Announcements

1. Announcements -Solutions to problem set 3 and from the questions pertaining to last Fridays lecture have been posted on the course website. -Problem set 4 (pertaining to last weeks lectures) is posted on the course website. -A reading assignment on DNA hybridization has been posted on the course website. Today: -Mutant analysis (screen vs. selection; reversion; suppression; mutation rate; mutagens). -Repair of mutations

2. Mutant analysis (AKA Genetic Analysis) The use of mutants to understand how a biological process normally works* • Start with “unknown” system (e.g., metabolic pathway, embryonic development, behavior, etc.) • Generate mutations that affect the “unknown” system (i.e., that “break” the “unknown” system) • Study the mutant phenotypes to reveal the functions of the genes • Map the genes • Identify the genes (more on this later) *See the Salvation of Doug article at the following site: http://bio.research.ucsc.edu/people/sullivan/savedoug.html

3. Conducting a mutant analysis with yeast Case study: analyzing the adenine biosynthetic pathway by generating and studying “ade” mutants Wild-type yeast can survive on ammonia, a few vitamins, a few mineral salts, some trace elements and sugar… They synthesize everything else they need, including adenine What genes does yeast need to synthesize adenine?

4. Adenine-requiring colonies (ade mutants) Treat wt haploid cells with a mutagen: plate cells -adenine plate m2 m1 sterile piece of velvet m3 Identifying yeast mutants that require adenine “complete” plate This is an example of a “genetic screen” Replica-plate

5. Identifying interesting mutations—screen vs. selection Screen Each member of the population is examined… does it fit the phenotype criteria that have been set up? Selection Individuals not meeting the criteria don’t survive (or are otherwise eliminated from the population) Example 1: Looking for a translator Russian  English Screen: read resumés Selection: advertise in Russian Example 2: Looking for wingless fly mutants Screen: Look at each fly… wings present? Selection: Open vial, let flies fly away Primary selection or screen is often followed by secondary selection or screen

6. Reversion and Suppressors Most genetic screens are “forward” screens - start with wild type organism and look for new phenotypes caused by mutation (e.g., screen for yeast ade mutants). Another approach - start with a mutant Look for reversion to wild type (or less mutant)-sometimes called “reverse” genetics. look for red eyes ww What kinds of mutations might you find?

7. or Reversion and Suppressors Mutations that restore function to the white gene (revertant). X X X w gene w gene w gene X X 2) Mutations that bypass (or suppress) the need for a white gene. plus suppressor mutation in some other gene What kinds of suppressor mutations might you imagine?

8. Y X red pigment Partial biosynthetic pathway for adenine in yeast ADE2 ADE1 Y adenine intermediate X ADE1 ade2 X adenine build up of

9. Ade+ revertant Revert ade- to Ade+… does RED revert to WHITE? it’s white Treat wt haploid cells with a mutagen: ade2 mutant -Ade plate Pretty good proof that one mutation (ade2) has two phenotypes

10. A working hypothesis… ade2 has reverted to ADE2 Growth on -ade Color mRNA sequence + white 5’..AUG....UAC....UGA..3’ ADE2 STOP! ade2 - red 5’..AUG....UAG....UGA..3’ revertant (ade2-R) + white 5’..AUG....UAC....UGA..3’

11. ADE2 x ade2-R :) ADE2 :) ade2-R meiosis Has the mutant gene changed back to WT? A test of the hypothesis… …do a cross: If ade2-R is “true revertant”… ade2-R = ADE2 the diploid is homozygous WT Spores from the diploid should be: All Ade+, white Most Ade+ revertants… like this. But some exceptions!

12. Diploids meiosis Some revertants behave differently… The cross: ADE2 x revertant Ade+, white Ade+, white Ade+, white Most spores: Ade+, white Some spores: ade-, red! In these revertants… • ade2 is still in mutated form • a new mutation somewhere else suppresses the ade2 phenotype! Interpretation?

13. Summary of revertant types ade revertants come in two varieties: “True” revertants Suppressors (aka “extragenic suppressors” or “second site suppressors”) ade2 mutant allele  ADE2 (wt) A mutation in a different gene eliminates the ade2 mutant phenotype Definition of suppressor? A mutation in a second gene that eliminates the mutant phenotype of a mutation in the first gene.

14. ADE2 ade2 TyrSTOP mutation sup3 SUP3 What is this suppressor? Linkage analysis… mapped to chr XV huh? SUP3 codes for tRNATyr To recap… 5’..AUG....UAG....UGA..3’ Ade+ white! WT Explanation?

15. Mutant tRNA (SUP3) Tyr Tyr AUG AUC 5’..AUG....UAG....UGA..3’ 5’..AUG....UAG....UGA..3’ ade2 mRNA ade2 mRNA How does SUP3 suppress ade2? WT tRNA (sup3) TyrSTOP The mutant tRNA suppresses the nonsense codon! --full-length ADE2 protein is made--Ade+, white colony!

