CHAPTER 29

DNA as Genetic Information

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Review site for basics of DNA and genetics:

http://vector.cshl.org/dnaftb

There will be a take-home bonus question today.

Studies of Heridity

• By geneticists - describe patterns of inheritance• traits (phenotypes)• heritable (passed from parents to offspring)• cytogeneticists knew that trait inheritance is associated with the cell nucleus and with chromosomes• biochemists knew that chromosomes are composed of DNA and protein

Q. What is the molecular/biochemical basis ofinheritance?

How is it known that DNAcontains genetic information

???

Parent trait Offspring trait

• Gene:- segment of DNA that contains all the information needed for regulated synthesis of an RNA or protein product.

• Genome:- the entire DNA sequence content of an organism (nuclear DNA)

Some Important Definitions

Biochemical Genetics

Archibald Garrod (1902) - an English doctor

Described “alkaptanurea” disease

Symptom: urine turns black when exposed to air

Found it was due to oxidation of homogentisic acid in urinehomogentisic acid = an intermediate in Phe degradation (p.897)

Phe Tyrhomogentisic

acidfurther

metabolites

Accumulation of homogentisic

acid

Biochemical Genetics

Archibald Garrod : important contributions

Described “alkaptanurea” disease

Deduced that it is due to a defective metabolic enzyme

Disease is a hereditary condition (ran in his patients’ families)

Led to concept of “inborn errors of metabolism”

A novel phenotype may reflects a discrete biochemical difference

Biochemical Genetics

“Real-World Biochemistry”

Aspartame

= a dipeptide: aspartyl-phenylalanine methyl ester

Aspartame is metabolized in the body to its components: aspartic acid, phenylalanine, and methanol. Like other amino acids, it provides 4 calories per gram. Since it is about 180 times as sweet as sugar, the amount of aspartame needed to achieve a given level of sweetness is less than 1% of the amount of sugar required. Thus 99.4% of the calories can be replaced.

Look on your diet soda cans and read the warning

Biochemical Genetics

Archibald Garrod : important contributions

Proposed that inheritance of a defective metabolic enzyme leads to inheritance of a phenotype (disease)

Parent trait Offspring traitdefectiveenzyme

• born in Wahoo, Ne• undergraduate degree at UNL• did graduate work at Cornell• got a faculty position at CalTech• ended up as the president of the Univ of Chicago

• did work in the 1930’s & 40’s on Drosophila eyesand on Neurospora (bread mold)• “one gene - one enzyme” hypothesis (1941)• awarded Nobel prize in 1958 (with research colleagues J. Lederberg and E. Tatum)

George W. Beadle

George W. Beadle

• Bread Mold: Neurospora crassa• can grow on minimal media

sucrose Inorganic salts biotin

• Beadle selected for nutritional mutants (auxotrophs)• irradiated fungal spores, grew these up on completemedia, and transferred part of the stock to minimal media

• He looked for mutants that can grow on complete media but NOT on minimal media

•These mutants are lacking an enzyme for the synthesis of an essential nutrient

Beadle’s Experiment- Part 1

These mutants may be lacking an enzyme for the synthesis of an essential nutrient

• Now have nutritional mutants (auxotrophs)• Figure out which pathway is defective

• By “feeding” experiments

• feed mutants with nutrients (one at a time) to supplement their defective pathway

•By trial and error, can identify which nutrient the mutant can’t make, and therefore which pathway is defective

•Beadle did this feeding experiment with amino acids to look for mutants in amino acid biosynthetic pathways.

Beadle’s Experiment- Part 2

Supplements: LysPro Ser Arg

Beadle’s Experiment- Part 3

•Using the methods in part1 and part 2, Beadle generated a collection of Arg auxotrophs•Then he used feeding experiments to identify which enzyme was defective in each mutant

Beadle’s Experiment Summary

•Beadle could identify mutants in specific steps of a pathway

•Assuming each mutant was defective in a single gene, Beadle postulated that the different mutant classes each lacked a different enzyme for Arg biosynthesis

•Therefore, he could show a one-to-one correspondance between mutation and absence of an enzyme.

• one gene specifies/encodes one enzyme

Beadle’s experiment gave riseto a new field called

Biochemical Genetics

But it left an open question:

What is the biochemical nature of a gene?

defectivegene

Parent trait

Offspring trait

defectiveenzyme

Frederick Griffith (1928) - a microbiologist

mutagenized

Staphylococcus pneumoniae

Smooth colonies(S strain)

Rough colonies(R strain)

•Produces a polysaccharide coat- slimy capsule - smooth•Capsule protects pathogen from being detected by the host’s immune system.

•Defective in polysaccharide synthesis - no capsule - rough•No capsule means no protection from being detected by the host.

Mouse dies and has S strain in its blood

Inoculate a mouse

Mouse survives and has no R strain in its blood

Inoculate a mouse

Wild-typevirulent

mutantnon-virulent

Something from heat-killed S passed into live R and transformed them into live S. Griffith called this the “Transforming principle”

Purifying the Transforming Principle (TP)

Avery, McCarty and McLeod - Biochemists (1944)

Heat-killed S bacteriaTP is probably not a protein

(heat treatment!)

Cell-free extract

Mouse dies and has live S in its blood

Mix with live R

Mouse dies and has live S in its blood

Mix with live R

Mouse dies and has live S in its blood

Mix with live R

Add a protease Add a nuclease

Very pure preparation of DNA

Still transforms! Lose transforming activityIt’s DNA!!!

Further Proof Required

• Good evidence that DNA is the transforming principle

• There is no proof yet that the DNA itself is stably inherited

• i.e. no proof that transforming DNA from dead S actually gets inside live R to turn it into S.

