5. The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. As a basis for understanding this concept: a. Students know the general structures and functions of DNA, RNA, and protein. Nucleic acids are polymers composed of monomers called nucleotides. Each nucleotide consists of three subunits: a five-carbon pentose sugar, a phosphoric acid group, and one of four nitrogen bases. (For DNA these nitrogen bases are adenine, guanine, cytosine, or thymine.) DNA and RNA differ in a number of major ways. A DNA nucleotide contains a deoxyribose sugar, but RNA contains ribose sugar. The nitrogen bases in RNA are the same as those in DNA except that thymine is replaced by uracil. RNA consists of only one strand of nucleotides instead of two as in DNA. The DNA molecule consists of two strands twisted around each other into a double helix resembling a ladder twisted around its long axis. The outside, or uprights, of the ladder are formed by the two sugar-phosphate backbones. The rungs of the ladder are composed of pairs of nitrogen bases, one extending from each upright. In DNA these nitrogen bases always pair so that T pairs with A, and G pairs with C. This pairing is the reason DNA acts as a template for its own replication. RNA exists in many structural forms, many of which play different roles in protein synthesis. The mRNA form serves as a template during protein synthesis, and its codons are recognized by aminoacylated tRNAs. Protein and rRNA make up the structure of the ribosome. Proteins are polymers composed of amino acid monomers. Different types of proteins function as enzymes and transport molecules, hormones, structural components of cells, and antibodies that fight infection. Most cells in an individual organism carry the same set of DNA instructions but do not use the entire DNA set all the time. Only a small amount of the DNA appropriate to the function of that cell is expressed. Genes are, therefore, turned on or turned off as needed by the cell, and the products coded by these genes are produced only when required. 5. b. Students know how to apply base-pairing rules to explain precise copying of DNA during semiconservative replication and transcription of information from DNA into mRNA. Enzymes initiate DNA replication by unzipping, or unwinding, the double helix to separate the two parental strands. Each strand acts as a template to form a complementary daughter strand of DNA. The new daughter strands are formed when complementary new nucleotides are added to the bases of the nucleotides on the parental strands. The nucleotide sequence of the parental strand dictates the order of the nucleotides in the daughter strands. One parental strand is conserved and joins a newly synthesized complementary strand to form the new double helix; this process is called semiconservative replication. DNA replication is usually initiated by the separation of DNA strands in a small region to make a “replication bubble” in which DNA synthesis is primed. The DNA strands progressively unwind and are replicated as the replication bubble expands, and the two forks of replication move in opposite directions along the chromosome. At each of the diverging replication forks, the strand that is conserved remains a single, continuous “leading” strand, and the other “lagging” complementary strand is made as a series of short fragments that are subsequently repaired and ligated together. RNA is produced from DNA when a section of DNA (containing the nucleotide sequence required for the production of a specific protein) is transcribed. Only the template side of the DNA is copied. RNA then leaves the nucleus and travels to the cytoplasm, where protein synthesis takes place. 5. c. Students know how genetic engineering (biotechnology) is used to produce novel biomedical and agricultural products. Recombinant DNA contains DNA from two or more different sources. Bacterial plasmids and viruses are the two most common vectors, or carriers, by which recombinant DNA is introduced into a host cell. Restriction enzymes provide the means by which researchers cut DNA at desired locations to provide DNA fragments with “sticky ends.” Genes, once identified, can be amplified either by cloning or by polymerase chain reactions, both of which produce large numbers of copies. The recombinant cells are then grown in large fermentation vessels, and their products are extracted from the cells (or from the medium if the products are secreted) and purified. Genes for human insulin, human growth hormone, blood clotting factors, and many other products have been

identified and introduced into bacteria or other microorganisms that are then cultured for commercial production. Some agricultural applications of this technology are the identification and insertion of genes to increase the productivity of food crops and animals and to promote resistance to certain pests and herbicides, robustness in the face of harsh environmental conditions, and resistance to various viruses.