16. Doesn’t the suppressor tRNA cause problems for cells? What reads the normal TYR codons, UAC? • Yeast has 8 tRNA-TYR genes • Only one of them has the suppressor mutation. What about genes that normally end in UAG? • Not all ORFs end with UAG. • For those that do, there’s still a competition between the suppressor tRNA and termination factor. Even so, a cell with a SUP mutation can be quite sick.

17. Mutant protein 1 WT protein 2 Mutant protein 1 Mutant protein 2 Another kind of suppression (unrelated to ade2) WT protein 1 WT protein 2 Restoration of function!

18. Red/White and Ade+/ade- One gene or two closely linked ones? 1. Isolate new red mutants: do they also require adenine? yes 2. If the adenine mutation is reverted to wild type (ADE) does the red color also revert to white? yes 3. If red is reverted to white, does ade- revert to Ade+?

19. W Can we get redwhite revertants that are still ade-? Some ideas ade3 ADE3 ade2 ADE1 X Adenine Y gene R red pigment In an ade2 strain (red)… LOF mutation of either gene R or ADE3 white colonies, but still ade-.

20. rev#1 Treat with mutagen rev#2 revert phenotype to white Making redwhite revertants Using complete media . . . ade2 mutant Some white colonies could be true revertants. Some mutations could be suppressors. Some could be in other genes of the pathway?

21. Growth without adenine distinguishes from Summary… ADE3 ade2 ADE1 W X Adenine Y gene R red pigment grow without adenine? color? In ade2 strain: white yes revert ade2  ADE2 mutate ADE3 to LOF white no mutate gene R to LOF white no

22. * * * use up 1 ATP, non-reversible step Final tally… How many mutants are like ade3? • 10 complementation groups are ade- and white • 2 are ade- and red. ADE2 ADE1 adenine gene R red How many are like “gene R”? • lots and lots, but “gene R” has never been identified! • Respiration defective cells can’t make red pigment. Respiration mutations are epistatic to red pigment!

23. Quiz Section this week: Genetic Analysis in Caenorhabditis elegans An introduction to C. elegans. . .

24. A bit of background on Caenorhabditis elegans • 1 mm long nematode worm. • 3.5 day generation time. • Predominantly internally selfing hermaphrodite (make sperm and oocytes). • Rare males arise spontaneously and can cross with hermaphrodite (male sperm fertilize hermaphrodite oocyte). • Moves by wriggling (like a snake).

25. C. elegans hermaphrodite head tail

26. C. elegans generates bends using dorsal and ventral muscle strips. worm movie

27. Inbreeding is important for model organism genetics • Outbred (wild) populations are genetically heterogeneous. • Highly inbred strain has little or no genetic variability.

28. Inbreeding makes strains homozygous for everything X X X X X X X X With each generation, ½ of the previously heterozygous alleles become homozygous.

29. Inbreeding is important for model organism genetics • Outbred (wild) populations are genetically heterogeneous. • Highly inbred strain has little or no genetic variability. • Mutant alleles behave simply - only change present in cross. • E. coli, yeast, fruit fly, C. elegans, zebrafish, mouse are highly inbred.

30. Mutant Analysis: generating mutants To conduct a mutant analysis begin with inbred WT strain, then treat with a mutagen to generate a large population of mutagenized animals Why mutagenize? FREQUENCY!! Spontaneous mutations are VERY RARE. Mutagenesis can increase frequency by about 10,000 fold.

31. rossover suppressor = X-chromosome with inversions… no recombination ethal (l) = recessive lethal (XlY males are dead) B l ar (B) = bar-shaped eyes; bar shape is DOMINANT X-rays x B l Bar-eyed female x Estimation of mutation rate: X-ray-induced mutations X-Rays (H. J. Muller’s X-linked “ClB” system in Drosophila) C l B How frequently do new mutations appear on this X-chromosome?

32. Pick Bar-eyed female progeny x wt look just at sons B B B B l l l l If no new mutations… If new lethal mutation… Estimation of mutation rate: X-ray-induced mutations How frequently do new mutations appear on this X? Bar-eyed female x 1 female/cross; repeat many times dead dead dead! viable no sons!

33. Spontaneous mutation rate (2/1000 X-chromosomes) no X-ray treatment Estimation of mutation rate: X-ray-induced mutations % X-linked recessive Lethal mutations X-ray dose Certain external agents (mutagens) can drastically increase mutation rates.

34. Spontaneous mutation rates • Measurement of spontaneous mutation rates: • 2 mutations per 1000 X-chromosomes • 2 mutations per 1,000,000 genes • Mutation rate = 2 x 10-6/gene/generation (from assumption of 1000 genes on X) i.e., you would only get ~2 mutants/1,000,000 animals analyzed from spontaneous mutations - using a mutagen can increase this rate to ~2 mutants/100 animals Very similar rate calculated for humans! Rough calculation: If 35,000 genes in human genome… 2 x 10-6 x 35000 = ~ 0.07 mutations per generation or 1 mutation (somewhere in the genome) per 14 gametes…

35. Some mutagens (electromagnetic radiation) Radiation - X-rays, -rays: ionizing radiation cause breaks in DNA  chromosomal rearrangements! • Ultraviolet light: non-ionizing radiation thymine dimers impede DNA polymerase

