a summary of DNA replication in eukaryotes



How is DNA replicated?

What triggers replication?

How are DNA strands replicated?

How long does replication take?

Watch this video for a summary of DNA replication in eukaryotes

Unit 1







Key Questions What if an error happens during replication?

How is DNA stored in the cell before and after replication?

What do the leading and lagging strands look like when they are being replicated?

Key Concepts DNA polymerase

primer

transcription

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PROGRESS

1.5 The Information in DNA Is Decoded by Transcription Prev Page

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DNA is essentially a storage molecule. It contains all of the instructions a cell needs to sustain itself. These instructions are found within genes, which are sections of DNA made up of specific sequences of nucleotides. In order to be implemented, the instructions contained within genes must be expressed, or copied into a form that can be used by cells to produce the proteins needed to support life. The instructions stored within DNA are read and processed by a cell in two steps: transcription and translation. Each of these steps is a separate biochemical process involving multiple molecules. During transcription, a portion of the cell's DNA serves as a template for creation of an RNA molecule. (RNA, or ribonucleic acid, is chemically similar to DNA, except for three main differences described later on in this concept page.) In some cases, the newly created RNA molecule is itself a finished product, and it serves an important function within the cell. In other cases, the RNA molecule carries messages from the DNA to other parts of the cell for processing. Most often, this information is used to manufacture proteins. The specific type of RNA that carries the information stored in DNA to other areas of the cell is called messenger RNA, or mRNA.

How does transcription proceed? Transcription begins when an enzyme called RNA polymerase attaches to the DNA template strand and begins assembling a new chain of nucleotides to produce a complementary RNA strand. There are multiple types of types of RNA. In eukaryotes, there are multiple types of RNA polymerase which make the various types of RNA. In prokaryotes, a single RNA polymerase makes all types of RNA. Generally speaking, polymerases are large enzymes that work together with a number of other specialized cell proteins. These cell proteins, called transcription factors, help determine which DNA sequences should be transcribed and precisely when the transcription process should occur.

Initiation

Figure 1: Transcription begins when RNA polymerase binds to the DNA template strand.

The first step in transcription is initiation. During this step, RNA polymerase and its associated transcription factors bind to the DNA strand at a specific area that facilitates transcription (Figure 1). This area, known as a promoter region, often includes a specialized nucleotide sequence, TATAAA, which is also called the TATA box (not shown in Figure 1)

Strand elongation


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Figure 2: RNA polymerase (green) synthesizes a strand of RNA that is complementary to the DNA template strand below it.

Once RNA polymerase and its related transcription factors are in place, the single-stranded DNA is exposed and ready for transcription. At this point, RNA polymerase begins moving down the DNA template strand in the 3' to 5' direction, and as it does so, it strings together complementary nucleotides. By virtue of complementary base- pairing, this action creates a new strand of mRNA that is organized in the 5' to 3' direction. As the RNA polymerase continues down the strand of DNA, more nucleotides are added to the mRNA, thereby forming a progressively longer chain of nucleotides (Figure 2). This process is called elongation.

EFigure 3: DNA (top) includes thymine (red); in RNA (bottom), thymine is replaced with uracil (yellow).

Three of the four nitrogenous bases that make up RNA — adenine (A), cytosine (C), and guanine (G) — are also found in DNA. In RNA, however, a base called uracil (U) replaces thymine (T) as the complementary nucleotide to adenine (Figure 3). This means that during elongation, the presence of adenine in the DNA template strand tells RNA polymerase to attach a uracil in the corresponding area of the growing RNA strand (Figure 4).Interestingly, this base substitution is not the only difference between DNA and RNA. A second major difference between the two substances is that RNA is made in a single-stranded, nonhelical form. (Remember, DNA is almost always in a double-stranded helical form.) Furthermore, RNA contains ribose sugar molecules, which are slightly different than the deoxyribosemolecules found in DNA. As its name suggests, ribose has more oxygen atoms than deoxyribose.

Figure 4: A sample section of RNA bases (upper row) paired with DNA bases (lower row). When this base-pairing happens, RNA uses uracil (yellow) instead of thymine to pair with adenine (green) in the DNA template below.

Thus, the elongation period of transcription creates a new mRNA molecule from a single template strand of DNA. As the mRNA elongates, it peels away from the template as it grows (Figure 5). This mRNA molecule carries DNA's message from the nucleus to ribosomes in the cytoplasm, where proteins are assembled. However, before it can do this, the mRNA strand must separate itself from the DNA template and, in some cases, it must also undergo an editing process of sorts

Figure 5: During elongation, the new RNA strand becomes longer and longer as the DNA template is transcribed. In this view, the 5' end of the RNA strand is in the foreground. Note the inclusion of uracil (yellow) in RNA.



Termination and editing

Figure 6: In eukaryotes, noncoding regions called introns are often removed from newly synthesized mRNA.

As previously mentioned, mRNA cannot perform its assigned function within a cell until elongation ends and the new mRNA separates from the DNA template. This process is referred to as termination. In eukaryotes, the process of termination can occur in several different ways, depending on the exact type of polymerase used during transcription. In some cases, termination occurs as soon as the polymerase reaches a specific series of nucleotides along the DNA template, known as the termination sequence. In other cases, the presence of a special protein known as a termination factor is also required for termination to occur.

Figure 7: In eukaryotes, a poly-A tail is often added to the completed, edited mRNA molecule to signal that this molecule is ready to leave the nucleus through a nuclear pore.

Once termination is complete, the mRNA molecule falls off the DNA template. At this point, at least in eukaryotes, the newly synthesized mRNA undergoes a process in which noncoding nucleotide sequences, called introns, are clipped out of the mRNA strand. This process "tidies up" the molecule and removes nucleotides that are not involved in protein production (Figure 6). Then, a sequence of adenine nucleotides called a poly-A tail is added to the 3' end of the mRNA molecule (Figure 7). This sequence signals to the cell that the mRNA molecule is ready to leave the nucleus and enter the cytoplasm.

What's next for the RNA molecule? More on transcription

How are polymerases different in prokaryotes and eukaryotes?•How is bacterial transcription unique?•How is transcription regulated?•

Once an mRNA molecule is complete, that molecule can go on to play a key role in the process known as translation. During translation, the information that is contained within the mRNA is used to direct the creation of a protein molecule. In order for this to occur, however, the mRNA itself must be read by a special, protein-synthesizing structure within the cell known as a ribosome.

Watch this video for a summary of eukaryotic transcription



How does transcription proceed?

What's next for the RNA molecule?

Watch this video for a summary of eukaryotic transcription

Unit 1







Key Questions What does RNA do in the cell?

What are introns and exons?

Key Concepts RNA

replication

transcription

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PROGRESS

1.6 The Information in DNA Determines Cellular Function via Translation Prev Page

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The ribosome assembles the polypeptide chain.

To manufacture protein molecules, a cell must first transfer information from DNA to mRNA through the process of transcription. Then, a process called translation uses this mRNA as a template for protein assembly. In fact, this flow of information from DNA to RNA and finally to protein is considered the central dogma of genetics, and it is the starting point for understanding the function of the genetic information in DNA.But just how does translation work? In other words, how does the cell read and interpret the information that is stored in DNA and carried in mRNA? The answer to this question lies in a series of complex mechanisms, most of which are associated with the cellular structure known as the ribosome. In order to understand these mechanisms, however, it's first necessary to take a closer look at the concept known as the genetic code.

What is the genetic code? More on translation

How did scientists discover how ribosomes work?•What are ribosomes made of?•Is prokaryotic translation different from eukaryotic translation?•

At its heart, the genetic code is the set of "rules" that a cell uses to interpret the nucleotide sequence within a molecule of mRNA. This sequence is broken into a series of three-nucleotide units known as codons (Figure 1). The three-letter nature of codons means that the four nucleotides found in mRNA — A, U, G, and C — can produce a total of 64 different combinations. Of these 64 codons, 61 represent amino acids, and the remaining three represent stop signals, which trigger the end of protein synthesis. Because there are only 20 different amino acids but 64 possible codons, most amino acids are indicated by more than one codon. (Note, however, that each codon represents only one amino acid or stop codon.) This phenomenon is known as redundancy or degeneracy, and it is important to the genetic code because it minimizes the harmful effects that incorrectly placed nucleotides can have on protein synthesis. Yet another factor that helps mitigate these potentially damaging effects is the fact that there is no overlap in the genetic code. This means that the three nucleotides within a particular codon are a part of that codon only — thus, they are not included in either of the adjacent codons.

Figure 1: In mRNA, three-nucleotide units called codons dictate a particular amino acid. For example, AUG codes for the amino acid methionine (beige).


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The idea of codons was first proposed by Francis Crick and his colleagues in 1961. During that same year, Marshall Nirenberg and Heinrich Matthaei began deciphering the genetic code, and they determined that the codon UUU specifically represented the amino acid phenylalanine. Following this discovery, Nirenberg, Philip Leder, and Har Gobind Khorana eventually identified the rest of the genetic code and fully described which codons corresponded to which amino acids.

Reading the genetic code Redundancy in the genetic code means that most amino acids are specified by more than one mRNA codon. For example, the amino acid phenylalanine (Phe) is specified by the codons UUU and UUC, and the amino acid leucine (Leu) is specified by the codons CUU, CUC, CUA, and CUG. Methionine is specified by the codon AUG, which is also known as the start codon. Consequently, methionine is the first amino acid to dock in the ribosome during the synthesis of proteins. Tryptophan is unique because it is the only amino acid specified by a single codon. The remaining 19 amino acids are specified by between two and six codons each. The codons UAA, UAG, and UGA are the stop codons that signal the termination of translation. Figure 2 shows the 64 codon combinations and the amino acids or stop signals they specify.

Figure 2: The amino acids specified by each mRNA codon. Multiple codons can code for the same amino acid.

What role do ribosomes play in translation? As previously mentioned, ribosomes are the specialized cellular structures in which translation takes place. This means that ribosomes are the sites at which the genetic code is actually read by a cell. Ribosomes are themselves composed of a complex of proteins and specialized RNA molecules called ribosomal RNA (rRNA).

Figure 3: A tRNA molecule combines an anticodon sequence with an amino acid.

During translation, ribosomes move along an mRNA strand, and with the help of proteins called initiation factors, elongation factors, and release factors, they assemble the sequence of amino acids indicated by the mRNA, thereby forming a protein. In order for this assembly to occur, however, the ribosomes must be surrounded by small but critical molecules called transfer RNA (tRNA). Each tRNA molecule consists of two distinct ends, one of which binds to a specific amino acid, and the other which binds to a specific codon in the mRNA sequence because it carries a series of nucleotides called an anticodon (Figure 3). In this way, tRNA functions as an adapter between the genetic message and the protein product. (The exact role of tRNA is explained in more depth in the following sections.)

What are the steps in translation? Like transcription, translation can also be broken into three distinct phases: initiation, elongation, and termination. All three phases of translation involve the ribosome, which directs the translation process. Multiple ribosomes can translate a single mRNA molecule at the same time, but all of these ribosomes must begin at the first codon and move along the mRNA strand one codon at a time until reaching the stop codon. This group of ribosomes, also known as a polysome, allows for the simultaneous production of multiple strings of amino acids, called polypeptides, from one mRNA. When released, these polypeptides may be complete or, as is often the case, they may require further processing to become mature proteins.

Initiation



Figure 4: During initiation, the ribosome (grey globe) docks onto the mRNA at a position near the start codon (red).

At the start of the initiation phase of translation, the ribosome attaches to the mRNA strand and finds the beginning of the genetic message, called the start codon (Figure 4). This codon is almost always AUG, which corresponds to the amino acid methionine. Next, the specific tRNA molecule that carries methionine recognizes this codon and binds to it (Figure 5). At this point, the initiation phase of translation is complete.

Figure 5: To complete the initiation phase, the tRNA molecule that carries methionine recognizes the start codon and binds to it.

Elongation

Figure 6: Within the ribosome, multiple tRNA molecules bind to the mRNA strand in the appropriate sequence.

Figure 7: Each successive tRNA leaves behind an amino acid that links in sequence. The resulting chain of amino acids emerges from the top of the ribosome.

The next step in translation, called elongation, begins when the ribosome shifts to the next codon on the mRNA. At this point, the corresponding tRNA binds to this codon and, for a short time, there are two tRNA molecules on the mRNA strand. The amino acids carried by these tRNA molecules are then bound together. After this binding has occurred, the ribosome shifts again, and the first tRNA, which is no longer connected to its corresponding amino acid, is released (Figure 6). Now, the third codon in the mRNA strand is ready to bind with the appropriate tRNA (Figure 7). Once again, the tRNA binds to the mRNA strand, the third amino acid is added to the series, the ribosome shifts, and the second tRNA (which no longer carries an amino acid) is released. This process is repeated along the entire length of the mRNA, thereby elongating the polypeptide chain that is emerging from the top of the ribosome (Figure 8).



Figure 8: The polypeptide elongates as the process of tRNA docking and amino acid attachment is repeated.

Termination Eventually, after elongation has proceeded for some time, the ribosome comes to a stop codon, which signals the end of the genetic message. As a result, the ribosome detaches from the mRNA and releases the amino acid chain. This marks the final phase of translation, which is called termination (Figure 9).

Figure 9: The translation process terminates after a stop codon signals the ribosome to fall off the RNA.

What happens after translation? For many proteins, translation is only the first step in their life cycle. Moderate to extensive post-translational modification is sometimes required before a protein is complete. For example, some polypeptide chains require the addition of other molecules before they are considered "finished" proteins. Still other polypeptides must have specific sections removed through a process called proteolysis. Often, this involves the excision of the first amino acid in the chain (usually methionine, as this is the particular amino acid indicated by the start codon).Once a protein is complete, it has a job to perform. Some proteins are enzymes that catalyze biochemical reactions. Other proteins play roles in DNA replication and transcription. Yet other proteins provide structural support for the cell, create channels through the cell membrane, or carry out one of many other important cellular support functions.

Watch this video for a summary of translation in eukaryotes

Unit 1



What is the genetic code?

Reading the genetic code

What role do ribosomes play in translation?

What are the steps in translation?

What happens after translation?

Watch this video for a summary of translation in eukaryotes







Key Questions What other functions does RNA have in the cell?

What happens to proteins after they are translated?

Who discovered the relationship between DNA and proteins?

Key Concepts mRNA

transcription

ribosome

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PROGRESS

2.1 Introduction: How Does DNA Move from Cell to Cell? Prev Page

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Cell division is the mechanism by which DNA is passed from one generation of cells to the next and ultimately, from parent organisms to their offspring. Although eukaryotes and prokaryotes both engage in cell division, they do so in different ways. In particular, eukaryotic cells divide using the processes of mitosis and meiosis. Mitosis is common to all eukaryotes; during this process, a parent cell splits into two genetically identical daughter cells, each of which contains the same number of chromosomes as the parent cell. Meiosis, on the other hand, only occurs in eukaryotic organisms that reproduce sexually. During meiosis, the cells needed for sexual reproduction divide to produce new cells called gametes. Gametes contain half as many chromosomes as the other cells in the organism, and each gamete is genetically unique because the DNA of the parent cell is shuffled before the cell divides. This helps ensure that the new organisms formed as a result of sexual reproduction are also unique.Unlike eukaryotes, prokaryotes (which include bacteria) undergo a type of cell division known as binary fission. In some respects, this process is similar to mitosis; it requires replication of the cell's chromosomes, segregation of the copied DNA, and splitting of the parent cell's cytoplasm. However, binary fission is less complex than mitosis due to the fact that prokaryotic cells have a simpler structure than eukaryotic cells. This unit concentrates primarily on the two types of cell division used by eukaryotes. It begins by explaining the major steps involved in mitosis, and it next examines the major similarities and differences between this process and meiosis. The unit then explores recombination and mutation — two of the primary reasons why daughter cells don't always contain the same DNA as their parent cells.

Unit 2

Introduction: How Does DNA Move from Cell to Cell?

Replication and Distribution of DNA during Mitosis

Replication and Distribution of DNA during Meiosis

DNA Is Constantly Changing through the Process of Recombination

DNA Is Constantly Changing through the Process of Mutation

Some Sections of DNA Do Not Determine Traits, but Affect the Process of Transcription: Gene Regulation

Key Questions Where can I learn more about what happens to DNA during cell division?

Key Concepts DNA

mitosis

meiosis

Essentials of Genetics Contents Unit 2: How Does DNA Move from Cell to Cell?

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PROGRESS

2.2 Replication and Distribution of DNA during Mitosis Prev Page

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Most cells grow, perform the activities needed to survive, and divide to create new cells. These basic processes, known collectively as the cell cycle, are repeated throughout the life of a cell. Of the various parts of the cell cycle, the division portion is particularly important, because this is the point at which a cell passes its genetic information to its offspring cells. In many situations, division also ensures that new cells are available to replace the older cells within an organism whenever those cells die. Prokaryotic cells, which include bacteria, undergo a type of cell division known as binary fission. This process involves replication of the cell's chromosomes, segregation of the copied DNA, and splitting of the parent cell's cytoplasm. The outcome of binary fission is two new cells that are identical to the original cell. In contrast to prokaryotic cells, eukaryotic cells may divide via either mitosis or meiosis. Of these two processes, mitosis is more common. In fact, whereas only sexually reproducing eukaryotes can engage in meiosis, all eukaryotes — regardless of size or number of cells — can engage in mitosis. But how does this process proceed, and what sorts of cells does it produce?