Major concepts:

1. In the 1930s the favored hypothesis suggested that the genetic material (the chemical substance that carried hereditary information) most probably was protein. Nucleic acids were considered too simple to provide much information and were thought to be structural molecules onto which the informational proteins were organized. Experiments performed in the 1940s and 1950s clearly argued for DNA as the genetic material, and effectively opened the current era of Molecular Biology.

2. Cells contain two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The main difference between the two is surprisingly simple; RNA lacks a single oxygen atom in a repeating sugar group that forms part of the backbone of the molecule. This chemical difference makes DNA an unusually stable molecule (capable of carrying information for generations) and RNA an unstable molecule (usually destroyed in a matter of minutes or at most hours).

3. Since cells have to pass an exact copy of their hereditary instructions from one generation to the next DNA must be faithfully copied before cells divide. The structure of DNA (in Watson & Crick famous understatement) "…suggests a possible copying mechanism for the genetic material."

DNA is the "genetic material"

In 1868 Friedrich Miescher, a Swiss physician, first isolated a molecule he called "nuclein". Miescher knew that the genetic material resided in nuclei, and was probably carried on the bodies which microscopists had called chromosomes. However, as you can imagine, seeing chromosomes does not explain how they work. Miescher wanted to understand them chemically. Nuclein we would call deoxyribonucleic acid (literally, an acid from nuclei which contains the sugar deoxyribose).

I'm sure that most people take it for granted that DNA is the genetic material. You have heard that DNA is the genetic material because it is inherited from one generation to the next. But what is the evidence that this is so? The early history of molecular biology is punctuated by a series of experiments so simple, and yet so elegant, that they have become known by the authors names which constitute almost a pantheon of biology--Avery, McCloud and McCarthy; Watson & Crick; Hershey-Chase; Meselson & Stahl; Jacob & Monod. Most of these experiments date from the first decade of modern molecular biology-the 1950s. Though they are over 40 years old, nearly every biology student learns about these experiments. They form the intellectual basis for much of modern biology, or at least of modern genetics.

Griffith and Avery, McCloud & McCarthy provide the first evidence for DNA as the genetic material

Much of the first half of the century microbiologists attempted to identify organisms responsible for disease. Fred Griffith was an Army doctor who in 1928 wanted to make a vaccine against Streptococcus pneumoniae, which caused a type of pneumonia. Since the time of Pasteur, about 50 years before, vaccines had been made using killed microorganisms which could be injected into patients to elicit the immune response of live cells without risk of disease. Though he failed in making the vaccine he stumbled on a demonstration of the transmission of genetic instructions by a process we now call transformation.

• He found that the bacterium had two forms when grown on agar plates, a smooth (S) and a rough (R) form. • The R bacteria were harmless, but the S bacteria were lethal when injected into mice. • Heat-killed S cells were also harmless-the same effect seen by Pasteur.

• However, surprisingly when live R cells were mixed with killed S cells and injected into mice the mice died, and the bacteria rescued from the mice had been "transformed" into S type.

This experiment strongly implied that genetic material had been transferred from the dead to the live cell. It was hard to be certain of this, or to know what the material was in this crude experiment.

Sixteen years later the team of Avery, MacLeod and McCarty revisited this experiment and attempted a more definitive experiment.

• A favorite hypothesis of the 1930s was that the genetic instructions in chromosomes was protein. • Chromosomes, as we shall see, are largely made of proteins, and biologists thought that DNA performed a

structural role as a scaffold to hold these informational proteins. • However, chemical analysis of DNA had begun to suggest that it might be a more interesting molecule than

had been supposed.

