Anatomy and Function of a Gene

1. Anatomy and Function of a Gene Dissection through mutation

2. Outline of Chapter 7 • What mutations are • How often mutations occur • What events cause mutations • How mutations affect survival and evolution • Mutations and gene structure • Experiments using mutations demonstrate a gene is a discrete region of DNA • Mutations and gene function • Genes encode proteins by directing assembly of amino acids • How do genotypes correlate with phenotypes? • Phenotype depends on structure and amount of protein • Mutations alter genes instructions for producing proteins structure and function, and consequently phenotype

3. Mutations: Primary tools of genetic analysis • Mutations are heritable changes in base sequences that modify the information content of DNA • Forward mutation – changes wild-type to different allele • Reverse mutation – causes novel mutation to revert back to wild-type (reversion)

4. Classification of mutations by affect on DNA molecule • Substitution – base is replaced by one of the other three bases • Deletion – block of one or more DNA pairs is lost • Insertion – block of one or more DNA pairs is added • Inversion 180o rotation of piece of DNA • Reciprocal translocation – parts of nonhomologous chromosomes change places • Chromosomal rearrangements – affect many genes at one time

5. Fig. 7.2

6. Spontaneous mutations influencing phenotype occur at a very low rate Mutation rates from wild-type to recessive alleles for five coat color genes in mice Fig. 7.3 b

7. General observations of mutation rates • Mutations affecting phenotype occur very rarely • Different genes mutate at different rates • Rate of forward mutation is almost always higher than rate of reverse mutation

8. Are mutations spontaneous or induced? • Most mutations are spontaneous. • Luria and Delbruck experiments - a simple way to tell if mutations are spontaneous or if they are induced by a mutagenic agent

9. Fig. 7.4

10. Replica plating verifies preexisting mutations Fig. 7.5 a

11. Fig. 7.5b

12. Interpretation of Luria-Delbruck fluctuation experiment and replica plating • Bacterial resistance arises from mutations that exist before exposure to bactericide • After exposure to bactericide, the bactericide becomes a selective agent killing the nonresistant cells, allowing only the preexisting resistant cells to survive. • Mutations do not arise in particular genes as a direct response to environmental change • Mutations occur randomly at any time

13. Chemical and Physical agents cause mutations • Deamination removes –NH2 group. Can change C to U, inducing a substitution to and A-T base pair after replication • Hydrolysis of a purine base, A or G occurs 1000 times an hour in every cell Fig. 7.6 a,b

14. X rays break the DNA backbone • UV light produces thymine dimers Fig. 7.6 c, d

15. Oxidation from free radicals formed by irradiation damages individual bases Fig. 7.6 e

16. Repair enzymes fix errors created by mutation Excision repair enzymes release damaged regions of DNA. Repair is then completed by DNA polymerase and DNA ligase Fig. 7.7a

17. Mistakes during replication alter genetic information • Errors during replication are exceedingly rare, less than once in 109 base pairs • Proofreading enzymes correct errors made during replication • DNA polymerase has 3’ – 5’ exonuclease activity which recognizes mismatched bases and excises it • In bacteria, methyl-directed mismatch repair finds errors on newly synthesized strands and corrects them

18. DNA polymerase proofreading Fig. 7.8

19. Methyl-directed mismatch repair Fig. 7.9

20. Unequal crossing over creates one homologous chromosome with a duplication and the other with a deletion 7.10 a

21. Transposable elements move around the genome and are not susceptible to excision or mismatch repair Fig. 7.10 e

22. Trinucleotide instability causes mutations • FMR-1 genes in unaffected people have fewer than 50 CGG repeats. • Unstable premutation alleles have between 50 and 200 repeats. • Disease causing alleles have > 200 CGG repeats. Fig. B(1) Genetics and Society

