Chapter 18

Molecular Genetics

Goals for this Chapter:1. Summarize the events

and experiments that led to the discovery of the structure of DNA

2. Explain how the interaction between DNA and proteins results in the accurate replication of genetic information

3. Design and construct models to simulate the structure and replication of DNA

Goals for this Chapter:4. Explain how genetic

information is encoded in DNA molecules

5. Describe the processes through which genetic information is expressed in living cells

6. Design and perform a simulation to illustrate the steps of protein synthesis

7. Explain some of causes and effects of DNA mutations

Goals for this Chapter:8. Describe how random

changes in nucleotide sequences provide a source of genetic variability

9. Explain how nucleotide sequences provide evidence that different species are related

10.Design and perform a simulation to illustrate the use of restriction enzymes and ligases to create recombinant DNA

Goals for this Chapter:• Explain how the insertion

of new DNA sequences into cells can transform organisms

• Describe some of the social, environmental, and ethical issues associated with genetic technologies

18.1 – DNA Structure and Replication

• In 1869, Friedrich Mieschner coined the term “nucleic acid” to describe the material found in the nuclei of cells

• However, it took almost a century for scientists to understand the DNA was the material that carried hereditary information

Isolating the Material of Heredity• In the early 1900s,

Phoebus Levene identified two compounds in chromosomes – proteins and DNA

• Scientists did not know what part (the DNA or protein) actually carried hereditary information

• Two major experiments led to the identification of DNA as hereditary material:

Griffith’s Transforming Principle

Griffith’s Transforming Principle• Griffith’s experiment

provided good evidence that DNA was the material responsible for passing on traits

• However, scientists were not prepared to accept this explanation until more evidence was gathered

Hershey & Chase• Hershey and Chase

performed an experiment in 1952 that used radioactive labeling of compounds to trace hereditary material

• They used two radioactive materials (sulfur-35, which would be found in proteins and phosphorus-32, which would be found in DNA) to label parts of a bacteriophage

Hershey and Chase• In the case

where the phosphorus marker on DNA was used, material was found inside the cell, while the sulfur markers on the proteins were not

What is DNA?• DNA is deoxyribonucleic

acid• It is a molecule used by

cells to carry genetic information

• The code in DNA is arranged into genes

http://www.pbs.org

What is Found in DNA?• DNA actually contains

both proteins and nucleic acids

• However, the proteins do not contain the genetic code

• Our genetic code is contained in the nucleic acids found within the DNA structure

http://www.accelrys.com

The Structure of DNA• DNA consists of 6

chemicals:1. Deoxyribose sugar2. Phosphate3. Adenine4. Cytosine5. Guanine6. Thymine

• The nitrogen bases are always found in complementary pairs

http://student.ccbcmd.edu

Chagraff’s Rule• In the 1940s, Edwin

Chagraff determined that although nucleotides were not found in equal amounts, there are roughly the same amounts of complementary bases

• For instance, if a sample of DNA has 15% thymine bases…

Watson & Crick• To understand how DNA

operates, its structure must be understood

• James Watson & Francis Crick determined the helical structure of DNA at Cambridge University in 1953

• Their analysis of X-ray diffraction patterns of crystallized DNA molecules allowed them to determine the structure of DNA

http://nitro.biosci.arizona.edu

http://genome.jgi-psf.org

Rosalind Franklin• Rosalind Franklin

provided the X-ray diffraction analysis of crystallized DNA to Watson & Crick

• Her work along with the work of Chagraff allowed Watson and Crick to develop the well-known double-helix model of DNA that we have today

A Closer Look at DNA• As you can

see, DNA is antiparallel, which means that the left hand strand runs the opposite direction of the right hand strand

mRNA vs. DNAmRNA

Genes and the Genome• Gene:

• Genome:

Placement of Genes• Genes are not equally

spaced on chromosomes• For instance, chromosome

4 is relatively long (200 million bases), but has about 800 genes

• Chromosome 19 has only 55 million bases in comparison, but has more than 1500 genes

The Replication of DNA• The DNA molecule can

make copies of itself• This is required to ensure

that two new cells that arise from mitosis have the same genetic code

• Replication occurs in a series of steps

Initiation• Replication starts at a

specific nucleotide sequence, called the replication origin

• Our chromosomes have multiple replication origins, while the circular DNA of bacteria only have a single replication origin