Avery, MacLeod and McCarty repeated Griffith's experiment using purified DNA molecules and got the same "transformation" of R cells into S cells. Since it is often the case that favored hypotheses are given up only very grudgingly, they did some critical control experiments. Knowing that many would claim that their DNA was contaminated with protein which was the true active principle, they treated the "DNA" with two enzymes:

• Protease: this degrades proteins to its monomers (amino acids)

• DNAse: this degrades DNA to its monomers (nucleotides) • Pre-treatment with protease had no effect on transformation, while pre-treatment with DNAse eliminated

transformation

The Hershey-Chase experiment

The second classical demonstration of DNA as the genetic material was an outgrowth of a group of molecular biologists who in the late 1940s and early 1950s started to use viruses that attack bacteria (bacteriophages--or literally "bacteria eaters") to study the nature of heredity. This group, the "phage group", at Indiana University included the very young James Watson who would later propose a structure for DNA along with Francis Crick.

Alfred Hershey was one of the founders of this group. One of his students, Martha Chase, performed an experiment which clearly distinguished between DNA and protein as the genetic material. The phage group had identified a series of bacteriophage and shown that they consisted of a combination of only DNA and protein. They had also shown that these viruses attacked cells from the outside, but that most of the virus never entered the cell. Later work showed that the viruses are like microscopic hypodermic needles, injected their genome into the inside of the bacteria

Hershey & Chase used a simple radiolabling protocol to distinguish between the DNA and protein portions of the viruses being the genetic material. They knew that protein includes the atom sulfur (in two of the amino acids) but that DNA does not, and that DNA contains phosphorus while the proteins of the viruses did not. They labeled viruses with either a radioactive form of sulfur (35S) or of phosphorus (32P) and used them to infect cells. After allowing the viruses to infect the cells they put them through a blender-this knocked the viruses off of the cells without affecting the cells. They found that the cells were still infected after this treatment. They looked for the radioactivity of the 35S or the 32P and found that some of the 32P went with the cells, while all of the 35S did not. This simple experiment clearly suggested that the nucleic acid, but not the protein, was the genetic material since the nucleic acid, and not the protein was transfered into cells when they were infected by viruses.

Notes: Structure and Function of DNA

DNA is an enormous polymer composed of monomers called nucleotides. A nucleotide is made up of 3 parts which are

1. phosphate group 2. a 5-carbon sugar (deoxyribose) 3. a nitrogen base

There are 4 nitrogen bases and thus 4 nucleotide monomers found in DNA. They are:

1. Adenine 2. Thymine 3. Guanine 4. Cytosine

Adenine always pairs with thymine using 2 hydrogen bonds and guanine pairs with cytosine using 3 hydrogen bonds. Early examination of DNA's chemical composition showed that the amounts of thymine was equal to that of adenine, and likewise the amounts of guanine and cytosine nucleotides were equal. This information would become a vital clue in deciphering the genetic code.

Many researchers did not believe DNA with only 4 different monomers was complicated enough to be the genetic material. They felt that proteins which seemed more complex must be the stuff of genes. DNA's structure was finally worked out by 2 groups of researchers.

Rosalind Franklin and her team used X-ray diffraction techniques on crystallized Dto show that it formed a helix, each coil consisting of 10 nucleotides.

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James Watson and Francis Crick used Franklin's data and the rest of the information gathered over the years about DNA and hypothesized that DNA was actually two strands. In each strand the nucleotides were bonded -- sugar to phosphate -- in a linear fashion like the letters of words strung together in a sentence. They also determined that

the DNA molecule must be a double helix with the complimentary nucleotides (A-T and C-G weakly bonded together with hydrogen bonds.

The DNA double helix structure is often compared to a twisted ladder. The rails or sides of the ladder are the sugar-phosphate backbones. The rungs or steps are the pairs of nitrogen bases.

The linear order of bases make up the genetic code. Members of the same species have nearly identical sequences. "Differences in base order become progressively greater as organisms become further separated in phylogeny." Sequences in bacteria are very different (but not entirely) from those found in animals.