What happens during mitosis? During mitosis, a eukaryotic cell undergoes a carefully coordinated nuclear division that results in the formation of two genetically identical daughter cells. Mitosis itself consists of five active steps, or phases: prophase, prometaphase, metaphase, anaphase, and telophase. Before a cell can enter the active phases of mitosis, however, it must go through a period known as interphase, during which it grows and produces the various proteins necessary for division. Then, at a critical point during interphase (called the S phase), the cell duplicates its chromosomes and ensures its systems are ready for cell division. If all conditions are ideal, the cell is now ready to move into the first phase of mitosis.

Prophase

Figure 1: During prophase, the chromosomes in a cell's nucleus condense to the point that they can be viewed using a light microscope.

Prophase is the first phase of mitosis. During this phase, the chromosomes inside the cell's nucleus condense and form tight structures. In fact, the chromosomes become so dense that they appear as curvy, dark lines when viewed under a microscope (Figure 1). Because each chromosome was duplicated during S phase, it now consists of two identical copies called sister chromatids that are attached at a common center point called the centromere.


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Figure 2: The mitotic spindle (white) begins to form outside the cell's nucleus.

Important changes also take place outside of the nucleus during prophase. In particular, two structures called centrosomes move to opposite sides of the cell during this phase and begin building the mitotic spindle. The mitotic spindle plays a critical role during the later phases of mitosis as it orchestrates the movement of sister chromatids to opposite poles of the cell (Figure 2).

Prometaphase After prophase is complete, the cell enters prometaphase. During prometaphase, the nuclear membrane disintegrates and the mitotic spindle gains access to the chromosomes. During this phase, a protein structure called the kinetochore is associated with the centromere on each sister chromatid. Stringlike structures called microtubules grow out from the spindle and connect to the sister chromatids at their kinetochores; one microtubule from one side of the spindle attaches to one sister chromatid in each chromosome, and one microtubule from the other side of the spindle attaches to the other sister chromatid (Figure 3a).

Metaphase Following prometaphase, metaphase begins. At the start of metaphase, the microtubules arrange the chromosomes in a line along the equator of the cell, known as the metaphase plate (Figure 3b). The centrosomes, on opposite poles of the cell, then prepare to separate the sister chromatids.

Figure 3: In metaphase (a), the microtubules of the spindle (white) have attached and the chromosomes have lined up on the metaphase plate. During anaphase (b), the sister chromatids are pulled apart and move toward opposite poles of the cell.

Anaphase After metaphase is complete, the cell enters anaphase. During anaphase, the microtubules attached to the kinetochores contract, which pulls the sister chromatids apart and toward opposite poles of the cell (Figure 3c). At this point, each chromatid is considered a separate chromosome.

Telophase

Figure 4: During telophase, two nuclear membranes form around the chromosomes, and the cytoplasm divides.

Finally, once anaphase is complete, the cell enters the last stage of the division process — telophase. During telophase, the newly separated chromosomes reach the mitotic spindle and a nuclear membrane forms around each set of chromosomes, thus creating two separate nuclei inside the same cell. As Figure 4 illustrates, the cytoplasm then divides to produce two identical cells.

Why is mitosis important? As previously mentioned, most eukaryotic cells that are not involved in the production of gametes undergo mitosis. These cells, known as somatic cells, are important to the survival of eukaryotic organisms, and it is essential that somatic parent and daughter cells do not vary from one another. With few exceptions, the mitotic process ensures that this is the case.



Therefore, mitosis ensures that each successive cellular generation has the same genetic composition as the previous generation, as well as an identical chromosome set.

Watch this historic video from 1960 to see mitosis in action

What happens during mitosis?

Why is mitosis important?

Watch this historic video from 1960 to see mitosis in action

Unit 2







Key Questions How do centromeres work?

What’s the difference between mitosis and meiosis?

Key Concepts chromosomes

replication

meiosis

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PROGRESS

2.3 Replication and Distribution of DNA during Meiosis Prev Page

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Like mitosis, meiosis is a form of eukaryotic cell division. However, these two processes distribute genetic material among the resulting daughter cells in very different ways. Mitosis creates two identical daughter cells that each contain the same number of chromosomes as their parent cell. In contrast, meiosis gives rise to four unique daughter cells, each of which has half the number of chromosomes as the parent cell. Because meiosis creates cells that are destined to become gametes (or reproductive cells), this reduction in chromosome number is critical — without it, the union of two gametes during fertilization would result in offspring with twice the normal number of chromosomes!Apart from this reduction in chromosome number, meiosis differs from mitosis in yet another way. Specifically, meiosis creates new combinations of genetic material in each of the four daughter cells. These new combinations result from the exchange of DNA between paired chromosomes. Such exchange means that the gametes produced through meiosis exhibit an amazing range of genetic variation.Finally, unlike mitosis, meiosis involves two rounds of nuclear division, not just one. Despite this fact, many of the other events of meiosis are similar to those that occur in mitosis. For example, prior to undergoing meiosis, a cell goes through an interphase period in which it grows, replicates its chromosomes, and checks all of its systems to ensure that it is ready to divide. Like mitosis, meiosis also has distinct stages called prophase, metaphase, anaphase, and telophase. A key difference, however, is that during meiosis, each of these phases occurs twice — once during the first round of division, called meiosis I, and again during the second round of division, called meiosis II.

What happens during meiosis I? As previously mentioned, the first round of nuclear division that occurs during the formation of gametes is called meiosis I. It is also known as the reduction division because it results in cells that have half the number of chromosomes as the parent cell. Meiosis I consists of four phases: prophase I, metaphase I, anaphase I, and telophase I.

Prophase I

Figure 1: Recombination is the exchange of genetic material between homologous chromosomes.

During prophase I, the chromosomes condense and become visible inside the nucleus. Because each chromosome was duplicated during the S phase that occurred just before prophase I, each now consists of two sister chromatids joined at the centromere. This arrangement means that each chromosome has the shape of an X. Once this chromosomal condensation has occurred, the members of each chromosome pair (called homologous chromosomes, because they are similar in size and contain similar genes), align next to each other. At this point, the two chromosomes in each pair become tightly associated with each other along their lengths in a process called synapsis. Then, while the homologous chromosomes are tightly paired, the members of each pair trade adjacent bits of DNA in a process called crossing over, also known as recombination (Figure 1). This trading of genetic material creates unique chromosomes that contain new combinations of alleles.


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At the end of prophase I, the nuclear membrane finally begins to break down. Outside the nucleus, the spindle grows out from centrosomes on each side of the cell. As in mitosis, the microtubules of the spindle are responsible for moving and arranging the chromosomes during division.

Metaphase I

Figure 2: Near the end of metaphase I, the homologous chromosomes align on the metaphase plate.

At the start of metaphase I, microtubules emerge from the spindle and attach to the kinetochore near the centromere of each chromosome. In particular, microtubules from one side of the spindle attach to one of the chromosomes in each homologous pair, while microtubules from the other side of the spindle attach to the other member of each pair. With the aid of these microtubules, the chromosome pairs then line up along the equator of the cell, termed the metaphase plate (Figure 2).

Anaphase I

Figure 3: During anaphase I, the homologous chromosomes are pulled toward opposite poles of the cell.

During anaphase I, the microtubules disassemble and contract; this, in turn, separates the homologous chromosomes such that the two chromosomes in each pair are pulled toward opposite ends of the cell (Figure 3). This separation means that each of the daughter cells that results from meiosis I will have half the number of chromosomes of the original parent cell after interphase. Also, the sister chromatids in each chromosome still remain connected. As a result, each chromosome maintains its X-shaped structure.

Telophase I

Figure 4: Telophase I results in the production of two nonidentical daughter cells, each of which has half the number of chromosomes of the original parent cell.

As the new chromosomes reach the spindle during telophase I, the cytoplasm organizes itself and divides in two. There are now two cells, and each cell contains half the number of chromosomes as the parent cell. In addition, the two daughter cells are not genetically identical to each other because of the recombination that occurred during prophase I (Figure 4).

Interkinesis At this point, the first division of meiosis is complete. The cell now rests for a bit before beginning the second meiotic division. During this period, called interkinesis, the nuclear membrane in each of the two cells reforms around the chromosomes. In some cells, the spindle also disintegrates and the chromosomes relax (although most often, the spindle remains intact). It is important to note, however, that no chromosomal duplication occurs during this stage.

What happens during meiosis II? During meiosis II, the two cells once again cycle through four phases of division. Meiosis II is sometimes referred to as an equational division because it does not reduce chromosome number in the daughter cells — rather, the daughter cells that result from meiosis II have the same number of chromosomes as the "parent" cells that enter meiosis II. (Remember, these "parent" cells already have half the number of chromosomes of the original parent cell thanks to meiosis I.)

Prophase II As prophase II begins, the chromosomes once again condense into tight structures, and the nuclear membrane disintegrates. In addition, if the spindle was disassembled during interkinesis, it reforms at this point in time.

Metaphase II

Figure 5: During metaphase II, the chromosomes align



along the cell's equatorial plate.

The events of metaphase II are similar to those of mitotic metaphase — in both processes, the chromosomes line up along the cell's equatorial plate, also called the metaphase plate, in preparation for their eventual separation (Figure 5).

Anaphase II

Figure 6: Anaphase II involves separation of the sister chromatids.

During anaphase II, microtubules from each spindle attach to each sister chromatid at the kinetochore. The sister chromatids then separate, and the microtubules pull them to opposite poles of the cell. As in mitosis, each chromatid is now considered a separate chromosome (Figure 6). This means that the cells that result from meiosis II will have the same number of chromosomes as the "parent" cells that entered meiosis II.

Telophase II

Figure 7: Telophase II results in the production of four daughter cells.

Finally, in telophase II, nuclear membranes reform around the newly separated chromosomes, which relax and fade from view. As soon as the cytoplasm divides, meiosis is complete. There are now four daughter cells — two from each of the two cells that entered meiosis II — and each daughter cell has half the normal number of chromosomes (Figure 7). Each also contains new mixtures of genes within its chromosomes, thanks to recombination during meiosis I.

Why is meiosis important? More about meiosis

Genes are packaged differently in mitosis and meiosis — but what is the effect of this difference?•

Meiosis is important because it ensures that all organisms produced via sexual reproduction contain the correct number of chromosomes. Meiosis also produces genetic variation by way of the process of recombination. Later, this variation is increased even further when two gametes unite during fertilization, thereby creating offspring with unique combinations of DNA. This constant mixing of parental DNA in sexual reproduction helps fuel the incredible diversity of life on Earth.

Watch this video for a summary of meiosis



What happens during meiosis I?

What happens during meiosis II?

Why is meiosis important?

Watch this video for a summary of meiosis

Unit 2







Key Questions How did sexual reproduction evolve?

What happens when meiosis goes wrong?

Key Concepts chromosome

meiosis

haploid

diploid

recombination

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PROGRESS

2.4 DNA Is Constantly Changing through the Process of Recombination Prev Page

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Recombination occurs when two molecules of DNA exchange pieces of their genetic material with each other. One of the most notable examples of recombination takes place during meiosis (specifically, during prophase I), when homologous chromosomes line up in pairs and swap segments of DNA. This process, also known as crossing over, creates gametes that contain new combinations of genes, which helps maximize the genetic diversity of any offspring that result from the eventual union of two gametes during sexual reproduction. Genetic diversity occurs because certain physical characteristics, like eye color, are variable; this variability is the result of alternate DNA sequences that code for the same physical characteristic. These sequences are commonly referred to as alleles. The various alleles associated with a specific trait are only slightly different from one another, and they are always found at the same location (or locus) within an organism's DNA. For example, no matter whether a person has blue eyes, brown eyes, or green eyes, the alleles for eye color are found in the same area of the same chromosome in all humans. The unique combination of alleles that all sexually reproducing organisms receive from their parents is the direct result of recombination during meiosis.

What happens during recombination? Genetic recombination is a complex process that involves alignment of two homologous DNA strands, precise breakage of each strand, equal exchange of DNA segments between the two strands, and sealing of the resultant recombined DNA molecules through the action of enzymes called ligases. Despite the complexity of this process, recombination events occur with remarkable accuracy and precision in the vast majority of instances. When recombination occurs during meiosis, the cell's homologous chromosomes line up extremely close to one another. Then, the DNA strand within each chromosome breaks in the exact same location, leaving two free ends. Each end then crosses over into the other chromosome and forms a connection called a chiasma. During this process, it is common for large sections of DNA containing many different genes to cross from one chromosome to another. Finally, as prophase I draws to a close and metaphase I begins, the crossing-over process concludes, and the homologous chromosomes prepare to separate. When the homologous chromosomes are later pulled apart during anaphase I, each chromosome carries new, unique allele combinations that are a direct result of recombination.

Does recombination occur in cells other than gametes? Beyond its role in meiosis, recombination is important to somatic cells in eukaryotes because it can be used to help repair broken DNA, even when the break involves both strands of the double helix. These breaks are known as double-stranded breaks, or DSBs. When DSBs happen, a homologous chromosome can serve as the template for synthesis of whatever portion of the genetic material has been lost as a result of the break. Then, once synthesized, this new DNA can be incorporated into the broken DNA strand, thereby repairing it. In effect, this is a form of recombination, because the broken-off area is replaced with new material from a homologous chromosome. Recombination can also be used in a similar way to repair smaller, single-stranded breaks. In general, recombination can occur any time homologous chromosomes pair up, whether they are freely floating in tandem or lined up on the metaphase plate during meiosis.

More on recombination

During recombination, what enzymes help break and rejoin DNA?•

Recombination isn't limited to eukaryotes, however. A special type of recombination called conjugation occurs in many prokaryotes, and it has been particularly well studied and characterized in E. coli bacteria. During conjugation, genetic material from one bacterium is transferred to another bacterium, and it is then recombined in the recipient cell. Recombination also plays important roles in DNA repair in prokaryotic organisms, just as it does in eukaryotic organisms.


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What happens during recombination?

Does recombination occur in cells other than gametes?

Unit 2







Key Questions How are recombination and sexual reproduction related?

Can errors in DNA be repaired?

How did Barbara McClintock discover recombination?

Key Concepts DNA

meiosis

mitosis

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PROGRESS

2.5 DNA Is Constantly Changing through the Process of Mutation Prev Page

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DNA is a dynamic and adaptable molecule. As such, the nucleotide sequences found within it are subject to change as the result of a phenomenon called mutation. Depending on how a particular mutation modifies an organism's genetic makeup, it can prove harmless, helpful, or even hurtful. Sometimes, a mutation may even cause dramatic changes in the physiology of an affected organism. Of course, in order to better understand the varying effects of mutations, it is first necessary to understand what mutations are and how they occur.

Where do mutations occur? Mutations can be grouped into two main categories based on where they occur: somatic mutations and germ-line mutations. Somatic mutations take place in non-reproductive cells. Many kinds of somatic mutations have no obvious effect on an organism, because genetically normal body cells are able to compensate for the mutated cells. Nonetheless, certain other mutations can greatly impact the life and function of an organism. For example, somatic mutations that affect cell division (particularly those that allow cells to divide uncontrollably) are the basis for many forms of cancer.Germ-line mutations occur in gametes or in cells that eventually produce gametes. In contrast with somatic mutations, germ-line mutations are passed on to an organism's progeny. As a result, future generations of organisms will carry the mutation in all of their cells (both somatic and germ-line).

What kinds of mutations exist? Mutations aren't just grouped according to where they occur — frequently, they are also categorized by the length of the nucleotide sequences they affect. Changes to short stretches of nucleotides are called gene-level mutations, because these mutations affect the specific genes that provide instructions for various functional molecules, including proteins. Changes in these molecules can have an impact on any number of an organism's physical characteristics. As opposed to gene-level mutations, mutations that alter longer stretches of DNA (ranging from multiple genes up to entire chromosomes) are called chromosomal mutations. These mutations often have serious consequences for affected organisms. Because gene-level mutations are more common than chromosomal mutations, the following sections focus on these smaller alterations to the normal genetic sequence.

Base substitution Base substitutions are the simplest type of gene-level mutation, and they involve the swapping of one nucleotide for another during DNA replication. For example, during replication, a thymine nucleotide might be inserted in place of a guanine nucleotide. With base substitution mutations, only a single nucleotide within a gene sequence is changed, so only one codon is affected (Figure 1).


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Figure 1: Only a single codon in the gene sequence is changed in base substitution mutation.

Although a base substitution alters only a single codon in a gene, it can still have a significant impact on protein production. In fact, depending on the nature of the codon change, base substitutions can lead to three different subcategories of mutations. The first of these subcategories consists of missense mutations, in which the altered codon leads to insertion of an incorrect amino acid into a protein molecule during translation; the second consists of nonsense mutations, in which the altered codon prematurely terminates synthesis of a protein molecule; and the third consists of silent mutations, in which the altered codon codes for the same amino acid as the unaltered codon.

Insertions and deletions

Figure 2: During an insertion mutation, the replicating strand "slips" or forms a wrinkle, which causes the extra nucleotide to be incorporated.

Insertions and deletions are two other types of mutations that can affect cells at the gene level. An insertion mutation occurs when an extra nucleotide is added to the DNA strand during replication. This can happen when the replicating strand "slips," or wrinkles, which allows the extra nucleotide to be incorporated (Figure 2). Strand slippage can also lead to deletion mutations. A deletion mutation occurs when a wrinkle forms on the DNA template strand and subsequently causes a nucleotide to be omitted from the replicated strand (Figure 3).

Figure 3: In a deletion mutation, a wrinkle forms on the DNA template strand, which causes a nucleotide to be omitted from the replicated strand.

Frameshift mutations



Figure 4: If the number of bases removed or inserted from a segment of DNA is not a multiple of three (a), a different sequence with a different set of reading frames is transcribed to mRNA (b).