23. Trinucleotide repeat in people with fragile X syndrom Fig. A, B(2) Genetics and Society

24. Mutagens induce mutations • Mutagens can be used to increase mutation rates • H. J. Muller – first discovered that X rays increase mutation rate in fruit flies • Exposed male Drosophila to large doses of X rays • Mated males to females with balancer X chromosome (dominant Bar eyed mutation and multiple inversions) • Could assay more than 1000 genes at once on the X chromosome

25. Muller’s experiment Fig. 7.11

26. Mutagens increase mutation rate using different mechanisms Fig. 7.12a

28. Fig. 7.12 b

29. Fig. 7.12 c

30. Consequences of mutations • Germ line mutations – passed on to next generation and affect the evolution of species • Somatic mutations – affect the survival of an individual • Cell cycle mutations may lead to cancer • Because of potential harmful affects of mutagens to individuals, tests have been developed to identify carcinogens

31. The Ames test for carcinogens using his- mutants of Salmonella typhimurium Fig. 7.13

32. What mutations tell us about gene structure • Complementation testing tells us whether two mutations are in the same or different genes • Benzer’s experiments demonstrate that a gene is a linear sequence of nucleotide pairs that mutate independently and recombine with each other • Some regions of chromosomes mutate at a higher rate than others – hot spots

33. Complementation testing Fig. 7.15 a

34. Fig. 7.15 b,c Five complementation groups (different genes) for eye color. Recombination mapping demonstrates distance between genes and alleles.

35. A gene is a linear sequence of nucleotide pairs • Seymour Benzer mid 1950s – 1960s • If a gene is a linear set of nucleotides, recombination between homologous chromosomes carrying different mutations within the same gene should generate wild-type • T4 phage as an experimental system • Can examine a large number of progeny to detect rare mutation events • Could allow only recombinant phage to proliferate while parental phages died

36. Benzer’s experimental procedure • Generated 1612 spontaneous point mutations and some deletions • Mapped location of deletions relative to one another using recombination • Found approximate location of individual point mutations by deletion mapping • Then performed recombination tests between all point mutations known to lie in the same small region of the chromosome • Result – fine structure map of the rII gene locus

37. How recombination within a gene could generate wild-type Fig. 7.16

38. Working with T4 phage

39. Phenotpyic properties of T4 phage Fig. 7.17 b

40. Complementation test to for mutations in different genes

41. Detecting recombination between two mutations in the same gene Fig. 7.17 d

42. Deletions for rapid mapping of point mutations to a region of the chromosome Fig. 7.18 a

43. Recombination mapping to identify the location of each point mutation within a small region Fig. 7.18 b

44. Fine structure map of rII gene region Fig. 7.18 c

45. What mutations tell us about gene function • One gene, one enzyme hypothesis: a gene contains the information for producing a specific enzyme • Beadle and Tatum use auxotrophic and prototrophic strains of Neurospora to test hypothesis • Genes specify the identity and order of amino acids in a polypeptide chain • The sequence of amino acids in a protein determines its three-dimensional shape and function • Some proteins contain more than one polypeptide coded for by different genes

46. Beadle and Tatum – One gene, one enzyme • 1940s – isolated mutagen-induced mutants that disrupted synthesis of arginine, an amino acid required for Neurospora growth • Auxotroph – needs supplement to grow on minimal media • Prototroph – wild-type that needs no supplement; can synthesize all required growth factors • Recombination analysis located mutations in four distinct regions of genome • Complementation tests showed each of four regions correlated with different complementation group (each was a different gene)

47. Fig. 7.20 a

48. Fig. 7.20 b

49. Interpretation of Beadle and Tatum experiments • Each gene controls the synthesis of an enzyme involved in catalyzing the conversion of an intermediate into arginine

50. Genes specify the identity and order of amino acids in a polypeptide chain • Proteins are linear polymers of amino acids linked by peptide bonds • 20 different amino acids are building blocks of proteins • NH2-CHR-COOH – carboxylic acid is acidic, amino group is basic • R is the side chain that distinguishes each amino acid Fig. 7.21 a