During cell division DNA preserves an organism's identity by passing exact copies of itself to each new cell. Mutations result if the DNA is changed in any way.

Replication of DNA

DNA is built to be copied. Its structure suits its function. The double strand is the key because it provides

• chemical stability, • ease of repair and the • ability to copy itself.

To replicate, the paired nitrogen bases are unzipped (hydrogen bonds broken between the two strands). Because A and T always match as do the bases C and G, it is easy to see that new complimentary bases can be paired up and linked into 2 additional strands.

Learn the following steps in the replication of DNA.

1. Expose the nitrogen bases by breaking the hydrogen bonds between them creating 2 separate strands. 2. Each separate strand can act as a template for a brand new strand. Energy rich nucleotides are paired to

those on the exposed chain(s). 3. Once paired with its complementary base the new nucleotide can be linked in place by a special enzyme

(DNA polymerase). The sugar or one nucleotide combines with the phosphate of the next in line. 4. Pairing and linking continues until the original two DNA chains have been copied resulting in 4 strands (2

double helixes). In other words where there was only one DNA molecule now there are two -- each identical to the other.

The DNA Code Revealed

Protein structure is encoded in the strands of DNA. Only one strand is needed for this. The other strand has no genetic meaning. The DNA found in each and every one of your cells represents a genetic library or instruction manual containing all of the instructions necessary to make a copy of you.

DNA's code is simple and elegant. The four nucleotides represent the letters of the genetic language. Only three letter words are permitted. Punctuation to start and stop each gene is also included, and must be 3 letters (nucleotides) long.

How many words are possible if you arrange four different letters in groups of three? The answer is 64. Some of the 64 possible combinations are AAA, AAT, AAC, AAG, ATA, AGA, ACA.

The genetic language actually needs just 22 words -- one for each amino acid found in proteins and 2 for punctuation (start and stop). Many amino acids have more than one code word (synonyms) so all 64 codes words are used. Each group of 3 nucleotides or code words in DNA is called a codon.

A Protein usually requires many amino acids to function properly so a gene (DNA) must have 3 times as many nucleotides as the protein. For example a protein with 100 amino acids is encoded by a segment of DNA with 300 nucleotides. DNA does not make proteins directly however. This task is the responsibility of RNA.

RNA and Transcription

RNA is a single stranded nucleic acid which is much shorter and less stable than DNA. It cannot make copies of itself, but it can translate its codons into a sequence of amino acids.

RNA's code is a series of nucleotides complementary to a single-stranded segment of DNA. The complement of a codon is an anticodon. The side of DNA used to transcribe a copy of RNA is actually a series of anticodons. Transcription is the process of making RNA from DNA.

To allow the cell the ability to distinguish between RNA and DNA two chemical differences are used.

1. RNA uses a different sugar group called ribose instead of deoxyribose. 2. RNA uses the nucleotide Uracil (U) instead of Thymine (T)

An example of RNA transcription by DNA is illustrated below.

Summary of differences between DNA and RNA

Size Strands Stability Sugar Nucleotides RNA small single - globular low ribose A-U-C-G DNA large double - helix high deoxyribose A-T-C-G

There are three varieties of RNA in a cell.

1. Messenger RNA (mRNA) -- carries the codons from the nucleus to locations of protein synthesis 2. Transfer RNA (tRNA) -- transport individual amino acids to the site of protein synthesis. There a 20

varieties of tRNA, one for each amino acid. Each tRNA has one anticodon to match the mRNA codon for the amino acid it carries.

3. Ribosomal RNA (rRNA) -- makes up part of the structure of ribosomes. rRNA is made in the nucleolus.

All three types of RNA are made by transcription from DNA.

Protein Synthesis - Translation

When a cell receives a signal to manufacture a specific protein several things must happen. The two main processes are:

1. Transcription: DNA makes mRNA in the nucleus. 2. Translation: mRNA makes protein in the cytoplasm using ribosomes and various other ingredients.