Insertion or deletion of one or more nucleotides during replication can also lead to another type of mutation known as a frameshift mutation. The outcome of a frameshift mutation is complete alteration of the amino acid sequence of a protein. This alteration occurs during translation because ribosomes read the mRNA strand in terms of codons, or groups of three nucleotides. These groups are called the reading frame. Thus, if the number of bases removed from or inserted into a segment of DNA is not a multiple of three (Figure 4a), the reading frame transcribed to the mRNA will be completely changed (Figure 4b). Consequently, once it encounters the mutation, the ribosome will read the mRNA sequence differently, which can result in the production of an entirely different sequence of amino acids in the growing polypeptide chain.To better understand frameshift mutations, let's consider the analogy of words as codons, and letters within those words as nucleotides. Each word itself has a separate meaning, as each codons represents one amino acid. The following sentence is composed entirely of three-letter words, each representing a three-letter codon:THE BIG BAD FLY HAD ONE RED EYE AND ONE BLU EYE.Now, suppose that a mutation eliminates the sixth nucleotide, in this case the letter "G". This deletion means that the letters shift, and the rest of the sentence contains entirely new "words":THE BIB ADF LYH ADO NER EDE YEA NDO NEB LUE YE.This error changes the relationship of all nucleotides to each codon, and effectively changes every single codon in the sequence. Consequently, there is a widespread change in the amino acid sequence of the protein. Lets consider an example with an RNA sequence that codes for a sequence of amino acids:AUG AAA CUU CGC AGG AUG AUG AUGWith the triplet code, the sequence shown in figure 5 corresponds to a protein made of the following amino acids: Methionine-Lysine-Leucine-Arginine-Arginine-Methionine-Methionine-Methionine

Figure 5: This sequence of mRNA codes for the amino acids methionine-lysine-leucine-arginine-arginine-methionine-methionine-methionine.

Now, suppose that a mutation occurs during replication, and it results in deletion of the fourth nucleotide in the sequence. When separated into triplet codons, the nucleotide sequence would now read as follows (Figure 6): AUG AAC UUC GCA GGA UGA UGA UG This series of codons would encode the following sequence of amino acids: Methionine-Asparagine-Phenylalanine-Alanine-Glycine-STOP-STOP



Figure 6: If the fourth nucleotide in the sequence is deleted, the reading frame shifts and the amino acid sequence changes to methionine-asparagine-phenylalanine-alanine-glycine-STOP-STOP

Each of the stop codons tells the ribosome to terminate protein synthesis at that point. Consequently, the mutant protein is entirely different due to the deletion of the fourth nucleotide, and it is also shorter due to the appearance of a premature stop codon. This mutant protein will be unable to perform its necessary function in the cell.

What causes mutations? Mutations can arise in cells of all types as a result of a variety of factors, including chance. In fact, some of the mutations discussed above are the result of spontaneous events during replication, and they are thus known as spontaneous mutations. Slippage of the DNA template strand and subsequent insertion of an extra nucleotide is one example of a spontaneous mutation; excess flexibility of the DNA strand and the subsequent mispairing of bases is another. Environmental exposure to certain chemicals, ultraviolet radiation, or other external factors can also cause DNA to change. These external agents of genetic change are called mutagens. Exposure to mutagens often causes alterations in the molecular structure of nucleotides, ultimately causing substitutions, insertions, and deletions in the DNA sequence.

What are the consequences of mutations? More on mutation

The sickle-cell trait: A beneficial mutation•

Mutations are a source of genetic diversity in populations, and, as mentioned previously, they can have widely varying individual effects. In some cases, mutations prove beneficial to an organism by making it better able to adapt to environmental factors. In other situations, mutations are harmful to an organism — for instance, they might lead to increased susceptibility to illness or disease. In still other circumstances, mutations are neutral, proving neither beneficial nor detrimental outcomes to an organism. Thus, it is safe to say that the ultimate effects of mutations are as widely varied as the types of mutations themselves.

Watch these videos for a summary of the different types of gene-level mutation

Unit 2



Where do mutations occur?

What kinds of mutations exist?

What causes mutations?

What are the consequences of mutations?

Watch these videos for a summary of the different types of gene-level mutation







Key Questions Is it possible to have too many mutations?

How can you have a disease-causing mutation but not have the disease?

Key Concepts DNA

replication

translation

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PROGRESS

2.6 Some Sections of DNA Do Not Determine Traits, but Affect the Process of Transcription: Gene Regulation

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The genetic code is universal and contains the instructions for all life on Earth. But the diversity of life relies on more than just the genetic code itself — it also relies on the variety of ways in which this code is used in different organisms. In much the same way that an orchestra depends upon a conductor to direct the individual musicians, all cells depend upon regulatory mechanisms to determine which of their genes are "turned on" and which are "turned off" at any given time. In other words, these regulatory mechanisms control gene expression.But why is this control necessary? To better understand the answer to this question, consider the example of a skin cell and a brain cell located within the same organism. Both of these cells contain the same set of genetic information, but each has a unique function within the organism. Both cells, for instance, carry the gene associated with skin pigmentation, but only the skin cell actually expresses this particular gene and produces the pigment. In order for this gene to be expressed by the skin cell, it must be transcribed into mRNA and then translated into protein — and regulatory mechanisms are what trigger the transcription of this particular gene to occur (or not occur, in the case of the brain cell). In fact, regulatory mechanisms are the reason why some genes are expressed in every cell in an organism regardless of type, but other genes are expressed by only certain types of cells under specific sets of circumstances.

Promoters and proteins In order to understand how regulatory mechanisms work, it's first necessary to understand that not all nucleotide sequences in a strand of DNA code for the production of proteins. Rather, some of these noncoding sequences serve as binding sites for the various protein molecules required to start or regulate the transcription process. For example, a group of nucleotides known as a promoter sequence lies near the beginning of most genes and provides a binding site for RNA polymerase to begin transcription. Similarly, other noncoding sequences near the promoter sequence function as protein binding sites that can either induce or block transcription. This basic system affects gene expression in both prokaryotes and eukaryotes, albeit in different ways.

How do prokaryotes regulate gene expression? In single-celled prokaryotes such as bacteria, multiple genes that work together often share the same promoter. Between the promoter sequence and these genes, there is a sequence called an operator at which a protein, known as a repressor, can bind and block transcription by blocking the binding of RNA polymerase. This system of promoter, operator, and gene(s) is called an operon.

Turning genes on One especially well-known operon is the lac operon found in E. coli bacteria. This operon contains the three genes E. coli cells need to break down lactose. (Lactose is a sugar molecule that these cells often use as a source of energy.) When lactose is not present in a bacterium's environment, the protein products of these three genes aren't needed. As a result, a repressor protein binds to the operator of the lac operon and blocks transcription of the three genes. In contrast, when lactose is present, a molecule of this sugar binds to the repressor protein and changes its shape. The shape change prevents the repressor from binding to the operator, thereby permitting transcription of the three genes in the lac operon to occur. In this case, lactose itself "turns on" the genes of the lac operon, which means that it acts as an inducer.

Turning genes off In prokaryotes, a similar system can also be used to turn genes off. Consider, for example, the E. coli trp operon, which contains the genes required to make the amino acid tryptophan. This operon functions much like the lac operon except for


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one major difference — specifically, the repressor protein in this system only binds with the operator sequence when tryptophan is present. Here, tryptophan binds with the repressor, thereby changing the repressor's shape such that it fits with the operator. This means that tryptophan acts as a co-repressor, because it helps turn the genes of the trp operon off.

Turning genes up or down Gene expression is not always an all-or-nothing process, however. Within prokaryotes, genes can be expressed to varying degrees. The exact degree of expression is controlled by a stack of molecules called CAP-cAMP, which binds to DNA upstream of the promoter (i.e. on the 5' side of the promoter, at some distance away) and causes RNA polymerase to bind to the promoter more efficiently. This enables cells to control the degree to which a gene is transcribed. By increasing the amount of cAMP available, a cell allows a greater degree of transcription to occur. By decreasing the amount of cAMP available, the cell allows a lesser degree of transcription to occur.

How do eukaryotes regulate gene expression? Gene expression is much more complicated in eukaryotic cells than it is in prokaryotic cells. This is due, in large part, to the fact that eukaryotic cells must differentiate into different cell types, and they also contain a greater number of genes than prokaryotic cells. Furthermore, the transcription and translation sites of eukaryotic DNA are separated from one another by the nuclear membrane. Given these complicating factors, eukaryotic cells employ a greater variety of control strategies than prokaryotic cells, and they do so at various steps in both transcription and translation. Nonetheless, each of these strategies begins at the level of DNA.

Control at the DNA level

Figure 1: Eukaryotic cells must tightly fold their DNA so that it fits within the cellular nucleus.

Eukaryotic cells contain a large amount of DNA, and they must tightly fold this DNA to fit it inside the cellular nucleus (Figure 1). One consequence of this folding, however, is that under normal circumstances, RNA polymerase cannot bind to promoter sequences and trigger transcription of the related genes. Thus, by selectively unfolding certain segments of their DNA at certain times, eukaryotic cells can control gene expression simply by making promoter sequences accessible to binding by RNA polymerase. In addition, some cells produce and transcribe multiple RNA copies of important genes, which results in the production of large amounts of protein product.

Control at the transcription level In eukaryotes, control at the level of transcription is specific and efficient. Eukaryotic cells do not have operator sequences like prokaryotic cells do; rather, different kinds of regulator sequences occur upstream of eukaryotic promoters and serve as sites for the binding of RNA polymerase. In some instances, enhancer sequences occur upstream of these regulator sequences and bind to activator proteins to further stimulate transcription. Silencer sequences that reduce transcription may also be present. These sequences bind to repressor proteins and turn transcription off by interfering with RNA polymerase binding.

Control via RNA splicing

More on gene expression

Can the central dogma be reversed?•How can the environment affect gene expression?•How does eukaryotic DNA unfold and open?•How, exactly, does RNA splicing occur?•

In some cases, transcription occurs, but the resulting mRNA is not translated exactly as it was created. This is the result of another control mechanism known as alternative splicing. Splicing is a normal process by which noncoding regions of a gene, known as introns, are cut out of a segment of mRNA. In alternative splicing, some of the coding regions are cut out as well, which results in the eventual creation of a different protein than originally coded for in the DNA. Specific conditions within a cell dictate which coding sequences to remove, and alternative splicing can result in the creation of many different proteins from only a single gene.

Control via RNA stability Sometimes, the stability of the mRNA molecule itself affects levels of eukaryotic gene expression. Once created, mRNA does not last forever; stable mRNAs will last long enough to be translated many times (thereby producing many protein molecules), but unstable mRNAs may not last long enough to be translated at all. The stability of an mRNA molecule depends upon its nucleotide sequence and the length of its poly-A tail, or the long sequence of adenines added to one end of the mRNA after transcription. The longer an mRNA's poly-A tail is, the more stable the mRNA molecule will be.

Control at the translation level



After a gene has been transcribed, control mechanisms can still regulate its expression during the translation process. Within eukaryotes, special repressor proteins can bind to mRNA molecules and physically block their translation. In addition, after translation, unneeded proteins may be marked for degradation by certain molecules before they have the opportunity to do their job.

Promoters and proteins

How do prokaryotes regulate gene expression?

How do eukaryotes regulate gene expression?

Unit 2







Key Questions What else is there to know about operons?

How do environmental influences affect gene expression?

What role does noncoding RNA play in gene expression?

How do genes express and regulate themselves?

Key Concepts intron

exon

splicing

transcription factor

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PROGRESS

3.1 Introduction: How Is Genetic Information Passed between Organisms? Prev Page

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Inheritance is the passing of traits from parents to offspring. Our modern understanding of inheritance comes from a set of principles proposed by Austrian monk and researcher Gregor Mendel in 1865. Interestingly, Mendel didn't arrive at these principles by studying human beings, but rather by studying the common pea plant, Pisum sativum. Although scientists now know that there are many exceptions to the patterns Mendel described, these principles describe the simplest mechanisms of inheritance. Moreover, because these so-called principles of Mendelian genetics hold true for organisms of many different types (including humans), they serve as the foundation for scientists' current understanding of heredity. This unit takes a closer look at the concept of inheritance. It begins with a description of Mendel's basic principles, each of which is illustrated with the fruit fly Drosophila melanogaster, an insect that is widely used in the field of modern genetics. The unit then examines how variability in inheritance patterns can help researchers understand and test relationships between genes. Finally, the unit concludes with a discussion of how inheritance can involve different mechanisms in different organisms, including bacteria.

Unit 3

Introduction: How Is Genetic Information Passed between Organisms?

Each Organism's Traits Are Inherited from a Parent through Transmission of DNA

Inheritance of Traits by Offspring Follows Predictable Rules

Some Genes Are Transmitted to Offspring in Groups via the Phenomenon of Gene Linkage

The Sex of Offspring Is Determined by Particular Chromosomes

Some Organisms Transmit Genetic Material to Offspring without Cell Division

Key Questions Where can I learn more about how DNA is inherited?

Key Concepts inheritance

Mendelian trait

linkage

Essentials of Genetics Contents Unit 3: How Is Genetic Information Passed between Organisms?

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PROGRESS

3.2 Each Organism's Traits Are Inherited from a Parent through Transmission of DNA

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Drosophila chromosome

Scientists first discovered chromosomes in the nineteenth century, when they were gazing at cells through light microscopes. But how did they figure out what chromosomes do? And how did they link chromosomes — and the specific genes within them — to the concept of inheritance? After a long period of observational studies through microscopes, several experiments with fruit flies provided the first evidence.

What is a gene? Physically, a gene is a segment (or segments) of a chromosome. Functionally, a gene can play many different roles within a cell. Today, most scientists agree that genes correspond to one or more DNA sequences that carry the coding information required to produce a specific protein, and that protein in turn carries out a particular function within the cell. Scientists also know that the DNA that makes up genes is packed into structures called chromosomes, and that somatic cells contain twice as many chromosomes as gametes (i.e., sperm and egg cells).But what were the key scientific discoveries that helped establish these principles? As it turns out, the connections between genes, chromosomes, DNA, and heredity were not recognized until long after researchers caught their initial glimpse of chromosomes. The following sections present an abbreviated summary of the major discoveries that revealed these connections.

The first words for genes: Elementen and gemmules

Gregor Mendel

Charles Darwin

Researchers began hypothesizing about the existence of genes as early as the mid-1800s — although they used different terminology than today's scientists when doing so. For example, during the 1860s, Austrian monk and scientist Gregor Mendel examined how certain physical characteristics of pea plants (e.g., seed color, seed shape, flower color, etc.), which he called traits, were passed down to successive generations. Mendel speculated that the cells that made up the pea plants


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contained material that carried the information about these traits from one generation to the next. Mendel called this material "elementen," and he proposed that during sexual reproduction, each parent contributed a form of elementen to the resulting offspring. This combination of parental elementen then determined which form of a trait was visible in the offspring.Around the same time, British biologist Charles Darwin independently proposed that traits could be passed on to successive generations in packets he referred to as "gemmules." Darwin also speculated that gemmules traveled from every body part to the sexual organs, where they were stored. The most remarkable feature of both Mendel's and Darwin's proposals is that neither of the two scientists knew about nucleotides or about any of the biochemical substances that are now widely recognized as DNA.After Mendel and Darwin put their ideas forward, several other scientists reported their own discoveries about the ways in which the appearance of the cellular nucleus changed during cell division. Although these scientists' observations connected genes to chromosomes, they still didn't use the word "gene" to represent what Mendel called "elementen" or what Darwin called "gemmules." The concept of the "chromosome," however, was rapidly becoming much clearer.

Describing chromosomes

Figure 1: Sample image from Walther Flemming's drawings of chromosome behavior during mitosis.

In 1882, German biologist Walther Flemming was the first person to describe what scientists now know as chromosomes. Flemming's elegant drawings showed how chromosomes aligned and were eventually pulled apart during mitosis (Figure 1). Then, in 1914, another German researcher named Theodor Boveri provided the first descriptions of meiosis, also supported by detailed drawings, except these drawings showed how the number of chromosomes in a parent cell was reduced by half in the resulting gametes.

Connecting heredity to chromosomes

Walter Sutton

Scientists now knew how chromosomes behaved during both mitosis and meiosis, but they still hadn't linked Mendel's ideas of heredity with these observations.Some thirty-five years after Mendel's work, however, American researcher Walter Sutton proposed a connection between trait inheritance and the path that chromosomes travel during meiotic cell division and gamete formation. In particular, when observing meiotic cells in the testes of the lubber grasshopper (Brachystola magna), Sutton noted that it was possible to distinguish and track the individual chromosomes in these cells. He also noticed that these chromosomes existed in pairs that could be distinguished from other pairs by their size, and that upon the union of two gametes during fertilization, the chromosomes in the newly fertilized cell maintained their original forms. Sutton therefore proposed that all chromosomes have a stable structure, or "individuality," that is maintained between generations. Bringing the idea full circle, Sutton also concluded that the association of paternal and maternal chromosomes in pairs after gamete fusion, and their subsequent separation during the reducing division of meiosis, "may constitute the physical basis of the Mendelian law of heredity." With these words, Sutton first articulated what is now known as the chromosome theory of inheritance.