36. Some mutagens (chemical mutagens) Chemical mutagens - Alkylating agents, e.g., ethylmethane sulfonate (EMS) C T EMS O6-ethyl-G G  base substitutions - intercalating agents, e.g., acridine orange cause frame shift mutations

37. Allele R Transposon insertion Allele r The wrinkled pea trait that Mendel studied was caused by a transposon insertion that inactivated a gene Transposons: jumping genomic segments of DNA Small pieces of DNA (a few hundred to a few kbp in length) that can move from one site in the genome to another. • ALL organisms have them (~45% of our genome: transposon remnants!) • Jumping genes, Selfish DNA • Mechanism for rapid evolutionary change Transposase gene Transposons can also cause mutations if they hop into or near genes

38. Mutation; repair of mutations What are the sources of spontaneous mutations? How are mutations repaired?

39. A A A T T T T T T T G G G C C C A A G T T T G G G new A A A A A C C C C C G G A A C G G A C C C C C T T G C A T G old no mutation! Spontaneous mutations probably exceeds 50,000/cell/day - Base alteration or loss - Replication errors yes corrected? replication

40. Damage control Experimentally observed mutation rate in E. coli (inside the cell): Expected error rate of E. coli DNA polymerases (from physical/chemical properties of the bases: Experimentally observed error rate of E. coli DNA polymerases (in the test tube): Conclusions: 1 mutation/1010 bases polymerized 1 mutation/105 bases polymerized 1 mutation/107 bases polymerized -DNA polymerases must possess a “proofreading” ability. -There must be yet another backup error detection system in the cell.

41. A T T T G C A T G A A C C G G C Damage control Proof-reading by DNA polymerase new correction A T T G C A T G old • DNA polymerase has 3 activities: • can add bases to 3’ end • the end must be base-paired • template must be available • can excise (remove) bases from 3’ end • Normally, addition rate >> excision rate • - can remove bases from 5’ end (involved in DNA replication and some forms of repair) (for optimal activity) (not covered in this course)

42. DNA pol C C 3’ end base-paired  extension rate high A A A A T T T G C A T G T T T T T T G G G C C C A A A T T T G G G A A A A C C C C G G G 3’ end base-paired again! Proof-reading (cont’d) 3’ end NOT base-paired  extension rate low probability of excision high Proof-reading corrects 99% of incorporation errors!

43. Damage control Experimentally observed mutation rate in E. coli (inside the cell): Expected error rate of E. coli DNA polymerases (from physical/chemical properties of the bases: Experimentally observed error rate of E. coli DNA polymerases (in the test tube): Conclusions: 1 mutation/1010 bases polymerized 1 mutation/105 bases polymerized 1 mutation/107 bases polymerized -DNA polymerases must possess a “proofreading” ability. -There must be yet another backup error detection system in the cell. mismatch repair system

44. GACGTACATG CTGCATGTAC “mismatched” base GACGTATATG CTGCATGTAC repair is biased; tends to restore normal sequence GACGTACATG CTGCATGTAC GACGTATATG CTGCATATAC repaired unrepaired Mismatch repair Proofreading catches many errors but some still slip by; how are they detected and repaired? GACGTACATG CTGCATGTAC

45. CH3 deoxyadenosine TGATCA ACTAGT TGATCA ACTAGT methylase (DAM) CH3 Mismatch repair Best understood in bacteria Identify mismatched bases in DNA Recognize the template strand Correct the OTHER strand mutS protein in E. coli use methylation state of DNA to identify template strand

46. DNA replication transiently hemimethylated template strand can be distinguished from newly synthesized strand DNA replication transiently hemimethylated template strand can be distinguished from newly synthesized strand Mismatch repair

47. mismatch mutH mutS mutL excise mismatch region TTACAAGGTCATGTTT 5'-CACG CCGATCTA-3’ 3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5' CH3 Mismatch repair—the mutSHL system mutS protein recognizes mismatch mutH protein recognizes parental strand mutL protein promotes mutH activity (make cut in new strand) 5'-CACGTTACAAGGTCATGTTTCCGATCTA-3’ 3'-GTGCAATGTTCCAGGACAAAGGCTAGAT-5' CH3 TTACAAGGTCCTGTTT re-synthesize DNA

48. Repair of UV light induced DNA damage 250nm 100 phr uvrA uvrB uvrC uvrD % surviving cells 10 0 0 6 12 Minutes of UV irradiation What genes are involved? E. coli WT cells Mutants defective in UV repair

49. Repair of UV light induced DNA damage in the dark # pyrimidine dimers/kb DNA Blue light (300-500nm) time UV light pulse 5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’ 3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’ Phr=photolyase (+ blue light) 5’-CACGTTACAAGGTCCTGTTTCCGATCT-3’ 3’-GTGCAATGTTCCAGGACAAAGGCTAGA-5’ 2 mechanisms of UV damage repair: light-dependent and light-independent. Pyrimidine dimers in the genome converted to small ss DNA fragments pyrimidine dimers ‘disappear’ 250nm

50. Phr=photolyase (+ blue light) Light-dependent UV repair mechanism