1. Reading (to get a head start on next Wed's lecture) : “Transposable genetic elements move from place to place in the genome” pp508-514 (Chp 14) Also: p548 (insertion sequences in bacteria) A practice problem set dealing with mutant categories and material from today's lecture is available on the website. Answers will be posted next Wednesday. Add to reading: (for today) pp240-241 of text

2. Approach for ovo D1 worked for Antp 32a5 and Ns 1 as well, two GOF mutant alleles that cause the antenna to develop as a leg. (I owe my own success to reverting dominant mutations) "revert" the dominance (one gets a recessive lethal in each case) …the resulting recessive lethal mutant chromosome(s) can be used in complementation tests

3. Categorized with respect to: Mutations (changes in DNA): (1) change in gene function relative to wildtype (2) molecular nature (Chp 7 and a bit of Chp 8) (you'll probably need for the MCATs)

4. Categorized with respect to: Mutations (changes in DNA): A) single base pair (point mutation) transition (Pur>Pur or Pyr>Pyr) transversion (Pur>Pyr) frameshift (reading frame changed) missense (amino acid difference) nonsense (translation terminates) B) more than a single base pair pseudopoint (cytologically invisible, and small enough to make it hard to distinguish genetically from a point) chromosomal (enough to see cytologically) e.g. transposon insertions (2) molecular nature (Chp 7 and a bit of Chp 8) effect on translation kind of base change

5. (2) molecular nature Categorized with respect to: Mutations (changes in DNA): (1) change in gene function relative to wildtype (3) Effect on organism

6. Mutations (changes in DNA): (3) Effect on organism a) Lethal (generally developmental) (most common) b) sterile (sterility is the genetic equivalent of death) c) homeotic (replacement by an inappropriate structure) d) visible (“morphological”) most useful as “genetic markers” if: easily distinguishable (high expressivity) reliably distinguishable (high penetrance) fully viable and fertile e) conditional [key for microbial (haploid) genetics but useful for all genetics] in many cases, molecular markers are supplanting (RFLP, PCR,Microarrays)

7. categories of conditional mutations: (new nutritional requirement for growth relative to wildtype ) 1. auxotrophic [prototroph] 2. host range(infectivity/virulence) parasite “nutrition” (including dominant lethals & steriles) 3. sex-limited/specific Sxl M1 dominant male lethal

8. categories of conditional mutations: (new nutritional requirement for growth relative to wildtype ) 1. auxotrophic [prototroph] 2. host range(infectivity/virulence) parasite “nutrition” (including dominant lethals & steriles) …varient allele at a different gene counteracts 3. sex-limited/specific (new nutritional requirement for growth relative to wildtype ) 1. auxotrophic [prototroph] 2. host range(infectivity/virulence) parasite “nutrition” Sxl M1 dominant male lethal 4. genetically suppressable: 3. sex-limited/specific (including dominant lethals & steriles) Sxl M1 dominant male lethal

9. categories of conditional mutations: …varient allele at a different gene counteracts a mut /a mut ; sup + /sup + = mutant phenotype a mut /a mut ; sup Mut /sup + = wildtype phenotype a mut /a mut ; sup mut /sup mut = wildtype phenotype (in haploids: a mut ; sup mut = wildtype phenotype) A mutation that makes a bad situation better is more likely to be informative than one that makes a bad situation worse (…and of course is likely to occur less frequently!) dominant vs. recessive suppression 4. genetically suppressable:

10. (new nutritional requirement for growth relative to wildtype ) categories of conditional mutations: 1. auxotrophic [prototroph] 2. host range(infectivity/virulence) parasite “nutrition” 3. sex-limited/specific (including dominant lethals & steriles) 4. genetically suppressable 5. temperature-sensitive (ts) 4. genetically suppressable

11. more useful in: mice or flies? cold-sensitive (lo temp. n.-p. ) heat-sensitive (hi temp non-permissive) 5. temperature-sensitive (ts) in microbes: generally caused by mutations destabilizing proteins and protein-protein interactions. in macrobes: causes not so obvious (interfere with temperature compensation?) condition that generates mutant phenotype = non-permissive) (vs. permissive) = restrictive categories of conditional mutations:

12. Glorious history of ts mutations: (1) validate 1gene:1enzyme ( 1 polypeptide ) concept concept based on nutritionally supplementable mutants mutants identified only because they were supplementable …hence not valid to conclude they are representative Horowitz & Leopold dried the blanket: isolated 161 ts growth (40C vs. 25C) E.coli mutants on minimal medium 124 (77%) grew at 40C on complete medium Delbrück wet blanket (1946) ALSO: ts muts. Identify vital genes (the 23%) in haploid orgnsms (not supplementable). (77%)

13. Glorious history of ts mutations: (2) genetically dissect bacteriophage T4 ( parasitizes E. coli ) …especially its assembly (“ morphogenesis ”) pathway at the restrictive temperature: different t.s. T4 mutants blocked at different steps in assembly (E.M. of phage lysates) 65 T4 genes by ts mutants genome sequencing: 55 more !! -- in vitro complementation of mutant extracts of phage grown at the nonpermissive temp. can one mutant extract provide the material/activity missing from the other mutant extract? …an assay for purification of specific gene products Add to reading: pp240-241 of text Fig."A"

14. ts mutations in “time-of-function” analysis of development determination of the “temperature-sensitive period” (TSP) permissive temp. throughout: normal development restrictive temp. throughout: abnormal development (aborts) to answer: When is a gene needed? (Fig. 20.6, p723) zyg-9: a C. elegans ts lethal subject zyg-9 mutant embryos to a 15 minutes pulse of high temperature start of pulse develop abort

15. fertilized egg (embryo) 1st instar larva 2nd instar larva 3rd instar larva Pupa ( metamorphosis ) 1 day 2 days 4 days adult ™ ¢ (reproductive in ~2 days) XX XY If a gene functions at different times to do different things, temperature shifts of ts mutants can reveal those various functions Drosophila life cycle @25C

16. determination of the “temperature-sensitive period” (TSP) by single temperature shifts up or down point (time) in development when temp. shifted embryo larva pupa adult ultimate phenotype all OK all mut permissive temp. throughout: normal development restrictive temp. throughout: abnormal development (aborts) restrictive unshifted permissive unshifted shift rstrctv to prmsv shift prmsv to restrv start : when begins drop end : when wildtype TSP