Confirming the chromosome theory of inheritance Though Sutton believed he had described evidence for the physical basis of Mendel's principles of inheritance, definitive proof was still lacking. Scientists thus needed an experimental system in which the inheritance of genetic traits could be linked directly to the movement of chromosomes. Such an opportunity presented itself soon thereafter, with a distinct mutation in the fruit fly Drosophila melanogaster.During the early years of the twentieth century, fruit flies were the model organism of choice for many genetic researchers, including those who worked in Thomas Hunt Morgan's famous "fly room" laboratory at Columbia University in New York City. Why fruit flies? For one, fruit flies breed quickly, so they are efficient organisms for scientists who want to follow traits in offspring through several generations. Also, the fruit fly has only four pairs of chromosomes, so these chromosomes can be easily recognized and tracked from one generation to the next. The Morgan lab therefore set out to examine patterns of



heredity through multiple series of breeding experiments with fruit flies, and in doing so, they hoped to discover exactly how heredity was or was not related to chromosomes. Eventually, the answer to this question became clear-all because of the appearance of a lone fly with unusually colored eyes.

Morgan's lab connects eye color with inheritance of sex chromosomes

Figure 2: Although white-eyed males were bred in several cycles with female flies, only male offspring were passed the unique trait.

Fruit flies normally have brilliant, red-colored eyes, although occasionally, male flies with white eyes would appear in Morgan's laboratory (Figure 2). Intrigued by these white-eyed males, Morgan's research team decided to follow this trait through multiple breeding cycles of white eyed males and red-eyed females. In doing so, the researchers noticed that the white-eyed trait was only passed onto other male flies. In fact, after the researchers conducted multiple rounds of breeding white-eyed males and red-eyed females without identifying a single white-eyed female, they began to suspect that white eye color was inherited along with the sex of the fly.This observation confirmed the chromosome theory proposed by Sutton. According to this theory, male flies should always inherit male characteristics by virtue of inheriting the "male" chromosome (denoted Y); likewise, female flies should always inherit "female" chromosomes (denoted X), which means that these flies should not display male characteristics. Thousands of matings had convinced the Morgan lab that white eyes were clearly a characteristic associated with only the Y chromosome.One day, however, the researchers in Morgan's lab encountered an unusual fly that challenged their conclusions regarding the relationship between sex and eye color. This exceptional fly was a white-eyed female that had resulted from a cross between two parents with red eyes. Where did this female's white-eye trait come from? How could this trait be explained? And did this fly disprove the basic premise of the chromosome theory?

The exception proves the rule In the Morgan lab's search to make sense of the white-eyed female, Lilian Vaughn Morgan (Thomas Morgan's wife) suggested that this exceptional fly might have an unusual chromosome composition. The research team seized upon this suggestion, and they soon examined some of the white-eyed female's cells under the microscope. In doing so, the scientists realized that Mrs. Morgan was right - the fly's cells did indeed appear to contain an extra chromosome. Specifically, these cells contained two X chromosomes as well as a single Y chromosome. The extra chromosome was determined to be the result of a defect during meiosis that caused a high frequency of nondisjunction. (Nondisjunction is the failure of two sister chromatids to separate during the second meiotic division.) Thus, when an egg containing two nondisjoined X chromosomes, each of which carried the mutant white gene, was fertilized by a sperm cell containing the Y chromosome, the product was an XXY female with white eyes. Rather than disproving the chromosome theory, this "exceptional" female actually provided strong experimental support that genes were in fact located on chromosomes.The Morgan lab's observations can be simplified as follows: • First observation: Flies normally have red eyes. • Second observation: Males sometimes have white eyes. • Third observation: Females never have white eyes. • Fourth observation, exception to the rule: A rare female has white eyes, and she also has an extra chromosome. • Conclusion: Traits are found on chromosomes. Morgan's lab also found that the trait for white eyes could appear even if a fly's father didn't have white eyes. This showed that flies could carry the white-eye trait even if they didn't show it themselves. The trait could vanish and reappear only in certain exceptional moments. This concept forms the basis of our modern understanding of the hereditary substance that exists on chromosomes but is not always apparent in the outward physical traits of an organism. Whereas Mendel called this substance "elementen" and Darwin called it "gemmules," researchers now use the more familiar term "gene."

Summary

Walther Flemming



When considered in view of all this information, the chromosome theory of inheritance was not the work of a single scientist. Rather, the theory was built on collaboration between multiple researchers working over a period of many decades. The seeds of this theory were first planted in the 1860s, when Gregor Mendel and Charles Darwin each proposed possible physical elements of heredity. It wasn't until several decades later, following Walther Flemming's discovery of chromosomes and description of their behavior during mitosis, that a probable mechanism for the transmission of traits was uncovered.Subsequently, Theodor Boveri and Walter Sutton's research strengthened the idea of a connection between chromosomes and hereditary elements. But direct evidence that explicitly demonstrated that traits exist on specific chromosomes wasn't delivered until the Morgan lab's experiments with fruit flies at the beginning of the twentieth century. Thus, after nearly fifty years of speculation, scientists were finally able to confirm what they had long suspected: chromosomes are indeed the physical carriers of hereditary information, and this information exists in the form of genes.

What is a gene?

The first words for genes: Elementen and gemmules

Describing chromosomes

Connecting heredity to chromosomes

Confirming the chromosome theory of inheritance

Summary

Unit 3







Key Questions What else can go wrong with chromosomes in meiosis?

How do meiosis and mitosis differ in the transmission of genes?

Key Concepts meiosis

gametes

chromosome

gene

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PROGRESS

3.3 Inheritance of Traits by Offspring Follows Predictable Rules Prev Page

Next Page Genes come in different varieties, called alleles. Somatic cells contain two alleles for every gene, with one allele provided by each parent of an organism. Often, it is impossible to determine which two alleles of a gene are present within an organism's chromosomes based solely on the outward appearance of that organism. However, an allele that is hidden, or not expressed by an organism, can still be passed on to that organism's offspring and expressed in a later generation.

Tracing a hidden gene through a family tree The family tree in Figure 1 shows how an allele can disappear or "hide" in one generation and then reemerge in a later generation. In this family tree, the father in the first generation shows a particular trait (as indicated by the black square), but none of the children in the second generation show that trait. Nonetheless, the trait reappears in the third generation (black square, lower right). How is this possible? This question is best answered by considering the basic principles of inheritance.

Figure 1: In this family pedigree, black squares indicate the presence of a particular trait in a male, and white squares represent males without the trait. White circles are females. A trait in one generation can be inherited, but not outwardly apparent before two more generations (compare black squares).

Mendel's principles of inheritance Gregor Mendel was the first person to describe the manner in which traits are passed on from one generation to the next (and sometimes skip generations). Through his breeding experiments with pea plants, Mendel established three principles of inheritance that described the transmission of genetic traits before genes were even discovered. Mendel's insights greatly expanded scientists' understanding of genetic inheritance, and they also led to the development of new experimental methods.One of the central conclusions Mendel reached after studying and breeding multiple generations of pea plants was the idea that "[you cannot] draw from the external resemblances [any] conclusions as to [the plants'] internal nature." Today, scientists use the word "phenotype" to refer to what Mendel termed an organism's "external resemblance," and the word "genotype" to refer to what Mendel termed an organism's "internal nature." Thus, to restate Mendel's conclusion in modern terms, an organism's genotype cannot be inferred by simply observing its phenotype. Indeed, Mendel's experiments revealed that phenotypes could be hidden in one generation, only to reemerge in subsequent generations. Mendel thus wondered how organisms preserved the "elementen" (or hereditary material) associated with these traits in the intervening generation, when the traits were hidden from view.

How do hidden genes pass from one generation to the next? Although an individual gene may code for a specific physical trait, that gene can exist in different forms, or alleles. One allele for every gene in an organism is inherited from each of that organism's parents. In some cases, both parents provide the same allele of a given gene, and the offspring is referred to as homozygous ("homo" meaning "same") for that allele. In other cases, each parent provides a different allele of a given gene, and the offspring is referred to as heterozygous ("hetero" meaning "different") for that allele. Alleles produce phenotypes (or physical versions of a trait) that are either dominant or recessive. The dominance or recessivity associated with a particular allele is the result of masking, by which a dominant phenotype hides a recessive phenotype. By this logic, in heterozygous offspring only the dominant phenotype will be apparent.

The relationship of alleles to phenotype: an example


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PROGRESS

3.4 Some Genes Are Transmitted to Offspring in Groups via the Phenomenon of Gene Linkage

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Black fly with short wings

Although Mendel's principle of independent assortment states that alleles of different genes will segregate independently into gametes, in reality, this is not always the case. Sometimes, alleles of certain genes are inherited together, and they do not appear to undergo independent assortment at all.Indeed, shortly after Mendel's discoveries about inheritance patterns became widely known, numerous researchers began to notice exceptions to his principles. For example, they realized that some crosses contradicted Mendel's principle of independent assortment, because these crosses produced organisms with certain phenotypes far more frequently than traditional Mendelian genetics predicted.Based on these findings, these scientists hypothesized that certain alleles of one gene were somehow coupled with certain alleles of another gene; however, they were not sure how this could occur. This phenomenon is now known as genetic linkage, and it generally describes an inheritance pattern in which two genes located in close proximity to each other on the same chromosome have a biased association between their alleles. This, in turn, causes these alleles to be inherited together instead of assorting independently. Genetic linkage is a violation of the Mendelian principle of independent assortment.

Independent assortment in test crosses To understand linkage, we must first compare it to an example of independent assortment of parental gametes. The best way to generate such an example is through a dihybrid test cross, which considers two different genes during a cross between two heterozygote parents. Mendel's principle of independent assortment predicts that the alleles of the two genes will be independently distributed into gametes. Thus, according to Mendel's principles, a dihybrid cross between two heterozygous fruit flies with brown bodies and red eyes (BbEe X BbEe) should yield offspring with nine possible genotypes (BBEE, BBEe, BBee, BbEE, BbEe, Bbee, bbEE, bbEe, and bbee) and four possible phenotypes (brown body with red eyes, brown body with brown eyes, black body with red eyes, and black body with brown eyes) (Figure 1, left). In this case, the ratio of phenotypes observed among the offspring is 9 (brown body, red eyes): 3 (brown body, brown eyes): 3 (black body, red eyes): 1 (black body, brown eyes) (Figure 1, right). This 9:3:3:1 phenotypic ratio is the classic Mendelian ratio for a dihybrid cross in which the alleles of two different genes assort independently into gametes.


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Figure 1: A classic Mendelian example of independent assortment: the 9:3:3:1 phenotypic ratio associated with a dihybrid cross (BbEe × BbEe).

In another example of Mendel's independent assortment principle, a test cross between a heterozygous BbEe fly and a homozygous bbee fly will yield offspring with only four possible genotypes (BbEe, Bbee, bbEe, and bbee) and four possible phenotypes (brown body with red eyes, brown body with brown eyes, black body with red eyes, and black body with brown eyes), as shown in Figure 2. Thus, in this case, the ratio of phenotypes observed among the offspring will be 1 (brown body, red eyes): 1 (brown body, brown eyes): 1 (black body, red eyes): 1 (black body, brown eyes).

Figure 2: This 1:1:1:1 phenotypic ratio is the classic Mendelian ratio for a test cross in which the alleles of the two genes assort independently into gametes (BbEe × bbee).

Exceptions to independent assortment In nature, some fruit fly traits like those described above assort independently, whereas others do not. As an example, consider the relationship between fruit fly body color and wing length. Here, the gene for wing length is represented by two alleles, V and v; the V allele codes for long wings, which is the dominant phenotype, and the v allele codes for short, misshapen wings (called vestigial wings), which is the recessive phenotype (Figure 3).

Figure 3: In fruit flies, the dominant V allele produces long wings, whereas the recessive v allele produces vestigial wings. Thus, flies with the genotype VV or Vv will have long wings, and flies with the genotype vv will have vestigial wings.

Figure 4: On the left is the expected phenotypic ratio of the offspring from a BbVv × bbvv cross (1:1:1:1). However, because the alleles BV and bv are linked, the observed phenotypic ratio is much different (5:1:1:5) than the expected ratio.

In order to observe the inheritance pattern associated with fruit fly body color and wing length, a test cross between a BbVv fly and a bbvv fly can be performed. The results of this cross, however, will not follow the classic 1:1:1:1 phenotypic ratio expected with independent assortment. Instead, the offspring of this particular cross will be present in a 5:1:1:5 ratio (5 brown body with long wings: 1 brown body with vestigial wings: 1 black body with long wings: 5 black body with vestigial wings). These results indicate that there is a bias toward brown body color and normal wings being inherited together (BV), as well as toward black body color and vestigial wings being inherited together (bv), from the parent with the BbVv genotype (Figure 4). Note that the parent with the bbvv genotype can only contribute bv alleles. What is the reason for this 5:1:1:5 non-Mendelian phenotypic ratio? It turns out that the body color and wing length genes are linked, which means they are located very close to each other on the same chromosome. The consequence of this is that these



gene alleles are much less likely to segregate independently into gametes. In addition, if two genes are linked in this way, then gametes are more likely to contain specific allele combinations. In this example, those combinations of alleles are BV and bv. As such, the heterozygous parent produces more BV and bv gametes than Bv and bV gametes. (Recall that the homozygous parent can only produce bv gametes.) This is why, when the BbVv fly is crossed with the bbvv fly, the resulting offspring are more likely to have BbVv and bbvv genotypes than Bbvv and bbVv genotypes, and the observed phenotypic ratio is 5:1:1:5. In fact, because the alleles do not assort independently into gametes during meiosis, Punnett squares like the ones shown in Figures 2 and 3 cannot be used to accurately predict inheritance patterns for crosses involving linked genes. To return to the fruit fly example, linkage means that the BbVv parent is more likely to produce gametes that match those contributed by its own parents: BV and bv. Therefore, offspring with parental genotypes (BbVv and bbvv) are more common than offspring with non-parental, or recombinant, genotypes (Bbvv and bbVv) after the test cross. This means the parental genotypes and their corresponding phenotypes are observed five times more often than the recombinant genotypes and their corresponding phenotypes.

Summary What is the lesson to be learned from the body color-wing length example? In short, whenever two genes are linked because of their location on a chromosome, their alleles will not segregate independently during gamete formation. As a result, test crosses involving alleles of linked genes will yield phenotypic ratios that stray from the classic Mendelian ratios. Also in the case of linked genes, the phenotypic ratio will show higher numbers of offspring with the parental genotypes than offspring with the recombinant genotypes.

Make your own fly Thomas Hunt Morgan

The fly geneticist and his remarkable findings•

Breeding flies is an exciting way to learn genetics. There are many possible allele combinations within a fruit fly, and you can explore them via the interactive image below. Just click on a genotype button from each category below to make your own customized fly (Drosophila melanogaster).

Unit 3




Key Questions Who discovered gene linkage?

What is sex linkage in flies?

How can we use linkage to map genes in a chromosome?

What do scientists like to argue about?

Key Concepts linkage



Independent assortment in test crosses

Exceptions to independent assortment

Summary

Make your own fly




complete linkage

physical linkage

incomplete linkage

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PROGRESS

3.5 The Sex of Offspring Is Determined by Particular Chromosomes Prev Page

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In humans and many other animal species, sex is determined by specific chromosomes. How did researchers discover these so-called sex chromosomes? The path from the initial discovery of sex chromosomes in 1891 to an understanding of their true function was paved by the diligent efforts of multiple scientists over the course of many years. As often happens during a lengthy course of discovery, scientists observed and described sex chromosomes long before they knew their function.

An idea inspired by the "X element" By the 1880s, scientists had established methods for staining chromosomes so that they could be easily visualized using a simple light microscope. With this staining method, scientists were able to observe cell division and to identify the steps that occurred during both mitosis and meiosis (Figure 1).

Figure 1: Cell division observed through the microscope (left) is redrawn to show the action of chromosomes (right). Arrows indicate the axis along which the cell divides.

The first indication that sex chromosomes were distinct from other chromosomes came from experiments conducted by German biologist Hermann Henking in 1891. While using a light microscope to study sperm formation in wasps, Henking noticed that some wasp sperm cells had 12 chromosomes, while others had only 11 chromosomes. Also, during his observation of the stages of meiosis leading up to the formation of these sperm cells, Henking noticed that the mysterious twelfth chromosome looked and behaved differently than the other 11 chromosomes. Accordingly, he named the twelfth chromosome the "X element" to represent its unknown nature. Interestingly, when Henking used a light microscope to study egg formation in female grasshoppers, he was unable to spot the X element.Based on his observations, Henking hypothesized that this extra chromosome, the X element, must play some role in determining the sex of insects. However, he was unable to gather any direct evidence to support his hypothesis.


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Figure 2: The darkling beetle,

Tenebrio molitor.

More than a decade after Henking's work, Nettie Stevens surveyed multiple beetle species and examined the inheritance patterns of their chromosomes. In 1905, while studying the gametes of the beetle Tenebrio molitor (Figure 2), Stevens noted an unusual-looking pair of chromosomes that separated to form sperm cells in the male beetles. Based on her comparisons of chromosome appearance in cells from male and female beetles, Stevens proposed that these accessory chromosomes were related to the inheritance of sex.Over time, other scientists studied the appearance of chromosomes in a wide variety of animal species, and it became clear that there was a relationship between the physical appearance and number of chromosomes in gametes and somatic cells from males and females of a given species.

The variety of sex determination systems

Figure 3: Example set of male human chromosomes. In the image, the X and Y chromosomes are indicated by arrows.

In humans, females inherit an X chromosome from each parent, whereas males always inherit their X chromosome from their mother and their Y chromosome from their father. Consequently, all of the somatic cells in human females contain two X chromosomes, and all of the somatic cells in human males contain one X and one Y chromosome (Figure 3). The same is true of all other placental mammals — males produce X and Y gametes, and females produce only X gametes (Figure 4). In this system, referred to as the XX-XY system, maleness is determined by sperm cells that carry the Y chromosome.

