1. Anatomy and Function of a Gene
Dissection through mutation

2. Outline of Chapter 7 What mutations are Mutations and gene structure
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. Figure 7.2
Fig. 7.2

6. Spontaneous mutations influencing phenotype occur at a very low rate
Figure 7.3 b
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. Figure 7.4
Fig. 7.4

10. Replica plating verifies preexisting mutations
Figure 7.5 a
Fig. 7.5 a

11. Figure 7.5 b
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
Hydrolysis of a purine base, A or G occurs 1000 times an hour in every cell
Deamination removes –NH2 group. Can change C to U, inducing a substitution to and A-T base pair after replication
Figure 7.6 a,b
Fig. 7.6 a,b

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

15. Oxidation from free radicals formed by irradiation damages individual bases
Figure 7.6 e
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
Figure 7.7a
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
Figure 7.8
Fig. 7.8

19. Methyl-directed mismatch repair
Fig. 7.9
Fig. 7.9

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

21. Transposable elements move around the genome and are not susceptible to excision or mismatch repair
Figure 7.10 b
Fig 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.
Figure B(1) Genetics and Society
Fig. B(1) Genetics and Society

23. Trinucleotide repeat in people with fragile X syndrom
Figure A, B(2) Genetics and Society
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
Figure 7.11
Fig. 7.11

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

27. Figure 7.12 b

28. Figure 7.12 b
Fig b

29. Figure 7.12 c
Fig 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
Figure 7.13
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
Figure 7.15 a
Fig a

34. Five complementation groups (different genes) for eye color.
Figure 7.15 b, c
Fig 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
Figure 7.16
Fig. 7.16

38. Working with T4 phage
Figure 7.17 a

39. Phenotpyic properties of T4 phage
Figure 7.17 b
Fig b

40. Complementation test to for mutations in different genes
Figure 7.17 c

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

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

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

44. Fine structure map of rII gene region
Figure 7.18 c
Fig 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. Figure 7.20 a
Fig a

48. Figure 7.20 b
Fig 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
Figure 7.21 a
Fig a

51. R is the side group that distinguishes each amino acid
Fig b
Figure 7.21 b

52. Figure 7.21 b

53. Figure 7.21 b
Fig b

54. N terminus of a protein contains a free amino group
C terminus of protein contains a free carboxylic acid group
Figure 7.21 c
Fig c

55. Genes specify the amino acid sequence of a polypeptide – example, sickle cell anemia
Mutant b chain of hemoglobin form aggregates that cause red blood cells to sickle
Figure 7.22 a
Fig a

56. Sequence of amino acids determine a proteins primary, secondary, and tertiary structure
Figure 7.23
Fig. 7.23

57. Some proteins are multimeric, containing subunits composed of more than one polypeptide
Figure 7.24
Fig. 7.24

58. How do genotypes and phenotypes correlate?
Alteration of amino acid composition of a protein
Alteration of the amount of normal protein produced
Changes in different amino acids at different positions have different effects
Proteins have active sites and sites involved in shape or structure

59. Dominance relations between alleles depend on the relation between protein function and phenotype
Alleles that produce nonfunctional proteins are usually recessive
Null mutations – prevent synthesis of protein or promote synthesis of protein incapable of carrying out any function
Hypomorphic mutations – produce much less of a protein or a protein with weak but detectable function; usually detectable only in homozygotes
Incomplete dominance – phenotype varies in proportion to amount of protein
Hypermorphic mutations – produces more protein or same amount of a more effective protein
Dominant negative – produces a subunit of a protein that blocks the activity of other subunits
Neomorphic mutations – generate a novel phenotype; example is ectopic expression where protein is produced outside of its normal place or time