“Unzipping the Helix”• An enzyme known as DNA

helicase unwinds the DNA at replication forks

• The action of helicase creates a “replication bubble” where the DNA has been unwound

• At each end of the “bubble” are replication forks that branch out to unpaired single strands

Elongation• Elongation:

• Elongation is carried out by DNA polymerase enzymes

• They act based on place placement of primers

“Primers”• An enzyme known as

primase places RNA primers at the sites where DNA replication is to begin

“Polymerases”• There are 2 significant

DNA polymerase enzymes• polymerase III attaches

base pairs to the exposed DNA strand in the 5’ to 3’ direction (the 5’ and 3’ refer to the carbons in the deoxyribose sugar)

• One strand that is created is continuous (known as the leading strand), while other strands (lagging strand) is replicated in short segments

• These short segments are known as Okazaki fragments, and they will be sealed later

“Polymerases”• The enzyme polymerase I

follows polymerase III and removes the RNA primers, replacing them with nucleotides

• The result is two strands of DNA that are identical to their parent

“Sealing the Deal”• At this point, the DNA still

has small “nicks” in it• Another enzyme, known

as ligase, repairs those nicks (assembles the Okazaki fragments into a single long DNA chain)

• The completion of the two new DNA strands is known as termination

Interactive Review

The Final Product• As a result, we are left

with two strands of DNA• DNA replication is

semiconservative – each new strand has part of the older parent strand

Gene Sequencing – Circa 1990s

• We can now map genes by using restriction enzymes to chop the DNA into small segments

• Each of these enzymes cuts at a specific DNA sequence

• This produces segments of varying lengths, known as RFLPs (Restriction Fragment Length Polymorphisms)

• The RFLPs are then marked with radioactive dyes

• Finally, the RFLPs are placed on a thin layer of gel through which a small electrical field is applied

• Within the gel, the RFLPs are pulled along by the electrical field

• The smaller, lighter fragments move the greatest distance

• This creates a distinct banding pattern

• These bands can then be used to map genes

• As well, this can be used for “DNA Fingerprinting” as each person’s pattern of bands is different

Modern Analysis• Mapping genes using gel

electrophoresis takes an incredibly long time

• Now, DNA is still cut into fragments, but four different colours of dyes are used

• A laser is run over the fragments and a computer records the reflected light

• Each of the colours corresponds to a different nitrogen base

• Therefore, genes can be now mapped by computer at a rate of over a thousand base pairs in a minute (rather than months of work by hand)

http://bioweb.wku.edu

The Human Genome Project• The first map of the

human genome was completed in 2000

• By 2003, a much more complete and comprehensive map was completed by an international team of scientists

18.2 – Protein Synthesis and Gene Expression

• In the same year that Watson and Crick published their model of DNA, Frederick Danger established that proteins consist of long chains of amino acids

• The sequence of the amino acids determines the shape and properties of the protein

• Ultimately, the interactions between proteins drives how cells operate

• Scientists began to wonder if the sequence in DNA was related to the sequence of amino acids in a protein

• It was soon shown that the genetic code in fact does determine the sequence of amino acids found in proteins

Gene Expression• Genetic information flows

from DNA to RNA to protein

• This is known as the “central dogma” of gene expression

DNA and Protein Synthesis• Although DNA contains very few

different structural components, it is responsible for coding for huge amounts of information (about 25, 000 genes in a human)

• The sequence of the base pairs is the key to coding for different proteins

• Because there are only 4 nitrogen bases and 20 amino acids, 3 bases together can code for different proteins (two bases can only code for 16, while three can code for 64 possible combinations)

Codons• A codon is a 3-base pair

segment of DNA• Each codon corresponds

to a particular amino acid, or it also may correspond to an initiator “go” or terminator “stop” command

mRNA• to produce

proteins, the DNA does not leave the nucleus

• a carrier molecule known as messenger RNA (mRNA) is used to carry the code to the ribosomes which produce protein

http://tigger.uic.edu

Transcription• The DNA strand

“unzips”, exposing the nucleotides

• Nucleotides in the mRNA are arranged using the complementary nucleotides on the DNA as a blueprint

• The mRNA chain fuses and is moved to the ribosome

• The DNA strands rejoin

http://fig.cox.miami.edu

Translation and Protein Synthesis• the single-stranded mRNA

attaches itself to the small ribosome like a ribbon

• initiator codons in the mRNA turn on protein synthesis

• transfer RNA (tRNA) molecules in the cytoplasm pick up amino acids and bring them to the mRNA