Figure 4: Sex determination in humans.

Figure 5: Sex determination in insects.

Many people do not realize, however, that the XX-XY sex determination system is only one of a variety of such systems within the animal kingdom. In fact, sex determination can be very different between different organisms. For example, in the



XX-XO system found in crickets, grasshoppers, and some other insects, sperm cells that lack an X chromosome (referred to as O) determine maleness. Here, females carry two X chromosomes (XX) and only produce gametes with X chromosomes. Males, on the other hand, carry only one X chromosome (XO) and produce some gametes with X chromosomes and some gametes with no sex chromosomes at all (Figure 5).

Figure 6: Sex determination in birds.

Despite the previous examples, males are not always the sex with the mismatched chromosome pair. For example, the ZZ-ZW sex determination system used in birds, snakes, and some insects relies upon females to carry the mismatched chromosome pair (ZW) and males to carry the identical pair (ZZ) (Figure 6). If the three systems discussed above are compared in side-by-side Punnett squares (Figure 7), it is easy to see that sex determination is simply a matter of gamete assortment. Determinations of male and female character arise from a variety of different gamete combination patterns, all of which are the result of gender coding in sexually reproducing organisms.

Figure 7: A side-by-side comparison of sex determination systems in humans, insects, and birds.

The variety of inheritance patterns described in this article illustrate that sex determination is a complex and varied feature among organisms. The XX-XY, XX-XO, and ZZ-ZW systems are only a sample of the wide variety of sex determination systems that scientists have documented in the wide world of living beings, however.

An idea inspired by the "X element"

The variety of sex determination systems

Unit 3







Key Questions How can environmental conditions determine sex in some animals?

What have honeybees taught scientists about sex determination?

What do transgenic mice reveal about sex reversal?

Key Concepts sex chromosomes

X chromosome

sex determination

XX-XO system

XX-XY system

ZZ-ZW system

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PROGRESS

3.6 Some Organisms Transmit Genetic Material to Offspring without Cell Division

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Prokaryotes, which include bacteria and single-celled microorganisms called Archaea, usually pass their chromosomal DNA on to their offspring asexually. In other words, a bacterial cell reproduces by simply replicating its chromosome and dividing into two daughter cells. The daughter cells that result from this division are genetically identical to each other and to the original parent cell. Thus, over time, asexual reproduction in bacteria can lead to a population of hundreds of thousands of cells, all of which are genetically identical to a lone original parent cell.Given their asexual method of reproduction, it is tempting to think that bacteria are sorely lacking in genetic variation, but this is not the case. Prokaryotic cells have developed a number of methods for recombining their genetic material, which, in turn, contributes to their genetic diversity. The three most common ways that bacteria diversify their DNA are transformation, conjugation, and transduction. However, not all types of bacterial cells are capable of engaging in all three processes.

Transformation Transformation is a process by which a susceptible or "competent" bacterial cell acquires new genetic material from its environment. There are two types of transformation: natural and artificial. But where does the environmental DNA required for transformation come from? And how does this DNA become part of a bacterium's genome? Natural transformation, as its name implies, is a natural mechanism used by some bacterial cells to take up DNA from the environment. This environmental DNA was, at one point, located in other bacteria. For instance, when bacteria die and disintegrate, their chromosomal DNA is released. Fragments of this DNA remain in the environment and are freely available to other living cells, including other bacteria. These naturally occurring DNA fragments can enter a living bacterium through its cell membrane, after contact with that membrane. If the DNA is double stranded, one of the strands will pass across the cell membrane into the cell, and the other strand will be dissolved, or hydrolyzed. Parts of the newly introduced single-stranded DNA molecule may then recombine with similar regions on the bacterial chromosome and become incorporated into the bacterium's genome. In contrast, during artificial transformation, DNA uptake by bacterial host cells occurs under certain laboratory conditions. In the lab, scientists often introduce foreign DNA into bacterial cells via transformation in order to study specific genes and their functions. Typically, these researchers use E. coli cells that have been chemically treated so that their outer cell membranes are permeable to foreign DNA. In addition, transformation can be induced by electroporation, a process in which the bacterial host cells are subjected to an electric field that allows molecules to pass more easily across the membrane. Heat shock is another way that transformation can occur, wherein host cells are exposed to extreme temperatures that also cause the cell membrane to temporarily allow molecules of foreign DNA into the cell. Within the lab environment, bacteria are also commonly transformed with sequences of DNA called plasmid vectors. These naturally occurring DNA molecules are circular, and they can replicate inside a bacterium independent of the bacterial chromosome (which can also be circular). Plasmid vectors can be used to clone, transfer, and manipulate genes. Often, these plasmids carry a gene for antibiotic resistance, which means that researchers can select for cells that are resistant to a given antibiotic in order to determine whether a bacterium has been successfully transformed.The following animation depicts the process of transformation:


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Conjugation Conjugation is a process by which one bacterium transfers genetic material to another bacterium through direct contact. During conjugation, one of the bacterial cells serves as the donor of the genetic material, and the other serves as the recipient. The donor bacterium carries a circular, double-stranded DNA sequence called the fertility factor, or F-factor. The F-factor allows the donor to produce a thin, tubelike protuberance called a pilus. The donor uses the pilus to contact the recipient. The pilus then shortens and draws the two bacteria together, at which time the donor bacterium transfers genetic material to the recipient bacterium. This genetic material is in the form of a plasmid, or a small, circular piece of non-chromosomal DNA. The newly transferred genetic material often provides the recipient bacterium with some sort of genetic advantage. In many cases, conjugation results in the transfer of a plasmid containing an antibiotic resistance gene.The following animation depicts two bacteria exchanging DNA via conjugation:

Transduction



Finally, transduction is a process by which a virus transfers genetic material from one bacterium to another bacterium. This process depends on a specific type of virus called a bacteriophage, which is capable of infecting bacterial cells and using them as hosts to produce more viruses. At the beginning of a transduction cycle, a bacteriophage injects its DNA into a host bacterium. The phage DNA then takes over the host cell's machinery and directs it to synthesize and assemble more phages. During this process, the host cell's DNA breaks into fragments; after that, the host cell replicates the phage DNA and assembles new phages. Occasionally, some of the bacterial host cell's DNA is included with the phage DNA during assembly. Once phage assembly is complete, the bacterial cell breaks open, and the newly assembled phages are released into the environment. This is called a lytic cycle because the original bacterial host cell is destroyed, or lysed. Later, when one of the newly released bacteriophages infects a new bacterium, any bacterial DNA that the phage contains may become incorporated into the genome of the new host.The original bacterial host cell is not always destroyed during transduction, however. In some cases, the phage DNA does not direct the host cell to produce more phages; instead, it incorporates itself into the chromosomal DNA of the bacterial host cell. This is called a lysogenic cycle. The phage DNA is then maintained within the bacterial chromosome through many generations of cell division. Eventually, at a point in the future when conditions are right, the phage DNA removes itself from the bacterial chromosome and initiates a lytic cycle. The following animation shows the process of transduction. The first part of the animation depicts a lytic cycle, and the second part shows a lysogenic cycle:

Transformation

Unit 3







Key Questions How is DNA organized in prokaryotes?

Key Concepts prokaryote

plasmid



Conjugation

Transduction

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PROGRESS

4.1 Introduction: How Do We Study the DNA Inside Cells? Prev Page

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Scientists began their studies of genes on a small scale. Typically they would conduct research on single genes, then use their results to hone in on the functions of these genes. Later, newer scientific techniques made it possible for researchers to analyze multiple genes in concert, and eventually to analyze all of an organism's genes at once.This unit explores some of the ways in which researchers can use modern laboratory techniques to monitor gene transcription, expression, and regulation on small and large scales alike. The unit begins with an explanation of two common processes for determining gene expression — Northern blot analysis and serial analysis of gene expression (SAGE) — both of which allow researchers to measure levels of messenger RNA in a sample, and thereby determine which genes are expressed in the sample and to what degree. Next, the unit examines some of the processes by which researchers have determined the actual sequence of nucleotides within genes. Here, the unit's primary focus is on the technique known as Sanger sequencing, wherein DNA replication is used to detect the presence and arrangement of individual nucleotides. When studying the function of a particular gene, scientists often need large amounts of the DNA sequence of interest in order to conduct their experiments. Thus, the next portion of this unit describes the sophisticated lab technique called the polymerase chain reaction (PCR), which enables researchers to rapidly generate multiple copies of genetic sequences. Thereafter, the unit describes the creation of a gene deletion model called a knockout mouse. By disabling or "knocking out" specific genes within these mice, researchers are able to uncover a wealth of information about the function of the missing genes. Lastly, the unit concludes with a look at microarray analysis, a technique that makes it possible to screen for vast amounts of genes at once, which permits more efficient examination of an organism's entire genome.

Unit 4

Introduction: How Do We Study the DNA Inside Cells?

The Order of Nucleotides in a Gene Is Revealed by DNA Sequencing

Scientists Can Make Copies of a Gene through PCR

Scientists Can Analyze Gene Function by Deleting Gene Sequences

Gene Expression Is Analyzed by Tracking RNA

Scientists Can Study an Organism's Entire Genome with Microarray Analysis

Key Questions Where can I learn more about gene expression and regulation?

Key Concepts DNA

DNA sequencing

PCR

Microarray

Gene knockout

Essentials of Genetics Contents Unit 4: How Do Scientists Study and Manipulate the DNA inside Cells?

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4.2 The Order of Nucleotides in a Gene Is Revealed by DNA Sequencing Prev Page

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All of the information needed to build and maintain an organism — whether it's a human, a dog, or a bacterial cell — is contained in its DNA. DNA molecules are composed of four nucleotides, and these nucleotides are linked together much like the words in a sentence. Together, all of the DNA "sentences" within a cell contain the instructions for building the proteins and other molecules that the cell needs to carry out its daily work.

How do researchers "read" gene sequences? Determining the order of the nucleotides within a gene is known as DNA sequencing. The earliest DNA sequencing methods were time consuming, but a major breakthrough came in 1975 with the development of the process called Sanger sequencing. Sanger sequencing is named after English biochemist Frederick Sanger, and it is sometimes also referred to as chain-termination sequencing or dideoxy sequencing. Some 25 years after its creation, the Sanger method was used to sequence the human genome, and, with the addition of many technological improvements and modifications, it remains an important method in laboratories across the world today.

How does Sanger sequencing work? Sanger sequencing is modeled after the natural process of DNA replication, and it uses dummy nucleotides to stop replication whenever a specific nucleotide is encountered. Because this truncated replication occurs over and over again, nucleic acids of varying lengths accumulate and can be used to determine the position of each nucleotide in the sequence.

Understanding DNA replication

Figure 1: DNA polymerase assembles nucleotides to make a new DNA strand.

In order to understand how Sanger sequencing works, it's first necessary to understand the process of DNA replication as it exists in nature. DNA is a double-stranded, helical molecule composed of nucleotides, each of which contains a phosphate group, a sugar molecule, and a nitrogenous base. Because there are four naturally occurring nitrogenous bases, there are four different types of DNA nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C). Within double-stranded DNA, the nitrogenous bases on one strand pair with complementary bases along the other strand; in particular, A always pairs with T, and C always pairs with G. Then, during DNA replication, the two strands in the double helix separate. This allows an enzyme called DNA polymerase to access each strand individually. As the DNA polymerase moves down the single-stranded DNA, it uses the sequence of nucleotides in that strand as a template for replication. Thus, whenever the DNA polymerase recognizes a T in the template strand, it adds an A to the complementary daughter strand it is building; similarly, whenever it encounters a C in the original strand, it adds a G to the daughter strand. This process happens along both strands simultaneously, resulting in the eventual production of two double-helical molecules, each of which contains one "old" strand and one "new" strand of DNA.


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Setting up the sequencing experiment The Sanger method relies upon a variation of the replication process described above in order to determine the sequence of nucleotides in a segment of DNA. Before Sanger sequencing can begin, however, researchers must first make many copies of, or amplify, the DNA segment they wish to sequence. This is done either by cloning the DNA or by triggering the polymerase chain reaction (PCR). Once the DNA has been amplified, it is heated so that the two strands separate, and a synthetic primer is added to the mixture. The primer's sequence is complementary to the first piece of target DNA, which means that the primer and the DNA target bind with each other. At this point, the target sequence is exposed to a solution that contains DNA polymerase and all of the nucleotides required for synthesis of the complementary DNA strand — along with one special ingredient.

Adding ddNTPs

Figure 2: The four ddNTPs.

As described above, the next major step in the Sanger process is to expose the target sequence to DNA polymerase and significant amounts of all four nucleotides. In their unbound form, nucleotides have three phosphate groups and are formally called deoxynucleotide triphosphates, or dNTPs (where the "N" is a placeholder for A, T, G, or C). During the construction of a new DNA strand, a molecule called a hydroxyl group (which contains an oxygen atom and a hydrogen atom) attaches to the sugar of the last dNTP in the strand and chemically binds to the phosphate group on the next dNTP. This binding causes the DNA chain to grow. In Sanger sequencing, however, a special type of "dummy" nucleotide is included with the regular dNTPs that surround the growing DNA strand. These special nucleotides are known as dideoxynucleotide triphosphates, or ddNTPs (Figure 2), and they lack the crucial hydroxyl group that is attached to the sugar of dNTPs. Therefore, whenever a ddNTP is added to a growing DNA strand, it is unable to chemically bind with the next nucleotide in the chain, and the DNA strand stops growing.When researchers carry out the Sanger process, they are manipulating many copies of the template strand at once, so an overabundance of dNTPs is required in order for DNA synthesis to proceed unimpeded on these copies until a ddNTP is added. Then, after the supply of dNTPs has been exhausted, the final result of the sequencing experiment is a group of new DNA strands of varying lengths. These strands all have a terminal ddNTP that indicates whether an A, T, G, or C occurs in that position on the template strand (Figure 3).

Figure 3: By adding together information about all of the truncated strands, researchers can determine the nucleotide sequence of the DNA target.

Reading the sequence: Now and then When Sanger sequencing was first introduced, four separate reagents were used, one for each type of ddNTP. The four reaction products were then separated by gel electrophoresis, a process that organizes DNA fragments in order of size. This enabled researchers to assess the lengths of the truncated strands in each sample. This was important, because the end of each truncated strand was used to determine the position at which a ddNTP was added to the strand, thereby halting DNA elongation. More recently, automation of the Sanger technique has made this process more efficient by combining all four ddNTP reactions in a single test tube. Each of the four ddNTPs in the tube is labeled with a different fluorescent color. Rather than being run on a gel and read manually, the reaction products are passed through a small tube containing a gel-like matrix. As the different-sized DNA fragments pass through the tube, a sequencing machine reads the fluorescent label at each position. Sequencing machines have vastly increased the speed and efficiency of DNA sequencing, and this technology continues to evolve at an astonishing rate.

How is DNA sequencing used by scientists? In recent years, DNA sequencing technology has advanced many areas of science. For example, the field of functional genomics is concerned with figuring out what certain DNA sequences do, as well as which pieces of DNA code for proteins and which have important regulatory functions. An invaluable first step in making these determinations is learning the nucleotide sequences of the DNA segments under study. Another area of science that relies heavily on DNA sequencing is comparative genomics, in which researchers compare the genetic material of different organisms in order to learn about their



evolutionary history and degree of relatedness. DNA sequencing has also aided complex disease research by allowing scientists to catalogue certain genetic variations between individuals that may influence their susceptibility to different conditions.

How can all people benefit from DNA sequencing? More about sequencing

DNA sequencing technologies•Sequencing the human genome•

At the individual level, biomedical research into the cause and course of common human diseases is primed to greatly improve health care. The application of DNA sequencing to the identification of disease-causing genetic variants will lead to improvements and expansion in genetic testing, as well as development of more targeted, personalized drug therapies in the years to come. Already today, the benefits of DNA sequencing can be seen in agriculture thanks to the production of disease-resistant plants and animals. In addition, microbial genome sequencing projects may someday lead to the development of new biofuels and pollutant-monitoring systems. DNA sequencing techniques are also used in forensic science, providing crucial evidence in criminal cases. In the United States, for instance, the Federal Bureau of Investigation (FBI) funds and operates a national database containing the genetic profiles of known offenders that can be searched whenever DNA evidence is obtained at a crime scene. According to the FBI, as of 2008, this database had profiles of over 6.5 million offenders and had assisted in almost 81,000 investigations.

Watch this video for a summary of the Sanger sequencing process

How do researchers "read" gene sequences?

How does Sanger sequencing work?

How is DNA sequencing used by scientists?

How can all people benefit from DNA sequencing?

Unit 4



Key Questions What are some other methods for DNA sequencing?

How much does gene sequencing cost?

How was the human genome sequenced?

What happens during DNA replication?

Who, exactly, discovered DNA?

How has the polymerase chain reaction (PCR) revolutionized biotechnology?



Watch this video for a summary of the Sanger sequencing process





What has genomics done for the biofuel industry?

What ethical problems does DNA sequencing raise?

How is sequencing done on a large scale?

Key Concepts Human Genome Project

bioinformatics

genome

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PROGRESS

4.3 Scientists Can Make Copies of a Gene through PCR Prev Page

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Many copies of DNA

Once scientists have zeroed in on a specific segment of DNA, how do they produce enough copies of that segment for their research? In most cases, the polymerase chain reaction, or PCR, is their method of choice for quickly generating a sufficient amount of identical genetic material for study and analysis.Prior to the development of PCR in the 1980s, the primary method for producing many copies of a gene was a relatively time-consuming process known as DNA cloning. This technique involved insertion of the gene of interest into living bacterial cells, which in turn replicated the gene along with their own DNA during the division and replication processes.