Further Proof Provided

Alfred Hershey Martha Chase

By virologists (1952)

Hershey and Chase clearly linked DNA and heridityBacteriophage - virus infects

bacteria

•Bacteriophage attach to a host cell via cell surface receptors• inject their DNA•Host cell now makes viral protein and more DNA•New virus assembles inside the cel•Cells lyse and phage are released.

Hershey and Chase prepared radioactively-labeled bacteriophage

DNA

rest of virus is protein

1) to make virus with labeled DNAMix: host bacteria

virus32P (radioactive)

Progeny viruses have radioactive DNA

2) to make virus with labeled proteinMix: host bacteria

virus35S (radioactive)

Progeny viruses have radioactive protein

Take these viruses and do a new infection to seewhat gets injected into a host cell

Further Proof Provided

• In 1952, Hershey and Chase, studying bacteriophages, labelled DNA with 32P and protein with 35S

• Bacteriophage progeny produced by infection of bacteria contained 32P (thus DNA from the original phage), but not 35S (from the protein)!

DNA Structure Model 1953

http://www.nature.com/genomics/human/

You should read Watson and Crick’s originalNature paper at the following site:

(Scroll to the bottom of the page and click on “full text”in the green Watson and Crick box)

DNA Structure Model 1953

WATSON, J. D. & CRICK, F. H. C.

Medical Research Council Unit for the Study of Molecular Structure of Biological

Systems, Cavendish Laboratory,Cambridge.

A Structure for Deoxyribose Nucleic Acid

We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest. …..

……… It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.

The Human Genome Project

What is the Human Genome Project?

• U.S. govt. project coordinated by the Department of Energy and the National Institutes of Health

• goals (1998-2003)– identify the approximate 100,000 genes in human DNA– determine the sequences of the 3 billion bases that

make up human DNA– store this information in databases– develop tools for data analysis– address the ethical, legal, and social issues that arise

from genome research

Why is the Department of Energy involved?

-after atomic bombs were dropped during War War II, Congress told DOE to conduct studies to understand the biological and health effects of radiation and chemical by-products of all energy production

-best way to study these effects is at the DNA level

Whose genome is being sequenced?

• the first reference genome is a composite genome from several different people

• generated from 10-20 primary samples taken from numerous anonymous donors across racial and ethnic groups

Benefits of HGP Research

• improvements in medicine• microbial genome research for fuel and

environmental cleanup• DNA forensics• improved agriculture and livestock• better understanding of evolution and human

migration• more accurate risk assessment

Ethical, Legal, and Social Implications of HGP Research

• fairness in the use of genetic information• privacy and confidentiality• psychological impact and stigmatization• genetic testing• reproductive issues• education, standards, and quality control• commercialization• conceptual and philosophical implications

Human Genome Project Information Websitehttp://www.ornl.gov/hgmis

For More Information...

Insights Learned from the Sequence

• What has been learned from analysis of the working draft sequence of the human genome? What is still unknown?

(information taken from Science, Nature, Wellcome Trust, and Human Genome News)

By the Numbers

• The human genome contains 3164.7 million nucleotide bases (A, C, T, and G).

• The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin (2.4 million bases).

• The total number of genes is estimated at 30,000 to 35,000, much lower than previous estimates of 80,000 to 140,000 that had been based on extrapolations from gene-rich areas as opposed to a composite of gene-rich and gene-poor areas.

• The order of almost all (99.9%) nucleotide bases are exactly the same in all people.

•The functions are unknown for over 50% of discovered genes.

The Wheat from the Chaff

• Less than 2% of the genome encodes for the production of proteins.

• Repeated sequences that do not code for proteins ("junk DNA") make up at least 50% of the human genome.

• Repetitive sequences are thought to have no direct functions, but they shed light on chromosome structure and dynamics. Over time, these repeats reshape the genome by rearranging it, thereby creating entirely new genes or modifying and reshuffling existing genes.

How It's Arranged

• The human genome's gene-dense "urban centers" are predominantly composed of the DNA building blocks G and C.

• In contrast, the gene-poor "deserts" are rich in the DNA building blocks A and T. GC- and AT-rich regions usually can be seen through a microscope as light and dark bands on chromosomes.

• Genes appear to be concentrated in random areas along the genome, with vast expanses of non-coding DNA between.

• Stretches of up to 30,000 C and G bases repeating over and over often occur adjacent to gene-rich areas, forming a barrier between the genes and the "junk DNA." These CpG islands are believed to help regulate gene activity.

How do we Compare to Other Organisms

• Humans have on average three times as many kinds of proteins as the fly or worm because of mRNA transcript "alternative splicing" and chemical modifications to the proteins. This process can yield different protein products from the same gene.

• Humans share most of the same protein families with worms, flies, and plants, but the number of gene family members has expanded in humans, especially in proteins involved in development and immunity.

Variations and Mutations

• Scientists have identified about 1.4 million locations where single-base DNA differences (SNPs) occur in humans. This information promises to revolutionize the processes of finding chromosomal locations for disease-associated sequences and tracing human history.

• The ratio of germline (sperm or egg cell) mutations is 2:1 in males vs females. Researchers point to several reasons for the higher mutation rate in the male germline, including the greater number of cell divisions required for sperm formation than for eggs.

Impact

The draft sequence already is having an impact on finding genes associated with disease.

Over 30 genes have been pinpointed and associated with breast cancer, muscle disease, deafness, and blindness.

Additionally, finding the DNA sequences underlying such common diseases as cardiovascular disease, diabetes, arthritis, and cancers is being aided by the human variation maps (SNPs).

These genes and SNPs provide focused targets for the development of effective new therapies.

There will be a take-home bonus question today.