Overview – Synthesis of Protein• DNA “unzips”• mRNA makes a

complementary copy of the DNA

• mRNA is taken to the ribosomes

• The ribosomes match the mRNA with tRNA that carry amino acids

• The amino acids form a chain, which becomes a protein

• the mRNA “stop” codon is read, and synthesis stops

Protein Synthesis Animation

The Genome and Proteome• Genomics is the study of

entire genomes and how the genes interact

• However, study of the proteome (the proteins produced by the genome) is often more important because they are the functional parts of the genome

Mutations and Genetic Recombination• Genomes are not constant• Mutations occur from time

to time• Mutations occurring in

body cells are called somatic cell mutations

• However, only mutations occurring in reproductive cells (germ line mutations) will be passed on to offspring

Errors and Mutations• If there are 3 billion base

pairs in the DNA of each of your cells, even 1 mistake in 1000 could cause up to 3,000,000 mutations during each replication

• However, mistakes in duplicating DNA are very infrequent

• This is because “proofreading” enzymes look for mismatched base pairs and make repairs

Types of Mutations• Point Mutation

• Silent Mutation

Types of Mutation• Mis-sense Mutation

• Nonsense Mutation

Types of Mutations• Frameshift Mutation

• Chromosomal Mutation

Causes of Mutations• Some mutations occur

naturally (spontaneous mutations)

• These mutations may be caused by incorrect base pairing by DNA polymerase during replication

• Some mutations, however, are caused by substances or events known as mutagens

Physical Mutagens• Physical mutagens are

events that change DNA sequences

Chemical Mutagens• Chemical mutagens are

chemicals that cause changes in the DNA sequence

• Many of these are carcinogenic (cancer-causing)

Mutations and Variation• Genetic variation is a

result of mutations• This is because changes

in the DNA are the only source of variation at a heritable level

• This variation can eventually become an adaptation if there is a change in the environment that favors that new variation

Mitochondrial DNA• Mitochondrial DNA

(mtDNA) is a short genome found in the mitochondria

• This may be a holdover from a time where mitochondria may have been free-living organisms

• mtDNA is always identical between mother and child, and can therefore be used to trace maternal lineage

Gene Recombinations• In a laboratory, restriction

and ligase enzymes can be used to put genes into small organisms such as bacteria to study individual genes

Restriction Enzymes (Endonucleases)

• Restriction enzymes, such as Eco R1, cut up DNA at specific sites

• These sites have “sticky ends” which tend to bond with “sticky ends” that are created by other restriction enzymes

DNA Fingerprinting• DNA fingerprinting is

carried out using RFLPs (Restriction Fragment Length Polymorphisms)

• These RFLPs, which are “chunks” of DNA are produced when restriction enzymes (which are found in bacteria) cut up DNA into small segments

• The length of the RFLPs differ from person to person

DNA Fingerprinting• The DNA fragments are

transferred to a gel that has a current run through it

• The current pulls the DNA fragments through the gel

• The smallest fragments move the furthest, so a set of bands is produced that is unique to each individual

Gel Electrophoresis Animation

Gene Sequencing

Ligase Enzymes• The ligase enzymes

are then used to reassemble the DNA segment, often in a vector plasmid

• These plasmids are then introduced to bacteria, which take in the new DNA and incorporate it into their own

Uses of Recombinant DNA• Bacteria can now be used

to produce many human products

• Insulin, erythropoietin, clotting factors, antibodies, GH, and proteins that fight cancers are all being produced by using bacteria

• The advantage to using bacteria is that they reproduce quickly (ensuring significant levels of the products), and they are cheap to grow

Oncogenes• most cancer cells show

nitrogen base substitution• cancer-causing genes

(oncogenes) seem to turn on cell division

• the oncogenes seem to be present in normal DNA strands, but they are not active

Regulator Genes• one theory to explain this

is that the oncogene must be transposed to the proper site on the chromosome to become active

• most genes on the chromosomes are structural genes, they produce required proteins

• these structural genes are controlled by regulator genes that produce proteins that turn other genes “on” or “off”

http://www.brooklyn.cuny.edu

• the most common oncogene, ras, is found in 50% of colon cancer cases and 30% of lung cancer cases

• Ras makes a protein that acts as a “on” switch for cellular division

• however, the oncogene produces a protein that prevents this gene from turning “off”

• this may occur if the regulator and structural genes, which are normally adjacent to each other, are separated

18.4 – Genetics and Society• Biotechnology allows us

to create new products and technologies from natural biological systems

• However, biotechnology has also raised a large number of ethical, social, and legal issues

Gathering and Managing Genetic Information• Computers now allow us

to analyze and store large amounts of genetic information

• There are computerized gene banks and DNA libraries that provide researchers access to large amounts of genetic information

DNA Microarray• Microarrays work in a 4-

step sequence:1.