What is PCR? The key element of PCR is heat. Throughout the PCR process, DNA is subjected to repeated heating and cooling cycles during which important chemical reactions occur. During these thermal cycles, DNA primers bind to the target DNA sequence, enabling DNA polymerases to assemble copies of the target sequence in large quantities. PCR makes it possible to produce millions of copies of a DNA sequence in a test tube in just a few hours, even with a very small initial amount of DNA. Since its introduction, PCR has revolutionized molecular biology, and it has become an essential tool for biologists, physicians, and anyone else who works with DNA.

How does PCR work?

Figure 1: The various components required for PCR include a DNA sample, DNA primers, free nucleotides called ddNTPs, and DNA polymerase.

PCR relies on several key chemical components (Figure 1):

A small amount of DNA that serves as the initial template or target sequence•A pair of primers designed to bind to each end of the target sequence•A DNA polymerase•Four dNTPs (i.e., dATP, dCTP, dGTP, dTTP)•A few essential ions and salts•

The PCR process then uses these ingredients to mimic the natural DNA replication process that occurs in cells. To automate this process, a machine called a thermocycler jump-starts each stage of the reaction by raising and lowering the temperature of the chemical components at specific times and for a preset number of cycles. Each cycle of PCR has three main steps, as described in the following sections.

Step 1: Denaturation


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Figure 2: When heated, the DNA strands separate.

During the first step in PCR, the starting solution is heated to the necessary temperature, usually between 90° and 100°C. As the heat builds, it breaks the bonds joining the two strands of the DNA double helix, thereby enabling the DNA to separate into two single strands. This "melting" of the DNA into single strands is called denaturation (Figure 2).

Step 2: Annealing

Figure 3: When the solution is cooled, the primers anneal.

After it is held for several minutes at the initial target temperature, the reaction mixture is quickly cooled, usually to between

30° and 65°C. The mixture is then held for less than one minute at this temperature. This gives the primers an opportunity to bind, or anneal, to their complementary sequences on the single strands of DNA (Figure 3).

Step 3: Extension

Figure 4: DNA polymerase attaches to each primer and assembles dNTPs to build a new strand.

During the final, or extension, stage of PCR, the sample is heated again, usually to between 60°and 75°C, and it is held at that temperature for less than one minute. At this point, the DNA polymerase begins making a new DNA strand by attaching to the primers and then adding dNTPs to the template strand, thereby creating a complementary copy of the target sequence (Figure 4).The number of new copies of the DNA sequence of interest doubles with each three-step cycle. Thus, if the PCR process is repeated 40 or 50 times, even small samples of template DNA can yield millions of identical copies (Figure 5).

Figure 5: The replication cycle repeats many times.

PCR is an incredibly versatile technique with many practical applications. Once PCR cycling is complete, the copied DNA molecules can be used for cloning, sequencing, mapping mutations, or studying gene expression.

Variations on conventional PCR Recently, PCR has proven useful in ways beyond merely copying and propagating identical segments of DNA. Today, geneticists rely on PCR to aid in the study of genes themselves.

Copying and quantifying DNA at the same time using real-time PCR One modification of conventional PCR allows researchers to copy a particular DNA sequence and quantify it simultaneously. Dubbed quantitative real-time PCR (qPCR), this technique makes it possible to measure the amount of DNA



produced during each PCR cycle. This refinement involves the use of fluorescent dyes or probes that label double-stranded DNA molecules. These fluorescent markers bind to the new DNA copies as they accumulate, making "real-time" monitoring of DNA production possible. As the number of gene copies increases with each PCR cycle, the fluorescent signal becomes more intense. Plotting fluorescence against cycle number and comparing the results to a standard curve (produced by real-time PCR of known amounts of DNA) enables scientists to determine the amount of DNA present during each step of the PCR reaction.

Quantifying RNA using reverse transcription PCR

More about gene copying

Who determined how DNA replicates?•Reversing the central dogma: How do you make DNA from RNA?rom RNA?•

Real-time PCR can also be used to calculate the amount of specific kinds of genetic material other than DNA, such as RNA. This extension of real-time PCR technology, called reverse transcription PCR (RT-PCR), combines real-time PCR with reverse transcription, the process that makes DNA from mRNA. RT-PCR can be used to determine how gene expression changes over time or under different conditions. For this reason, this technique is sometimes used to verify microarray data.

Watch this video for a summary of the PCR process

What is PCR?

How does PCR work?

Variations on conventional PCR

Watch this video for a summary of the PCR process

Unit 4





Key Questions What is DNA cloning?

Key Concepts primer

DNA polymerase

DNA sequencing

recombinant DNA technology





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PROGRESS

4.4 Scientists Can Analyze Gene Function by Deleting Gene Sequences Prev Page

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One way to understand the function of a gene is to observe a biological system that lacks that gene. But what is the best system to use? When studying human genes, researchers typically employ biological systems that approximate these genes and their functions as closely as possible. In particular, researchers often turn to mice because of all the various model organisms most commonly used in the lab (e.g. fruit flies and yeast), mice have the genome that most closely resembles that of humans. Consequently, manipulation of genes within the mouse genome has proven an effective method for learning about human gene functioning. Indeed, experimentally removing or altering certain genes within a mouse allows for the examination of a biological system with specific gene alterations.

What can mice reveal about human gene function? On the outside, humans and mice look nothing alike. However, human and mouse chromosomes share many of the same genes. In fact, 99% of the 20,000 to 25,000 genes in humans have a similar mouse counterpart. This high degree of genetic similarity between humans and mice offers researchers a unique approach for understanding human gene functioning. Ethical considerations prevent researchers from genetically engineering humans that lack a given gene for the sole purpose of learning how that gene functions. However, because so many mouse genes are similar to human genes, geneticists can generate knockout mice in which the mouse counterpart of a human gene of interest is deleted or disrupted. The term "knockout mouse" may at first conjure images of a mouse boxing champion or beauty queen. However, geneticists use the term to refer to a mouse that has been genetically engineered such that at least one of its genes is functionally inactivated (i.e. the inactivated gene is "knocked out"). If a mouse gene has a high degree of similarity to a human gene, researchers can predict that the two genes carry out related functions. Therefore, by generating a knockout mouse without a gene of interest, these scientists may be able to determine the functions carried out by the related human gene. In some cases, knockout mice exhibit phenotypes that mimic symptoms associated with human disease, including cancer, diabetes, obesity, cardiovascular disease, and neurodegenerative disease. In these cases, a knockout mouse is referred to as a mouse model of that form of human disease.

Making a knockout mouse: Step by step How are genes deleted or disrupted in a knockout mouse? In the past, making a knockout mouse was a major undertaking. Today, however, a combination of new molecular biology techniques and increased knowledge of the mouse genome has reduced the time and effort required to make a knockout mouse. Nevertheless, there still are a number of steps involved in engineering one of these creatures.

Step 1: Generating a targeting vector The first step in making a knockout mouse is identifying the region of the gene that will be deleted. Because the entire mouse genome sequence is already known, it is relatively simple to look up the chromosomal location and nucleic acid sequence of the gene of interest. Once the segment of the gene that will be deleted has been mapped out, the nucleic acid sequences of the DNA segments that appear on the chromosome before and after that gene must also be identified.Once these tasks are completed, a targeting vector specifically tailored to the gene of interest is made (Figure 1). A targeting vector is a long stretch of DNA made up of smaller pieces of DNA that have been joined together. Next, in order to make the targeting vector detectable, the scientists insert a marker gene into the middle of the vector; this marker is in some way able to "report" when it is present in a cell.


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Figure 1: The targeting vector is designed to contain both neomycin-resistant and a ganciclovir-sensitive (TK) sequences.

Currently, the neomycin-resistance gene, called NeoR, is a popular marker gene of choice for generating knockout mice. The antibiotic neomycin is toxic to mouse cells because they do not normally contain the NeoR gene. However, when the NeoR gene is added to mouse cells, these cells can survive in the presence of neomycin. Within the targeting vector, the NeoR gene is located between two other pieces of DNA: the "right arm" and the "left arm" of the targeting vector. The right arm of the targeting vector contains DNA with a nucleic acid sequence that matches the stretch of DNA immediately before the gene segment that will be deleted. The left arm of the targeting vector contains DNA with a nucleic acid sequence that matches the stretch of DNA immediately after the gene segment that will be deleted. The right and left arms of the targeting vector facilitate homologous recombination between the targeting vector and the target gene, thereby enabling the NeoR gene to replace the target gene segment.The targeting vector also contains one additional piece of DNA, called a negative selection marker gene. This gene is located at the right end of the targeting vector, after the right arm. The thymidine kinase (TK) gene from the herpes simplex virus is the most commonly used negative selection marker gene. Normally, mouse cells can grow in the presence of the antiviral drug ganciclovir. The TK gene is considered a "cell suicide gene," however, as cells containing the TK gene convert ganciclovir into a lethal toxin.Why is it necessary to include a cell suicide gene as part of the targeting vector? The reason is purely a matter of identification - specifically, the TK gene helps researchers locate cells that have correctly replaced the targeted gene segment with the NeoR gene. Often, mouse cells randomly insert the targeting vector in the wrong chromosomal location. If random insertion occurs, both the NeoR gene and the TK gene are inserted into the genome. As a result, the cells are resistant to neomycin, but they die in the presence of ganciclovir. In comparison, when the targeted gene segment is correctly replaced, the TK gene is not inserted into the chromosome along with the NeoR gene, so the resultant cells are resistant to both neomycin and ganciclovir. Therefore, the presence of the TK gene in the targeting vector allows researchers to efficiently screen for mouse cells that have correctly replaced the targeted gene segment by growing these cells in the presence of both neomycin and ganciclovir.

Step 2: Inserting the target sequence and selecting cells with the insertion After the targeting vector is made, it is used to knock out one copy of the target gene in mouse embryonic stem (ES) cells (Figure 2). But why must mouse ES cells be used? Why can't the targeting vector be introduced into any type of mouse cell?

Figure 2: The targeting vector is inserted into the ES cell genome, and disables the target gene.

It is certainly possible to use the targeting vector to knock out one of the two copies of the target gene in a standard somatic cell. However, unless that cell is an ES cell, the knockout mutation cannot be incorporated into a growing embryo. Therefore, it would not be possible to study the effects of the knockout mutation in a developing mouse.What makes ES cells so special? Primarily, it is their ability to become any one of the different adult cell types. When injected into a mouse embryo, the ES cells themselves are capable of maturing into some of the tissues of the developing mouse.



But how, exactly, are targeting vectors delivered into ES cells? Most often, a technique called electroporation is used. When ES cells are electroporated, a brief pulse of an electrical field is applied to the outside of the cells, creating a momentary increase in plasma membrane permeability and allowing the uptake of foreign DNA into the ES cells.After the ES cells have been electroporated, they are grown in the presence of neomycin to select for those particular cells that have taken up the targeting vector. Next, the neomycin-resistant cells are grown in the presence of ganciclovir to select for those that have inserted the targeting vector at the correct location within the mouse genome.

Step 3: Identifying ES cells with the correct gene knocked out

Figure 3: In a cell culture dish, only a portion of ES cells will contain the targeting vector. These "knockout ES cells" will survive exposure to neomycin and ganciclovir (shown in red), while the other cells will die (pink). The surviving cells will then be used for the next step.

Additional experiments using standard molecular biology techniques help researchers determine whether the target gene has been fully knocked out in the ES cells that are resistant to both neomycin and ganciclovir. After these experiments are complete, only the ES cells that have had one copy of the target gene knocked out remain (Figure 3). These ES cells are heterozygous for the knockout mutation. Although these cells will grow and divide in culture, they cannot form an embryo that will develop into a mouse on their own.

Step 4: Injecting heterozygous knockout ES cells into a developing embryo and transferring the embryo into a mouse

Figure 4: Knockout ES cells are then injected into a fresh mouse embryo containing normal cells.

Figure 5: A chimeric mouse contains both normal cells and genetically manipulated "knockout" cells. Coat color can reflect this with a spotted pattern.

How, then, do the heterozygous knockout ES cells grown in culture develop into a mouse? The first step involves injection of these cells into a developing mouse embryo (Figure 4). This step allows the heterozygous knockout ES cells to become part of the developing embryo. Then, because they are ES cells, the heterozygous knockout cells are incorporated throughout the embryo and are capable of becoming any type of tissue within the developing mouse. Therefore, like a patchwork quilt, the developing embryo contains a mixture of its own original cells and the heterozygous knockout cells. Because of this cellular mixing, the resultant mouse is called a chimeric mouse (Figure 5).How can researchers tell whether the heterozygous knockout cells have been incorporated into the mouse embryo? And how can the knockout cells be distinguished from the original mouse cells? The primary means for determining the success of knockout cells has to do with the coat color of the mice involved. The original ES cells that are electroporated with the targeting vector come from two parents with black coats, which is the dominant coat color in mice. In contrast, the original cells of the developing embryo come from two parents with white coats, which is the recessive coat color. When the heterozygous knockout cells are successfully incorporated into the developing embryo, the resulting mouse will have patches of heterozygous knockout cells and patches of original cells, so its coat will have both black and white patches of fur.Still, only some of the cells that make up the chimeric mouse carry the knockout mutation, and those cells are heterozygous for the mutation. In order to learn about the function of the target gene, a mouse that is homozygous for the knockout mutation must be studied.

Step 5: Mating chimeric mice to yield homozygous knockout mice



Figure 6: Mating a chimeric (spotted) mouse with a normal (white) mouse can yield a knockout mouse, among other normal mice. All mice born from this mating can be screened to verify the gene has been knocked out.

How can researchers use chimeric mice to produce homozygous knockout mice? The answer lies in the reproductive tissues of chimeric mice, some of which are made up of heterozygous knockout cells. As a result, some of the gametes from chimeric mice carry knockout mutations. To produce a mouse that is homozygous for the target gene knockout, chimeric mice capable of passing the knockout mutation on to their offspring must be identified.These chimeric mice can be identified by crossing them with normal white mice. If black offspring are produced from such a cross, a chimeric mouse is capable of passing the knockout mutation on to its offspring. When this is the case, 50% of the black offspring are heterozygous for the knockout mutation in all of their cells (Figure 6). Standard molecular biology techniques can be used to determine which of the black offspring are heterozygous for the knockout mutation. Then, to produce a homozygous knockout mouse, a heterozygous knockout male is mated to a heterozygous knockout female. Twenty-five percent of the resulting offspring will be homozygous knockout mice, which can, again, be readily identified using standard molecular biology techniques.

Step 6: Phenotypic characterization of homozygous knockout mice After the homozygous knockout mice have been created, researches must then characterize the phenotypes associated with the loss of the target gene. In theory, every measurable phenotype must be examined in order to determine every possible function of the knocked-out gene. The measurement of a given phenotype in a knockout mouse would then be compared to measurements of the same phenotype in a wild-type mouse (a mouse that has not been genetically engineered for specific traits) in order to identify the functions that are altered in the knockout mouse.Phenotypes such as size, weight, metabolism, behavior, bone development, neurological function, reproduction, and aging can be easily measured. If the knocked-out gene is required for development, however, it may not be possible to produce homozygous knockout mice. In this case, researchers may study heterozygous knockout mice, or they may instead turn to other types of knockout mice, such as conditional knockout mice (in which the target gene is inactivated in response to a specific stimulus) or tissue-specific knockout mice (in which the target gene is inactivated in only one or several tissues).

Large-scale mouse knockout projects Large-scale knockout mouse projects

KOMP•EUCOMM•NorCOMM•TIGM•See a timeline tracking the events leading the first knockout mouse•

In September 2006, the U.S. National Institutes of Health (NIH) initiated a five-year, $52 million project called the Knockout Mouse Project (KOMP). Together with the Texas A&M Institute of Genomic Medicine (TIGM), the North American Conditional Mouse Mutagenesis Project (NorCOMM) in Canada, and the European Conditional Mouse Mutagenesis Program (EUCOMM), KOMP set the goal of knocking out every one of the 20,000 mouse protein-encoding genes within five years.Similar to the mouse knockout consortia mentioned above, a number of labs have also collaborated to establish standardized methods for the phenotypic characterization of knockout mice. The European Union Mouse Research for Public Health and Industrial Applications (EUMORPHIA) group developed the first set of standard phenotyping protocols, which was validated among several different labs. The European Mouse Phenotypic Resource for Standardized Screens (EMPReSS) then established the primary phenotypic screen used by the European knockout mouse labs, one that comprises a subset of the standard protocols of EUMORPHIA.

Future directions Future studies of knockout mice will continue to yield new and unexpected discoveries regarding the function of mouse genes and their human counterparts. An increased understanding of human gene function will also lead to the design of more effective treatments for human disease.



The steps involved in making a knockout mouse.

Unit 4


Key Questions What is EUMORPHIA?



What can mice reveal about human gene function?

Making a knockout mouse: Step by step

Large-scale mouse knockout projects

Future directions






What is EMPReSS?

Which scientists won the Nobel Prize for the creation of the first knockout mouse?

Key Concepts knockout mouse

genome

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PROGRESS

4.5 Gene Expression Is Analyzed by Tracking RNA Prev Page

Next Page Even though nearly every cell in an organism's body contains the same set of genes, only a fraction of these genes are used in any given cell at any given time. It is this carefully controlled pattern of what is called "gene expression" that makes a liver cell different from a muscle cell, and a healthy cell different from a cancer cell. But how can researchers determine which genes are "turned on" and when?

How do scientists measure gene expression?

