Chp 8: from 258 (Nonoverlapping...) to 261 (…Cracking) &from 285 (8.6) to 293 (end of "essential concepts)Chp 14: Using deletions to locate genes (496-498)Chp 20: all except (for the time being) 724-25 (cloning) & RNA Interference (726-728).Reading for lectures 17-19Are mutant a and mutant b alleles (i.e. genetic alternatives)?high-resolutionsegregation testfor recombination:alleles as alternativeunits of segregation(recombination)complementation testfor functionalleles as alternativecis-acting units of functionmutant amutant bmutant amutant bMendel said that genes are the units of segregation, which led to the “beads on a string” model of genes & chromosomes:a & b may appear to be allelicby the functional test(failure to complement)…yet NOT allelicby the SEGREGATION test(nonparental alleles recoveredby meiotic recombination)results mayconflictgenetic map(linear like chromosome)Benzer’s question:How is complementation between mutantsrelated to recombination between mutants? (segregation)Need a selective genetic system(one with high resolving power for small map distances)Need a selective genetic system(one with high resolving power for small map distances)Rfa-b = NP pfu from hybrid / total pfu from hybridpfu = “plaque-forming units”hybrid = mixed infectionBenzer’s system made measuring 0.0001 cM (1x10-6) easyPhage are small, but plaques are often larger than fruit flies!How do phage help with measuring small Rfs?Use selective systems to easily measure NP pfu concentrationwithout complication from the much larger number of P pfu…and by the way, as an added bonus for mapping,phage happen to have a MUCHgreater rate of recombination per unit DNA than fruit flies or garden peasIn rII, smallest non-0 recombination ratemeasured was 0.02 cM (mutants 1 bp apart)(2 NP/10,000 total) In my first effort at fine-structure mapping in flies,I measured 0.007 cM (one recombinant) for adistance (I only later found out to be) 3,100 bp(1 NP/14,286 total)Among Hershey’s T4 collectionwere rapid lysis (r) mutants--- produced distinctivelylarge plaques.They arose spontaneously(1:10-4) and fell into severaldifferent complementation groupsthat mapped at different placesOne complementation groupwas rIIand Benzer discovered somethingspecial about it that madeit perfect for studying genetic fine structure.(1) rII vs. rII+ easily distinguished based on plaque morphology(2) extremely rare (<10-7) rII+ recombinants easy to recoverFig. 7.20(1) Characterize mutants: fell into two complementation groups, rIIA & rIIBvery close on a genetic map.Complementation test: phenotype of the hybridQuestion: did he test by plaque morphologyor ability to growth on K(!)?infect 1:1 mixtureat “multiplicity of infection”of ~3 phage:1 bacteriumBUT WHAT did Benzer do FIRST?!p.229Fig. 7.20 (still)NON-permissivehostdetermine that mutants are recessivemutants 1&2in same “gene”mutants 1&2in different “genes”alternativesNo growth(AND no recombination!)growthYes, but extremely tedious(almost never do)p.229Fig. 7.20 (still)(for complementation test)(1) Characterize mutants: fell into two complementation groups, rIIA & rIIBvery close on the genetic map.Make recombinantsby mixed infectionof the permissivehostAssay progenypfu concentrationon permissive (=total)and nonpermissive(=50% of nonparental)hostsp.229Fig. 7.20 (still)(quantitative testor qualitative test)(“unchanged in the hybrid”)…or NO: segregational alleles, so only…rIIA1 & rIIA2no progenylyse K(!)YESp.229Fig. 7.20 (still)(for recombination test)Determine reversion raterII-ArII-BThe first intragenic rII map8 rII mutants mapped originallyOriginal 8aftermany more1,612 INDEPENDENT mutants mapped for Fig. 7.21(and ultimately >3000)B -- A(1) How did he accumulate 3000+ rII mutations that he knew were independently generated? (i.e. NOT progeny of the same mutant parent --- the geneticist’s worst nightmare!)TWO IMPORTANT QUESTIONS:(2) How did he map all 3000 mutants againsteach other? Certainly not by doing all pair-wise combinationsrIImutant ~10-4rIIwildtyperII mottledplaque ~10-6mixture ofrII- and rII+(but remember: very low m.o.i. used for plaques …. chance of double infection <10-13)fairly rareis this rII- a relative?…these rII- mutants had to have arisenafter this killing center was founded.(1) How did he accumulate 3000+ rII mutations that he knew were independently generated? (i.e. NOT progeny of the same mutant parent --- the geneticist’s worst nightmare!)TWO IMPORTANT QUESTIONS:(2) How did he map all 3000 mutants againsteach other? Certainly not by doing all pair-wise combinationsTest each new rII mutant for revertablility(one of many of Benzer’s key insights)(1) revertables (2) nonrevertables (<<10-6)Fell into two clear classes:(single base-pair changes; “point mutants”?)(multi-base-pair deletions?)And these two classes of mutants behaved differentlyin recombination tests (tests for segregational allelism)Revertables respected a segregational allelism rule that nonrevertables violate:If two mutants are segregational alleles, they won’tbe able to generate a wildtype functional allele by recombinationrII-1/rII-2--> no rII+ (and of course no rll-1&-2) ---------(rev-a)-------------------------------------------(rev-a)------------------…more often, rev-a was not a segregational allele of either.-----------(b)----------------------------------------------------------(c)-------------Any revertable (a) can be a segregational alleleof at most only one of ANY two mutants (b & c)that are not segregational alleles of each otherAny revertable (a) can be a segregational alleleof at most only one of ANY two mutants (b & c)that are not segregational alleles of each other------[…….nonrev……….]---------------------(b)----------------------------------------------------------(c)-------------A nonrevertable can map to two or more different pointson the genetic map at once(be completely linked to those points; 0 genetic distance)What about nonrevertables?Any revertable (a) can be a segregational alleleof at most only one of ANY two mutants (b & c)that are not segregational alleles of each other------------[…….nonrev……….]-------------[…nonrev-d..]------------------------------------------------[…nonrev-e…]--A nonrevertable can be a segregational alleleof both of two other mutants, revertable or not,that are not segregational alleles of each
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