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CHAPTER 29 DNA as Genetic Information Review site for basics of DNA and genetics: http://vector.cshl.org/dnaftb There will be a take-home bonus question today. All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777 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 of inheritance? Parent trait Offspring trait How is it known that DNA contains genetic information ??? Some Important Definitions • 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) 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 urine homogentisic acid = an intermediate in Phe degradation (p.897) Phe Tyr homogentisic acid Accumulation of homogentisic acid further metabolites 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: aspartylphenylalanine 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 defective enzyme Offspring trait George W. Beadle • 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 eyes and 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 • 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 complete media, 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: Pro Ser Arg Lys 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 rise to a new field called Biochemical Genetics Parent trait defective gene defective enzyme Offspring trait But it left an open question: What is the biochemical nature of a gene? Frederick Griffith (1928) - a microbiologist Staphylococcus pneumoniae Wild-type mutant virulent non-virulent mutagenized Smooth colonies (S strain) •Produces a polysaccharide coat - slimy capsule - smooth •Capsule protects pathogen from being detected by the host’s immune system. Inoculate a mouse Mouse dies and has S strain in its blood Rough colonies (R strain) •Defective in polysaccharide synthesis no capsule - rough •No capsule means no protection from being detected by the host. Inoculate a mouse Mouse survives and has no R strain in its blood 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 bacteria Mix with live R TP is probably not a protein (heat treatment!) Cell-free extract Mix with live R Very pure preparation of DNA Mouse dies and has live S in its blood Mouse dies and has live S in its blood Mix with live R Add a protease Add a nuclease Still transforms! Lose transforming activity Mouse dies and has live S in its blood It’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 By virologists (1952) Alfred Hershey Martha Chase Hershey and Chase clearly linked DNA and heridity Bacteriophage - 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 radioactivelylabeled bacteriophage DNA rest of virus is protein 1) to make virus with labeled DNA Mix: host bacteria virus 32P (radioactive) Progeny viruses have radioactive DNA 2) to make virus with labeled protein Mix: host bacteria virus 35S (radioactive) Progeny viruses have radioactive protein Take these viruses and do a new infection to see what 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 You should read Watson and Crick’s original Nature paper at the following site: http://www.nature.com/genomics/human/ (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 For More Information... Human Genome Project Information Website http://www.ornl.gov/hgmis 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.