Figure 1: Siamese cats have colored "points" because of a temperature-sensitive pigmentation gene. In cooler areas of a cat's body (nose and paws), this gene is expressed to a greater degree.

Gene expression is dynamic, and the same gene may act in different ways under different circumstances. For example, imagine that two organisms have similar genotypes but different phenotypes. What is the cause of this variation in phenotype? Could the difference stem from differing regulation of gene expression? Could temperature affect expression of the organism's DNA (Figure 1)? Might some other factor be responsible? To go about answering these types of questions, researchers often use laboratory techniques such as a Northern blot or serial analysis of gene expression (SAGE). Both of these techniques make it possible to identify which genes are turned on and which are turned off within cells. Subsequently, this information can be used to help determine what circumstances trigger expression of various genes. Both Northern blots and SAGE analyses work by measuring levels of mRNA, the intermediary between DNA and protein. Remember, in order to activate a gene, a cell must first copy the DNA sequence of that gene into a piece of mRNA known as a transcript. Thus, by determining which mRNA transcripts are present in a cell, scientists can determine which genes are expressed in that cell at different stages of development and under different environmental conditions.

Northern blots: What are they, and how do they work?


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Figure 2: A Northern blot allows comparison of mRNA levels, which are a direct reflection of gene expression. This blot above shows the tracking of mRNA levels in a cell sample at various intervals after exposure to a drug. At 15 minutes after exposure, mRNA levels are low (red arrow, small black mark); at two hours after exposure, these levels have increased (blue arrow, larger black mark).

The quantity of mRNA transcript for a single gene directly reflects how much transcription of that gene has occurred. Tracking of that quantity will therefore indicate how vigorously a gene is transcribed, or expressed. To visualize differences in the quantity of mRNA produced by different groups of cells or at different times, researchers often use the method known as a Northern blot. For this method, researchers must first isolate mRNA from a biological sample by exposing the cells within it to a protease, which is an enzyme that breaks down cell membranes and releases the genetic material in the cells. Next, the mRNA is separated from the DNA, proteins, lipids, and other cellular contents. The different fragments of mRNA are then separated from one another via gel electrophoresis (a technique that separates molecules by passing an electrical current through a gel medium containing the molecules) and transferred to a filter or other solid support using a technique known as blotting. To identify the mRNA transcripts produced by a particular gene, the researchers next incubate the sample with a short piece of single-stranded RNA or DNA (also known as a probe) that is labeled with a radioactive molecule. Designed to be complementary to mRNA from the gene of interest, the probe will bind to this sequence. Later, when the filter is placed against X-ray film, the radioactivity in the probe will expose the film, thereby making marks on it. The intensity of the resulting marks, called bands, tells researchers how much mRNA was in the sample, which is a direct indicator of how strongly the gene of interest is expressed (Figure 2).

Is it possible to study the expression of multiple genes simultaneously? Until recently, scientists studied gene expression by looking at only one or very few gene transcripts at a time. Thankfully, new techniques now make large-scale studies of gene expression possible. One such technique is SAGE (serial analysis of gene expression). A method for measuring the expression patterns of many genes at once, SAGE not only allows scientists to analyze thousands of gene transcripts simultaneously, but it also enables them to determine which genes are active in different tissues or at different stages of cellular development.

How does SAGE work? SAGE identifies and counts the mRNA transcripts in a cell with the help of short snippets of the genetic code, called tags. These tags, which are a maximum of 14 nucleotides long, enable researchers to match an mRNA transcript with the specific gene that produced it. In most cases, each tag contains enough information to uniquely identify a transcript. The name "serial analysis" refers to the fact that tags are read sequentially as a continuous string of information.The basic steps of the SAGE technique are outlined below.

Capturing mRNA To begin a SAGE analysis, researchers must first separate the mRNA in a sample from the other cellular contents. To do this, they attach long strips of thymine nucleotides to tiny magnetic beads. When researchers flush the contents of a cell over the beads, these thymine strips form complementary base pairs with the poly-A tails of the mRNA molecules. Thus, when the flushing process is complete, the mRNA transcripts from the sample are captured because they are attached to the magnetic beads, while the other contents of the cells flush past the beads and are discarded.



Rewriting mRNA into cDNA

Figure 3: Reverse transcription converts mRNA into cDNA.

mRNA is more fragile than DNA, which makes it difficult to handle and analyze. To solve this problem, researchers often convert mRNA samples into complementary DNA sequences, or cDNA. This is done by reversing the natural process a cell uses to make mRNA from DNA, a method known as reverse transcription. The reverse transcription process doesn't use DNA polymerase or RNA polymerase; instead, it employs a special enzyme called reverse transcriptase. This enzyme makes cDNA sequences that are complementary to each mRNA transcript, essentially creating a converted form of the same sequence (Figure 3). This new single-stranded cDNA is then converted into a double-stranded cDNA molecule.

Cutting tags from each cDNA To begin the next portion of SAGE, the researchers use a cutting enzyme to slice off short segments of nucleotides, called tags, at designated positions in each cDNA molecule. Next, two tags from each cDNA are combined into a single unit. These tags then become the representative for the gene they came from, and they act as a unique identifier in the form of a stand-in. Without having to process the entire mRNA sequence thereafter, scientists can use these shorter tag sequences to keep track of whether a specific gene was expressed in mRNA form.

Linking tags together in chains for sequencing After the different tags have been made from each mRNA sequence, they are next linked together into long chains called concatemers. These concatemers therefore contain representatives of mRNAs from a group of genes. Linking the tags together in a concatemer is important, because it means that researchers will later be able to read thousands of tags at once during the analysis portion of the SAGE procedure.

Copying and reading the chains Although the researchers now possess concatemers representing the genes expressed in the sample, they need multiple copies of these concatemers if they wish to run the molecules through a sequencing machine. Thus, just before sequencing, the concatemers are inserted into bacteria, and through their own replication process these bacteria make millions of copies of each concatemer chain. This step increases the volume of material, and it therefore ensures that there is a baseline amount of material necessary for a sequencing machine. After that, researchers use a sequencing machine to decode and read the long string of nucleotides in each chain.

Identifying and counting the tags Finally, a computer processes the data from the sequencing machine and compiles a list of tags. By comparing the tags to a sequence database, the researchers can identify the mRNA (and ultimately the gene) that each tag came from. By subsequently counting the number of times each tag is observed, the researchers can also estimate the degree to which a particular gene is expressed: the more often a tag appears, the greater the level of gene expression.

What can researchers learn from SAGE? Compared to other techniques for measuring gene expression, SAGE offers a significant advantage because it measures the expression of both known and unknown genes. Sometimes, when analyzing SAGE data, computers cannot find matches for certain tags in their sequence databases. What does this mean? Interestingly, a lack of matches indicates that the mRNA used to produce these tags is associated with genes that have not been studied before. In this way, SAGE has been used to discover new genes involved in a variety of diseases.

Are there other ways to measure gene expression? More about measuring gene expression

What is a Western blot?•What is RT-PCR?•

In addition to Northern blot tests and SAGE analyses, there are several other techniques for analyzing gene expression. Most of these techniques, including microarray analysis and reverse transcription polymerase chain reaction (RT-PCR), work by measuring mRNA levels. However, researchers can also analyze gene expression by directly measuring protein levels with a technique known as a Western blot.

Unit 4



Key Questions What do we call the entire set of mRNA in a cell or organism?

What does the transcriptome reveal about the genome and gene function?

Key Concepts



How do scientists measure gene expression?

Northern blots: What are they, and how do they work?

Is it possible to study the expression of multiple genes simultaneously?

How does SAGE work?

What can researchers learn from SAGE?

Are there other ways to measure gene expression?





RNA

mRNA

Northern blot

Southern blot

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PROGRESS

4.6 Scientists Can Study an Organism's Entire Genome with Microarray Analysis

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To compare all the genes of one organism to those of another organism, we must first know how to define the entire gene sequence of each organism. However, looking at all of an organism's genes can be quite daunting. Sometimes, a better option is to consider only those genes expressed by an organism, because these genes may represent just a portion of all the genetic material that the organism contains. That is, an organism may only use a small fraction of its entire genetic sequence, otherwise known as its genome.

What is the genome? What is genomics? The genome is the set of all genes, regulatory sequences, and noncoding information within an organism's DNA. Thus, the study of genomics considers all of the genetic material contained within and shared between organisms. As genomic techniques advance, researchers continue to accumulate vast amounts of information about DNA sequences from scores of organisms. Indeed, genomic analysis methods enable researchers to analyze genetic data on a scale never before seen in the biological sciences. One genomic technique that has been widely used for large-scale genome comparisons is the DNA microarray.

What is a DNA microarray? Whereas Northern blots allow scientists to measure the expression of one or several genes at a time, DNA microarrays permit gene expression analysis on a massive scale. In fact, microarray analysis enables researchers to look at expression patterns across all of the genes in an entire genome — and to do so in a single procedure. As a result, it is now possible to monitor the activity of tens of thousands of genes simultaneously. Microarrays are particularly useful when researchers know that certain genes are being transcribed into mRNA, but they aren't sure exactly what those genes are. For example, scientists know that the expression of certain genes differs depending on environmental conditions, but how can they directly observe which genes vary under which conditions? In short, they can use microarray analysis.

How does microarray analysis work? In order to conduct microarray analysis (and therefore determine which genes in a sample are active), researchers must first isolate mRNA from a target sample, convert it into complementary DNA (cDNA), and label the cDNA with a fluorescent dye. The fluorescently labeled cDNA is then added to a glass slide or silicon chip upon which thousands of tiny dots of single-stranded DNA have been arranged in a grid pattern. No bigger than the period at the end of this sentence, each dot of DNA in the grid corresponds to a different gene. If any fluorescent cDNA binds to any one of these dots, researchers know that the corresponding genes are active in the sample. But how, exactly, does this process work? To better understand how microarray analysis is carried out, consider the example experiment described in the following sections.

Growing bacteria under two different conditions

Figure 1: The two temperature conditions for E. coli: normal (left) and heat-exposed (right).

Gene expression in colonies of E. coli bacteria can change when these colonies are exposed to short periods of intense heat. But exactly how do the patterns of gene expression differ? What genes are expressed under one condition, and not the other?


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The best way to go about answering this question is to do a microarray analysis of the genomes in each experimental condition: normal temperature and heat-exposed. The first step is to create the two conditions by exposing one colony of E. coli to normal temperatures and the other colony to a short burst of high temperature (Figure 1).

Converting RNA

Figure 2: mRNA is converted to fluorescently-labeled cDNA.

After this treatment is complete, the bacterial cells are removed from both culture plates and mRNA is extracted from them. Reverse transcriptase and fluorescently-labeled nucleotides are then added to the two test tubes containing the extracted mRNA. Specifically, each tube receives nucleotides marked with a particular fluorescent color: red or green. By using one fluorescent color for the tube of normal RNA and another color for the heat-exposed RNA, researchers can follow the genetic material from each colony during later stages of analysis.Within each test tube, the newly synthesized, fluorescently-labeled cDNA strands form complementary DNA strands with the original mRNA strands (Figure 2). Next, the mRNA is specifically degraded so that only the cDNA copy of the mRNA message is left behind. At this point, the cDNA that was synthesized in each tube is associated with its corresponding color (red or green). Remember, this sample-specific labeling means that the scientists will easily be able to track which cDNA came from normal cells and which cDNA came from heat-exposed cells during later phases of the microarray process.

Applying solution to the DNA chip Next, the fluorescently-labeled cDNA samples from normal and heat-exposed cells are combined into a single solution and applied to a microarray chip, also known as a DNA chip. The DNA chip is covered with a grid of small dots, each with multiple single-stranded pieces of DNA attached to it. Each single strand represents a specific gene sequence.

Figure 3: Fluorescently-labeled cDNAs from the normal and high temperature samples bind to complementary DNA strands on the DNA chip.

After the cDNA is applied to the microarray chip, the cDNA molecules will bind to any complementary strands that exist on the chip (Figure 3). As different genes are on different dots, some of the cDNA in the sample binds to certain dots, some binds to other dots, and some does not bind to any dots whatsoever. This binding identifies which genes were expressed in the original bacterial colonies, because the bound cDNA is joining with partner strands that are already preprogrammed onto the microarray chip. Then, any unbound cDNA is washed away from the chip with a careful rinse, so the only cDNA molecules left on the chip are those that found complementary partners on the chip and bound with them.

Imaging the chip

Figure 4: Scanning the microarray chip.

The chip is then scanned with a special laser that detects the fluorescent molecules attached to each cDNA strand. A single dot will "light up" if cDNA is attached to a complementary sequence on that dot (Figure 4). Here, because of the sample-specific fluorescent labeling, green dots reflect genes that are highly expressed in the normal temperature sample, and red dots reflect genes that are highly expressed in the heat-exposed sample. When red and green fluorescent molecules exist in equal amounts on the same dot, the dot will appear yellow, so yellow dots reflect genes that are expressed at equal levels in normal and heat-exposed samples.



Analyzing the imaged data

Figure 5: Multiple gene chips are needed to scan the entire genome for expression patterns.

Because each gene chip comes with a map of the genes represented by each dot, the pattern of green, red, and yellow dots can be easily translated into gene names. In addition, a computer connected to the scanning light can detect and measure the intensity of the color at each dot. By comparing the intensity of the fluorescent signals, researchers can estimate the relative abundance of each mRNA transcript. Moreover, because each chip used in this experiment surveys 6,000 different genes, the experiment can be repeated using different gene chips until every gene in the bacterium has been surveyed (Figure 5).Consequently, the raw data generated from multiple microarray chips look like sparkling patterns of red, green, and yellow dots. For the above experiment with bacterial colonies, these data tell a story of gene expression across the entire E. coli genome under two different environmental temperatures (Figure 6).

Figure 6: A photograph of real microarray chip data, arranged in a grid. Multiple chips, such as the ones shown here, reveal expression data for an entire genome.

Watch a summary video of the microarray experiment

Unit 4



Key Questions What does whole-genome microarray look like?

How have microarrays aided in the treatment of cancer?



What is the genome? What is genomics?

What is a DNA microarray?

How does microarray analysis work?

Watch a summary video of the microarray experiment





How are microarrays used in comparative methylation hybridization?

Key Concepts genome

microarray

Genome Wide Association Study (GWAS)

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PROGRESS

5.1 Introduction: How Does Inheritance Operate at the Level of Whole Populations?

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Population genetics involves the study of populations at the genetic level. A population is made up of members of the same species that interbreed and live in the same area at the same time. The collective set of all the alleles that exist within a population is referred to as its gene pool. Changes in the gene pool pave the way for species adaptation, the emergence of new species, and, ultimately, evolution.Population genetics involves the study of populations at the genetic level. A population is made up of members of the same species that interbreed and live in the same area at the same time. The collective set of all the alleles that exist within a population is referred to as its gene pool. Changes in the gene pool pave the way for species adaptation, the emergence of new species, and, ultimately, evolution. This unit provides an introduction to the vast and complex field of population genetics. It begins with the concept of the gene pool, descriptions concerning the emergence of variations in the gene pool, and the task of measuring these variations. The unit concludes with an explanation of genetic variation in human populations, as well as a look at the technological advances that have allowed scientists to explore the human genome.

Unit 5

Introduction: How Does Inheritance Operate at the Level of Whole Populations?

The Collective Set of Alleles in a Population Is Its Gene Pool

The Variety of Genes in the Gene Pool Can Be Quantified within a Population

The Genetic Variation in a Population Is Caused by Multiple Factors

Genomics Enables Scientists to Study Genetic Variability in Human Populations

Key Questions Where can I learn more about genes at a population level?

Key Concepts Hardy-Weinberg equation

Hardy-Weinberg equilibrium

Natural selection

Essentials of Genetics Contents Unit 5: How Does Inheritance Operate at the Level of Whole Populations?

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5.2 The Collective Set of Alleles in a Population Is Its Gene Pool Prev Page

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The fact that genes exist in alternate forms, called alleles, forms the basis for the study of population genetics. Populations are made up of members of the same species that interbreed. Population geneticists study the variation that naturally occurs among the genes within a population. The collection of all the genes and the various alternate or allelic forms of those genes within a population is called its gene pool.

What is variation in a gene pool? Genetic variation within a population is measured according to the number of different alleles of all genes and the frequency with which they appear. Variation is high when there are many different allelic forms of all genes and when there are many different combinations of those alleles. However, genetic variation is constantly changing. Different allelic forms of a single gene can appear and disappear from time to time within a single group of organisms. This means that the gene pool of a population is dynamic and can change at any moment for a variety of reasons. In addition, the rate of change within a gene pool can vary at different points in time.

Figure 1: The concept of a gene pool includes multiple alleles in a group of organisms.

Consider for a moment a population of the fruit fly (Drosophila melanogaster). The collective set of genes in the fruit fly population includes a variety of allelic forms: B and b, V and v, W and w, and more (Figure 1). Accounting for all of these alleles and their relative proportions within the population are measures of genetic variation. Scientists, however, cannot always determine which alleles are present in a population based on outward appearance (i.e. phenotypes) of the organisms that comprise it. Some phenotypes associated with certain alleles are masked or deemphasized when other alleles are present, or are less visible under certain environmental conditions. For this reason, it is important to understand a gene pool as the collective set of alleles whose phenotype may or may not be observable.

Figure 2: Genetic variation within three butterfly species. Three different butterfly species (top row) show distinct wing colors and patterns. When individuals from the same three species are born in a different season, they


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each show different wing color and pattern phenotypes (bottom row). This is a reflection of the variation that exists in the gene pool.