Translation involves the transfer of the genetic code from RNA to protein. Proteins are synthesized in the cytoplasm where ever there are ribosomes. Ribosomes are considered the smallest organelles. Their only job is to put together amino acids into polypeptides.

To make a protein you need a variety of components found in the cell.

Ribosomes Messenger RNA (~one for every gene) Transfer RNA (20 types) Amino Acids (20 types) ATP Various enzymes

To assemble a polypeptide, a simple linear strand of amino acids, follow the five steps.

Step 1. mRNA is made and is transported out of the nucleus Step 2. mRNA combines with a ribosome Step 3. initiator tRNA is charged with a met (methionine) amino acid and diffuses to the mRNA-ribosome

complex. Its codon hydrogen-bonds with the start codon (AUG) on the mRNA. Step 4. The rest of the amino acids are added in order one at a time as the ribosome moves from codon to

codon until- Step 5. one of three stop codons is encountered and the finished polypeptide drifts away

Molecular biology is useful in many fields. DNA technology is utilized in solving crimes. It also allows searchers to produce banks of DNA, RNA and proteins, while mapping the human genome. Tracers are used to synthesize specific DNA or RNA probes, essential to localizing sequences involved in genetic disorders. With genetic engineering, new proteins are synthesized. They can be introduced into plants or animal genomes, producing a new type of disease resistant plants, capable of living in inhospitable environments (i.e. temperature and water extremes,...). When introduced into bacteria, these proteins have also produced new antibiotics and useful drugs.

Techniques of cloning generate large quantities of pure human proteins, which are used to treat diseases like diabetes. In the future, a resource bank for rare human proteins or other molecules is a possibility. For instance, DNA sequences which are modified to correct a mutation, to increase the production of a specific protein or to produce a new type of protein can be stored . This technique will be probably play a key role in gene therapy.

Plasmids are similar to viruses, but lack a protein coat and cannot move from cell to cell in the same fashion as a

virus. Plasmid vectors are small circular molecules of double stranded DNA derived from natural plasmids that occur in bacterial cells. A piece of DNA can be inserted into a plasmid if both the circular plasmid and the source of DNA have recognition sites for the same restriction endonuclease. The plasmid and the foreign DNA are cut by this restriction endonuclease (EcoRI in this example) producing intermediates with sticky and complementary ends. Those two intermediates recombine by base-pairing and are linked by the action of DNA ligase. A new plasmid containing the foreign DNA as an insert is obtained. A few mismatches occur, producing an undesirable recombinant. The new plasmid can be introduced into bacterial cells that can produce many copies of the inserted DNA . This technique is called DNA cloning

Process by which a plasmid is used to import recombinant DNA into a host cell for cloning.

Many diseases are caused by gene alterations. Our understanding of genetic diseases was greatly increased by information gained from DNA cloning. In DNA cloning, a DNA fragment that contains a gene of interest is inserted into a cloning vector or plasmid.

The plasmid carrying genes for antibiotic resistance, and a DNA strand, which contains the gene of interest, are both cut with the same restriction endonuclease. The plasmid is opened up and the gene is freed from its parent DNA strand. They have complementary "sticky ends." The opened plasmid and the freed gene are mixed with DNA ligase, which reforms the two pieces as recombinant DNA.

Plasmids + copies of the DNA fragment produce quantities of recombinant DNA.

This recombinant DNA stew is allowed to transform a bacterial culture, which is then exposed to antibiotics. All the cells except those which have been encoded by the plasmid DNA recombinant are killed, leaving a cell culture containing the desired recombinant DNA.

DNA cloning allows a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. This technique is the first stage of most of the genetic engineering experiments: production of DNA libraries, PCR, DNA sequencing, et al.

Example of the technique of DNA cloning into a plasmid.: Insertion of the gene coding for insulin into a bacterial plasmid, which in turn carries the gene into a replicating bacterial cell that produces human insulin.

