Reading for lectures 17 19 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 Are mutant a and mutant b alleles i e genetic alternatives complementation test for function alleles as alternative cis acting units of function high resolution segregation test for recombination alleles as alternative units of segregation recombination results may conflict mutant a mutant b a b may appear to be allelic by the functional test failure to complement mutant a mutant b yet NOT allelic by the SEGREGATION test nonparental alleles recovered by meiotic recombination Mendel said that genes are the units of segregation which led to the beads on a string model of genes chromosomes genetic map linear like chromosome Benzer s question How is complementation between mutants related 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 hybrid pfu plaque forming units hybrid mixed infection Phage 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 concentration without complication from the much larger number of P pfu Benzer s system made measuring 0 0001 cM 1x10 6 easy and by the way as an added bonus for mapping phage happen to have a MUCH greater rate of recombination per unit DNA than fruit flies or garden peas In rII smallest non 0 recombination rate measured 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 a distance I only later found out to be 3 100 bp 1 NP 14 286 total Among Hershey s T4 collection were rapid lysis r mutants produced distinctively large plaques They arose spontaneously 1 10 4 and fell into several different complementation groups that mapped at different places One complementation group was rII and Benzer discovered something special about it that made it 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 recover Fig 7 20 1 Characterize mutants fell into two complementation groups rIIA rIIB very close on a genetic map Complementation test phenotype of the hybrid Question did he test by plaque morphology or ability to growth on K infect 1 1 mixture at multiplicity of infection of 3 phage 1 bacterium p 229 Fig 7 20 still NON permissive host mutants 1 2 in same gene alternatives No growth mutants 1 2 in different genes growth AND no recombination BUT WHAT did Benzer do FIRST determine that mutants are recessive for complementation test Yes but extremely tedious almost never do p 229 Fig 7 20 still 1 Characterize mutants fell into two complementation groups rIIA rIIB very close on the genetic map p 229 Fig 7 20 still Make recombinants by mixed infection of the permissive host Assay progeny pfu concentration on permissive total and nonpermissive 50 of nonparental hosts quantitative test or qualitative test YES or NO segregational alleles so only rIIA1 rIIA2 unchanged in the hybrid no progeny lyse K for recombination test Determine reversion rate p 229 Fig 7 20 still The first intragenic rII map rII A 8 rII mutants mapped originally rII B after many more Original 8 1 612 INDEPENDENT mutants mapped for Fig 7 21 and ultimately 3000 B A TWO IMPORTANT QUESTIONS 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 2 How did he map all 3000 mutants against each other Certainly not by doing all pair wise combinations mixture of but remember very low m o i used for 13plaques chance of double infection 10 rII and rII rII mottled plaque 10 6 these rII mutants had to have arisen after this killing center was founded rIImutant 10 4 fairly rare is this rII a relative rIIwildtype TWO IMPORTANT QUESTIONS 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 2 How did he map all 3000 mutants against each other Certainly not by doing all pair wise combinations Test each new rII mutant for revertablility one of many of Benzer s key insights Fell into two clear classes 1 revertables single base pair changes point mutants 2 nonrevertables 10 6 multi base pair deletions And these two classes of mutants behaved differently in recombination tests tests for segregational allelism If two mutants are segregational alleles they won t be able to generate a wildtype functional allele by recombination rII 1 rII 2 no rII and of course no rll 1 2 Revertables respected a segregational allelism rule that nonrevertables violate Any revertable a can be a segregational allele of at most only one of ANY two mutants b c that are not segregational alleles of each other b c rev a more often rev a was not a segregational allele of either rev a Any revertable a can be a segregational allele of at most only one of ANY two mutants b c that are not segregational alleles of each other What about nonrevertables b c nonrev A nonrevertable can map to two or more different points on the genetic map at once be completely linked to those points 0 genetic distance Any revertable a can be a segregational allele of at most only one of ANY two mutants b c that are not segregational alleles of each other What about nonrevertables nonrev d nonrev e nonrev A nonrevertable can be a segregational allele of both of two other mutants revertable or not that are not segregational alleles of each other With rII it was easy to determine if any two mutants were or were not segregational alleles i e would or would not produce wildtype recombinants from the hybrid Infect liquid B culture with any two rII mutant phage then spot a drop of that mixture on top of a lawn of K ask do the progeny from lysed B clear the lawn of K Map of nonrevertables based on their segregational allelism revertables point mutants nonrevertables deletions So how does the large scale map generated by recombination between mutants in different genes as complementation groups compare with the fine structure map generated by
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