2.

3.

4.

• The microarrays allow scientists to study the action of thousands of genes at once

• Scientists can use these to compare the expression of genes in different environments

Public Benefits of Genetic Research

• Most of the important benefits of genetic research are the development of new treatments for genetic disorders

• It is also now possible to study how genes affect the activity of medications

• As well, the information gathered from the Human Genome Project is publicly available for anyone who wants to perform research

Ownership of Genetic Information• Many companies have

patented genes and genetically modified organisms

• This presents some controversy, as some people do not believe that one should be able to patent a living thing

Biotechnology Products• Many products can be

produced through genetic modification of organisms

• Transgenic organisms are organisms that have a gene from a different species spliced into their genome

• Ex:

Medicinal Bacteria• In 1982, insulin produced

by transgenic bacteria was approved for medical use

• Bacteria are ideal for the production of hormones because they are easy to put genes into, and they are cheap to grow

• Bacteria can also be used for bioremediation (cleanup of environmental toxins by living things)

Transgenic Plants• Recombinant crops now

account for more than half of the corn and canola produced in North America

• Transgenic plants can also be grown in new places

• Sometimes, the new transgenic plants can combine nutritional value of more than one plant in one (such as golden rice, which is sent to developing countries)

Cloning

Assessing Risks• When considering proposals

for approving transgenic products in Canada, the following criteria are used:

1. Potential social, environmental, and economic costs and benefits

2. The process by which the product is made, including the source of the genetic material

3. The biological characteristics of the transgenic product

4. The potential health effects of the product

Objections to the use of GMOs1. Environmental threats

(such as herbicide-resistant “superweeds”

2. Health effects (not enough research is done on long-term effects of consuming transgenic products)

3. Social and economic issues (is the cost of research better spent somewhere else?)

Diagnosis and Treatment of Genetic Disorders• Prenatal screening can

be carried out using either:

1. Amniocentesis

2. Chorionic Villus Sampling

Treating Human Genetic Disorders

• We now have a complete map of the human genome (it was completed in 2000)

• Therefore, it is now possible to locate damaged genes based on their DNA sequence

• But how can this be done?

Viral Transduction & Transformation

• Viruses are simply strands of DNA or RNA within a protein shell

• They work by injecting their genetic material into a cell’s genome

• When the cell reads its own DNA, it also then reads the virus DNA

• As a result, more virus particles are formed

The Virus “Life Cycle”

• However, we can use this to our advantage

• If a therapeutic gene is spliced into viral DNA, then the virus will insert the therapeutic gene into the cell’s DNA as well

• As a result, the new cell will have a functional gene that has replaced the damaged gene

• In theory, if germ-line cells were targeted by these viruses, then modifications could be passed on to the next generation

Biological Warfare• biological warfare has

been used since 600 BC• generally, a bioweapon is

considered to be a disease-causing living organism, or the toxins produced by a living organism

• one of the favored biological agents is anthrax, a bacteria (bacillus antractis) that forms spores which protect it from environmental factors

http://www.safebiology.com

Anthrax• if inhaled into the lungs,

anthrax bacteria can be fatal

• however, anthrax cannot be passed from one human to another http://www3.niaid.nih.gov

http://www.postgradmed.com

“Good” & “Bad” Agents:• Some organisms, such as HIV,

do not make good biological weapons (they need to enter the body in ways that make it difficult to deliver)

• Others produce symptoms and death so quickly that they are not easily spread (the agent “burns” itself out)

• The “best” organisms to use for biological weapons would be those that can be delivered easily (usually this means an airborne agent), and have a high mortality rate

• Smallpox is such an example, because it is easily transmitted and kills many of the people it is infected with

• As well, by using genetic engineering, viruses such as smallpox can become even more deadly by preventing successful immunization

• It is also possible that the genes could be manipulated so that a virus that is normally not easily transmitted could become airborne (such as Ebola)

http://www.lewrockwell.com

http://webs.wichita.edu

Global Guide to Bioweapons