Tropical butterflies provide an excellent example of genetic variation within species. In these butterflies, temperature and light can influence gene expression. Consequently, wing color and pattern can vary depending on the season during which a butterfly is born. The top row of Figure 2 shows examples of three different butterfly species. The bottom row shows butterflies from the same three species, but these individuals were born under different temperature and light conditions than those in the top row. How can this happen? The differences in wing colors and patterns of butterflies of the same species reflect the underlying genetic variability within a population. Even though each butterfly within a species has the potential to develop the wide variety of colors and patterns shown above, its environment influences the phenotypic expression of its genetic characteristics. The gene pool of each species, therefore, contains a collection of many different alleles whose phenotype may or may not be observable.

Can gene pools vary within populations? From the perspective of a geneticist, a population is a group of organisms of the same species that interbreed. This may mean that the group of organisms all live in the same area, or that they can travel over long distances to mate. Over time, a population's gene pool may change. A variety of different factors can account for these changes, including migration of new individuals into the population, death of a large number of individuals within the population, or environmental factors that favor certain traits over others. The factors that affect the composition of the gene pool are shaped both by the physical environment and by time. Consequently, the definition of populations can be refined to include groups of organisms whose genetic makeup can change over time, and groups of organisms that tend to interbreed.

What is variation in a gene pool?

Can gene pools vary within populations?

Unit 5






Key Questions How can mutation cause genetic variation?

Key Concepts genetic variation

species

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PROGRESS

5.3 The Variety of Genes in the Gene Pool Can Be Quantified within a Population

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Most populations have some degree of variation in their gene pools. By measuring the amount of genetic variation in a population, scientists can begin to make predictions about how genetic variation changes over time. These predictions can then help them gain important insights into the processes that allow organisms to adapt to their environment or to develop into new species over generations, also known as the process of evolution.Genetic variation is usually expressed as a relative frequency, which means a proportion of the total population under study. In other words, a relative frequency value represents the percentage of a given phenotype, genotype, or allele within a population. Relative phenotype frequency is the number of individuals in a population that have a specific observable trait or phenotype. To compare different phenotype frequencies, the relative phenotype frequency for each phenotype can be calculated by counting the number of times a particular phenotype appears in a population and dividing it by the total number of individuals in the population. Relative genotype frequency and relative allele frequency are the most important measures of genetic variation. Relative genotype frequency is the percentage of individuals in a population that have a specific genotype. The relative genotype frequencies show the distribution of genetic variation in a population. Relative allele frequency is the percentage of all copies of a certain gene in a population that carry a specific allele. This is an accurate measurement of the amount of genetic variation in a population.

Examining allele frequencies A gene that can occur in two forms is said to have two alleles. Body color in fruit flies is an example of a gene with two alleles: a dominant allele for brown body color, and a recessive allele for black body color. The brown body color allele can be represented as "B" and the black body color allele as "b." The allele frequencies for a gene with two alleles are usually represented by the letters p and q, where the relative frequency of the B allele is p and the relative frequency of the b allele is q. Symbolically, these relative allele frequencies can be represented as: relative frequency (B) = p and relative frequency (b) = q

Remember the Punnett square?

Figure 1: A Punnett square showing how p and q alleles combine.

If B and b are the only two alleles of a gene, then possible genotypes can be predicted by arranging the alleles in a Punnett square, in their p and q representation (Figure 1). This exercise can help to visualize the computation of relative allele frequencies and their corresponding relative genotype frequencies in a population.The possible combinations can be represented mathematically as:


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[p × p] + [2 × p × q] + [q × q]or

p2 + 2pq + q2

How can relative frequencies be used to study populations? The mathematical expression p2 + 2pq + q2 can be used as a platform for understanding both allele frequencies and genotype frequencies in real populations. For instance, if a population does not change over time, then scientists can make certain predictions about its relative allele frequencies, and about its relative genotype frequencies. In other words, if they have information about its relative genotype frequencies, they may also make predictions about its relative allele frequencies. When a population is in equilibrium, the BB homozygotes (individuals that carry the same two dominant B alleles) will have a relative genotype frequency of p2: freq (BB) = p2. Similarly, bb homozygotes (individuals that carry the same two recessive b alleles) will have a relative genotype frequency of q2: freq (bb) = q2. Finally, the Bb heterozygotes (individuals that carry both the dominant B allele and the recessive b allele) will have a relative genotype frequency of 2pq: freq (Bb) = 2pq.In a stable population, the sum of all these relative genotype frequencies remains equal to 1 over successive generations. This is a mathematical way of expressing that the sum of all relative genotype frequencies always equals 1 because if one relative genotype frequency increases, another will decrease in tandem, and alleles become redistributed rather than increasing in proportion to the population. Therefore, this relationship can be expressed mathematically as follows:

This equation is known as the Hardy-Weinberg equation, and it defines a population in which relative allele frequencies do not change over successive generations. Such a population is said to be in equilibrium. This state of equilibrium represented by the Hardy-Weinberg equation is an ideal model against which to compare observed changes in relative allele and genotype frequencies in natural populations.

How is the Hardy-Weinberg equation used? The Hardy-Weinberg equation describes a population at equilibrium. This can only occur in the absence of disturbing factors and when mating between individuals is completely random. When mating is random in a large population, both the relative genotype and allele frequencies will remain constant. Hardy-Weinberg equilibrium in a population can be disturbed by a number of forces, including mutations, nonrandom mating, migration and genetic drift (random changes in alleles from one generation to the next). These forces drive evolutionary change because they add to or take away from the relative allele frequencies in a population. For instance, mutations can disrupt the equilibrium of relative allele frequencies by introducing new alleles into a population. Nonrandom mating can influence relative genotype frequencies within the mating group, because mate choice of the parents can cause a bias toward certain combinations of alleles among their progeny. Migration causes a phenomenon called gene flow that occurs when breeding between two populations leads to the transfer of alleles into a new population, thereby altering the equilibrium of relative allele frequencies. Genetic drift, which typically occurs at a higher rate in small populations, takes place when relative allele frequencies increase or decrease by chance. Since all of these disruptive forces commonly occur in nature, the Hardy-Weinberg equilibrium rarely stays constant. Typically, populations can exist in equilibrium for short periods of time, but rarely stay there in perpetuity. Therefore, Hardy-Weinberg equilibrium describes an idealized state of a population, and genetic variations in nature can be measured as changes from this ideal. The Hardy-Weinberg equation is therefore a tool for measuring real genetic variation in a population over time.

This is just one model A population at Hardy-Weinberg equilibrium exhibits constant relative allele and genotype frequencies over successive generations. However, the converse is not necessarily true: a population that exhibits stable relative allele and genotype frequencies over time may not be in Hardy-Weinberg equilibrium. For example, if the heterozygous genotype provides an advantage (e.g. allows individuals to mate more successfully or provides resistance to disease), it will remain prevalent in the population, yet it will not occur in the proportion predicted by the Hardy-Weinberg equation.

Examining allele frequencies

Remember the Punnett square?

How can relative frequencies be used to study populations?

Unit 5




Key Questions How can the environment affect allelic frequencies?

Key Concepts genetic variation

Hardy-Weinberg equilibrium



How is the Hardy-Weinberg equation used?

This is just one model



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PROGRESS

5.4 The Genetic Variation in a Population Is Caused by Multiple Factors Prev Page

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Genetic variation describes naturally occurring genetic differences among individuals of the same species. This variation permits flexibility and survival of a population in the face of changing environmental circumstances. Consequently, genetic variation is often considered an advantage, as it is a form of preparation for the unexpected. But how does genetic variation increase or decrease? And what effect do fluctuations in genetic variation have on populations over time?

Mating patterns are important When a population interbreeds, nonrandom mating can sometimes occur because one organism chooses to mate with another based on certain traits. In this case, individuals in the population make specific behavioral choices, and these choices shape the genetic combinations that appear in successive generations. When this happens, the mating patterns of that population are no longer random. Nonrandom mating can occur in two forms, with different consequences. One form of nonrandom mating is inbreeding, which occurs when individuals with similar genotypes are more likely to mate with each other rather than with individuals with different genotypes. The second form of nonrandom mating is called outbreeding, wherein there is an increased probability that individuals with a particular genotype will mate with individuals of another particular genotype. Whereas inbreeding can lead to a reduction in genetic variation, outbreeding can lead to an increase.

Random forces lead to genetic drift Sometimes, there can be random fluctuations in the numbers of alleles in a population. These changes in relative allele frequency, called genetic drift, can either increase or decrease by chance over time. Typically, genetic drift occurs in small populations, where infrequently-occurring alleles face a greater chance of being lost. Once it begins, genetic drift will continue until the involved allele is either lost by a population or is the only allele present at a particular gene locus within a population. Both possibilities decrease the genetic diversity of a population. Genetic drift is common after a population experiences a population bottleneck. A population bottleneck arises when a significant number of individuals in a population die or are otherwise prevented from breeding, resulting in a drastic decrease in the size of the population. Genetic drift can result in the loss of rare alleles, and can decrease the size of the gene pool. Genetic drift can also cause a new population to be genetically distinct from its original population, which has led to the hypothesis that genetic drift plays a role in the evolution of new species.

Distribution How does the physical distribution of individuals affect a population? A species with a broad distribution rarely has the same genetic makeup over its entire range. For example, individuals in a population living at one end of the range may live at a higher altitude and encounter different climatic conditions than others living at the opposite end at a lower altitude. What effect does this have? At this more extreme boundary, the relative allele frequency may differ dramatically from those at the opposite boundary. Distribution is one way that genetic variation can be preserved in large populations over wide physical ranges, as different forces will shift relative allele frequencies in different ways at either end. If the individuals at either end of the range reconnect and continue mating, the resulting genetic intermixing can contribute to more genetic variation overall. However, if the range becomes wide enough that interbreeding between opposite ends becomes less and less likely, and the different forces acting at either end become more and more pronounced, and the individuals at each end of the population range may eventually become genetically distinct from one another.

Migration Migration is the movement of organisms from one location to another. Although it can occur in cyclical patterns (as it does in birds), migration when used in a population genetics context often refers to the movement of individuals into or out of a


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defined population. What effect does migration have on relative allele frequencies? If the migrating individuals stay and mate with the destination individuals, they can provide a sudden influx of alleles. After mating is established between the migrating and destination individuals, the migrating individuals will contribute gametes carrying alleles that can alter the existing proportion of alleles in the destination population.Here is an example of migration affecting relative allele frequency:

The overall effect How do populations respond to all these forces? As relative allele frequencies change, relative genotype frequencies may also change. Each genotype in the population usually has a different fitness for that particular environment. In other words, some genotypes will be favored, and individuals with those genotypes will continue to reproduce. Other genotypes will not be favored: individuals with those genotypes will be less likely to reproduce. What type of genotype would be unfavorable? Unfavorable genotypes take many forms, such as increased risk of predation, decreased access to mates, or decreased access to resources that maintain health. Overall, the forces that cause relative allele frequencies to change at the population level can also influence the selection forces that shape them over successive generations. For example, if moths with genotype aa migrate into a population composed of AA and Aa individuals, they will increase the relative allele frequency of a. However, if the aa genotype has a clear disadvantage to survival (e.g. vulnerability to predation), eventually the changes brought about by the initial migration will be reversed. Here is an example of how a specific genotype is less favorable than another genotype:



Summary Genetic variation in a population is derived from a wide assortment of genes and alleles. The persistence of populations over time through changing environments depends on their capacity to adapt to shifting external conditions. Sometimes the addition of a new allele to a population makes it more able to survive; sometimes the addition of a new allele to a population makes it less able. Still other times, the addition of a new allele to a population has no effect at all, yet the new allele will persist over generations because its contribution to survival is neutral.

Mating patterns are important

Random forces lead to genetic drift

Distribution

Migration

The overall effect

Summary

Unit 5






Key Questions How can genetic variation influence evolution?

What is an example of genetic drift?

Key Concepts genetic drift

population bottleneck

migration/gene flow

Hardy-Weinberg equation

artifical selection

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PROGRESS

5.5 Genomics Enables Scientists to Study Genetic Variability in Human Populations

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Thinking about population genetics often brings to mind visions of animals in the wild being swept along by the tide of natural catastrophes, soil depletion, or predation. However, over the past ten years the field of population genetics has undergone major renovations because of recent advances in gene sequencing and screening technologies. These technological innovations have allowed scientists to tackle bigger and broader questions related to population trends, and to study genetic variation on a much broader scale than ever before possible with older methods, such as test crosses, random sampling, and field work. Today, discoveries can be facilitated by the ever-expanding field of genomics, which is the use of large databases for the purpose of studying genetic variation on a large scale across many different organisms.

What is genomics?

Figure 1: Genome size is the total number of base pairs in an organism. While the number of genes in an organism's DNA (red bars) varies from species to species (numbers at right), it is not always proportional to genome size (blue bars). Note how many genes a fruit fly can squeeze out of its relatively small genome.

Until recently, the term genome was used to describe the complete set of chromosomes that made up a given species. Today, scientists use the term genome to refer to the complete set of DNA sequences derived from each chromosome of a given species. Genomics is a relatively new and ever-expanding field dedicated to the study of defining genomes in this more specific way. The direct analysis of the genome of an organism, or the genomes of a group of organisms, is now possible through advances in the efficiency of DNA sequencing and large-scale genetic screening. These new high-throughput methods allow researchers to collect vast amounts of information about genetic variation in very short periods of time.Genomics has also shown that the size of a genome (i.e. the number of nucleotide pairs it contains) is not necessarily proportional to the number of genes contained within it. Some organisms, like the fruit fly, fit a considerable number of genes into a relatively small genome, whereas humans and mice possess many extra "unused" nucleotide pairs that do not encode genes (Figure 1).

See how human genomes compare to others


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How many genes does it take to build a human being?Although early reports suggested that human chromosomes might contain as many as 100,000 different genes, we now know that the 24 different human chromosomes altogether contain 20,000-25,000 different genes. However, it is likely that many of those genes are not absolutely required.

How can we study human genetic variation?Humans can also be the focus of population genetics studies, as they too have been subject to the forces of change over long periods of time. Recently, the DNA sequence of the entire human genome was deciphered in a massive effort called The Human Genome Project (HGP). This project sequenced the DNA of each human chromosome from end to end, determined the DNA sequence of every human gene, and mapped the precise location of every human gene to a particular region of a human chromosome. With this information in hand, scientists now have a baseline definition of every human gene. With this baseline, they are beginning to study how the DNA sequences of human genes can vary among individuals and populations. In fact, scientists can currently study the variability of those genes (i.e. all allelic forms) in different populations around the globe. Early results from these studies indicate that humans are identical over the vast majority of their genome. The apparently striking phenotypic variation among human beings around the world can be accounted for by only an exceptionally small number of genetic differences. Genes that code for skin color, facial features, or body size represent a small fraction of the DNA that comprises the total human genome.

Variation in the human genome: SNPs More on human populations

See a model of recent human evolution•What are some human allelic variations?•What is the Human Genome Project (HGP)?•The HGP: The contributions of Francis Collins and Craig Venter•

After the completion of the HGP in 2003, researchers began to pinpoint locations within the genome that varied among individuals. These scientists discovered that the most common type of DNA sequence variation found in the human genome is the single nucleotide polymorphism (SNP, pronounced "snip"). There are approximately 10 million SNPs in the human genome. A worldwide effort known as the HapMap Project is mapping SNPs and other genetic variants in human populations around the world. By mapping the distribution of SNPs among different human populations, researchers can begin to learn which types of variation are most common in certain regions of the world. This information will help explain human origins and disease risks as well as how they relate to environmental conditions, both past and present. To date, the HapMap project has identified over 3.1 million SNPs across the human genome that are common among individuals of African, Asian, and European ancestry.The HapMap database has also helped foster a new type of research in personalized medicine called the genome-wide association study (GWAS). With these studies, the distribution of SNPs is determined in hundreds, or even thousands, of people with and without a particular disease. Comparisons between diseased and non-diseased groups of individuals help



determine which SNPs co-occur with disease symptoms. With this information in hand, scientists can carry out statistical analyses to help predict whether a certain SNP is associated with a specific disease, with the hope of identifying individuals who may be at risk. For example, in a recent study conducted in the United Kingdom, researchers genotyped 2,000 individuals who had one of seven common disorders. Next, those individuals were compared to 3,000 genotyped control individuals who did not have the common disorders. With these comparisons, the researchers identified new genetic markers associated with increased risk for disorders such as heart disease and diabetes. In the future this study will be expanded to include 36,000 more individuals, and it will focus on 14 more health-related disorders as well as individual responses to certain drugs. Using these types of studies, scientists can sample large numbers of people and make meaningful predictions regarding disease risk for individuals based on the presence or absence of genetic markers within their genome.

Genomics and biological discovery Genomic data can support discovery in diverse areas of biology, including medicine, systematics, and conservation biology. Like many histories, the history of genomics is fraught with conflict, disagreement, and excitement. The personalities and ideas that have shaped genomics even included a race between publicly funded and corporate genome sequencing groups that resulted in a tie at the finish line. Several subspecialty areas of genomics are also expanding as the community of scientists within them grows. These relatively new areas of genomic investigation include: epigenomics (the study of DNA modification), transcriptomics (the study of cellular RNA content), and proteomics (the study of proteins that characterize a particular cell).

What is genomics?

See how human genomes compare to others

How many genes does it take to build a human being?

How can we study human genetic variation?

Variation in the human genome: SNPs

Genomics and biological discovery

Unit 5






Key Questions How is a GWAS conducted?

What are some models of human origins based on genetic data?

Can we use SNP data to study human evolution?

What are the four main categories of genomics research?

Key Concepts GWAS

SNP

genomics

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