Plasmid: Plasmids are small circles of DNA found in bacteria cells, separate from the bacterial chromosome and smaller than it. They are able to pass readily from one cell to another, even when the cells are clearly from different species, far apart on the evolutionary scale. Consequently, plasmids can be used as vectors, permitting the reproduction of a foreign DNA by using the bacterial replicating system.

cDNA: Human genes composed of coding and non- coding sequences. The copy of the coding sequences is called cDNA. It can be obtained from the reverse transcription of messenger RNA. The transcription and translation of the insulin cDNA will allow the production of a functional insulin molecule.

Transfer of the Insulin gene into a plasmid vector.

• The plasmid is cut across both strands by a restriction enzyme, leaving loose, sticky ends to which DNA can be attached.

• Special linking sequences are added to the human cDNA so that it will fit precisely into the loose ends of the opened plasmid DNA ring.

• The plasmid containing the human gene, also called a recombinant plasmid, is now ready to be inserted into another organism, such as a bacterial cell.

Cloning the Insulin gene

The recombinant plasmids and the bacterial cells are mixed up. Plasmids enters the bacteria in a process called transfection. With the recombinant DNA molecule successfully inserted into the bacterial host, another property of plasmids can be exploited - their capacity to replicate. Once inside a bacterium, the plasmid containing the human cDNA can multiply to yield several dozen copies.When the bacteria divide, the plasmids are divided between the two daughter cells and the plasmids continue to reproduce. With cells dividing rapidly (every 20 minutes), a bacterium containing human cDNA (encoding for insulin, for example) will shortly produce many millions of similar cells (clones) containing the same human gene.

DNA cloning is a technique that allows the wholesale production of a specific DNA sequence. DNA containing a gene of interest is inserted into the purified DNA genome of a self replicating element, which can be a plasmid, a virus or in this case, a yeast artificial chromosome (YAC).

A YAC can be considered as a functional artificial chromosome (self replicating element), since it includes three specific DNA sequences that enable it to propagate from one cell to its offspring:

• TEL: The telomere which is located at each chromosome end, protects the linear DNA from degradation by nucleases.

• CEN: The centromere which is the attachment site for mitotic spindle fibers, "pulls" one copy of each duplicated chromosome into each new daughter cell.

• ORI: Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks.

It also contains few other specific sequences like:

• A and B: selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome.

• Recognition site for the two restriction enzymes EcoRI and BamHI.

While DNA cloning into a plasmid allows the insertion of DNA fragment of about 10,000 nucleotide base pairs, DNA cloning into a YAC allows the insertion of DNA fragments up to 1,000,000 nucleotide base pairs . Why is it so important to be able to clone such large sequences? To map the entire human genome (3x1,000,000,000 nucleotide base pairs) it would require more than 100,000 plasmid clones. In principle, the human genome could be represented in about 10,000 YAC clones.

Techniques for cloning genomic DNA into yeast artificial chromosomes (YAC) make it possible to analyze very long DNA sequences like human genes.

Cloning human genomic DNA into a YAC:

1. Genomic DNA is partially digested by the restriction enzyme EcoRI. Very large DNA fragments are obtained.

2. The YAC is digested by the two restriction enzymes EcoRI and BamHI. 3. Those two elements recombine at the EcoRI sites and are covalently linked by the DNA ligase. 4. A recombinant YAC vector, a yeast artificial chromosome with genomic DNA inserted, is produced. This

vector can be used to infect yeast cells and generated an unlimited number of copies.

Polymerase Chain Reaction (PCR)

The elegant technique of PCR, by which fragments of DNA can be made to replicate very rapidly, is illustrated.

Figure Legend:

Polymerase chain reaction (PCR), is a common method of creating copies of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule into many billions of molecules.

In one application of the technology, small samples of DNA, such as those found in a strand of hair at a crime scene, can produce sufficient copies to carry out forensic tests.