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What do we mean (what have we meant) by "a gene": Reading for lectures 15-17 (We F27, Fr F29, We M5) 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). How do we recognize if A given heritable difference in phenotype (appearance) is caused by the simplest possible difference in genotype (breeding behavior) SIMPLEST DIFFERENCE: ….a difference with respect to one gene ….different alleles (forms) of one gene ….a "monogenic traite" A definition of what we mean by "gene": the thing that exists in two different forms (alleles) Two different operational tests for allelism: Mendel's "segregational test" The gene as the unit of segregation -- breeding behavior of the hybrid (1:2:1) (=genotype of the hybrid)) Recall that a conflict arose Green's/Lewis'/Benzers' "complementation test" The gene as the cis-acting unit of function --- phenotype of the hybrid recessive lozenge-shaped-fly-eye "alleles" in trans: recessive lozenge-shaped-fly-eye "alleles" in trans: recombination lz(g) lz(g) lz(BS) lz(BS) hybrid looks mutant (same defective unit of function = functional alleles) …and yet: wt lz(g) lz(BS) wt hybrid looks mutant (phenotype - function) …and yet: wt wt rare wildtype gametes rare wildtype gametes hence can't be segregational alleles (breeding behavior of hybrid) (also: wt wt lz(BS) lz(g) ) hence can't be segregational alleles (breeding behavior of hybrid) 1 Are mutant a and mutant b alleles (i.e. genetic alternatives)? Do we go with Mendel or high-resolution segregation test for recombination a + + b OR a b Genes as defined as cis-acting units of function have multiple parts that are separable by recombination complementation test for function phenotype of hybrids breeding behavior of hybrids mutant a mutant b The complementation test is generally favored: (Green/Lewis) /Benzer? mutant a mutant b a + a OR + b b ? Different alleles of the same gene may be defective for different reasons Segregation test is a useful preliminary test (especially when we can't make the hybrids we would like) ? Are a & b alternative units of segregation during meiosis? Are a & b alternative cis-acting units of function during development? i.e. are they segregational alleles? i.e. are they functional alleles? If: mutant alleles with similar phenotypic effects map to approximately the same point Then: those mutants are likely to be alleles of the same gene Are mutant a and mutant b alleles (i.e. genetic alternatives)? Are mutant a and mutant b alleles (i.e. genetic alternatives)? complementation test for function phenotype of hybrids complementation test for function phenotype of hybrids --- could we ever be misled by the apparent failure of these recessive mutants to complement in trans? mutant a mutant b a + + b OR a b ? mutant phenotype wildtype phenotype (complement) (don’t complement) not alleles alleles wildtype Are mutant a and mutant b alleles (i.e. genetic alternatives)? When alleles FAIL TO COMPLEMENT we may need the cis control: BECAUSE: If the two mutants are individually recessive alleles of different genes, but dominant in combination the cis control will also be mutant! OR wildtype phenotype a b ? mutant phenotype (complement) (don’t complement) not alleles alleles mutant AND same recessive mutants in CIS IF: recessive mutants in TRANS mutant a mutant b mutant a mutant b + + mutant a mutant b a + + b wildtype Are mutant a and mutant b alleles (i.e. genetic alternatives)? complementation test for function phenotype of hybrids mutant a mutant b + + …could mutants be recessive individually mutant a and in different cis-acting mutant b units of genetic function (i.e. not be functional alleles), a + a ? OR but interact in combination to + b b appear dominant together) mutant wildtype phenotype phenotype a + a + (complement) (don’t complement) + b + b ? not alleles alleles wildtype phenotype complementation test for function phenotype of hybrids mutant phenotype don’t appear to complement … then we can trust the trans result trans 䍫㻃cis, hence a & b are functional alleles of the same gene …orientation of the genetic information matters for genes 2 Are mutant a and mutant b alleles (i.e. genetic alternatives)? recessive mutants in TRANS same recessive mutants in CIS mutant a mutant b + + wildtype Phenotype … we can trust the trans result mutant a mutant b Are mutant a and mutant b alleles (i.e. genetic alternatives)? CIS/TRANS test CIS/TRANS test complementation test for function phenotype of hybrids complementation test for function phenotype of hybrids mutant phenotype don’t appear to complement But nobody bothers with the cis control for recessive mutants when doing complementation tests despite what all the textbooks say (not even Benzer did it, as we will see) Cis test is too hard in most cases, and in most cases unnecessary. …then why am I wasting your time with this? Are mutant a and mutant b alleles (i.e. genetic alternatives)? CIS/TRANS test for functional allelism The complete cis/trans test will allow us to determine allelism even if one or both of the mutants are not recessive! Remember: the “complementation test” per se is limited to recessive mutants. Most mutants are recessive, but some of the most useful & interesting are not. CIS phenotype CIS phenotype 䍫㻃TRANS phenotype, hence functional alleles = TRANS phenotype, hence NOT functional alleles …and all that matters is whether the cis vs. trans phenotypes are the same or different, not what the phenotypes of each actually are! complementation test for function alleles as alternative cis-acting units of function Conflict favored Intuitively, a better operational definition of alternative forms of genes high-resolution segregation test for recombination: alleles as alternative units of segregation (recombination) Raises questions: If one can get recombination between functional alleles (alternative forms of a gene), then how do the genetic maps one can therefore construct within single genes compare to the genetic maps that can be (had been) constructed between genes? --- WHAT IS THE NATURE OF GENETIC FINE STRUCTURE? …and what is the basic (minimal) unit of recombination (i.e. what are true segregational alleles)? Fig. 7.20 p228 Phage kill bacteria for a living (and replicate in the process). geneticists count “centers of klling” (plaques) 3 (centers of infection) “multiplicity of infection” Must have a phenotypic difference between pure-breeding lines to do genetics Phage phenotypes (differences): based on growth plaque morphology large/small (growth rate) clear/turbid smooth edged/rough edged growth conditions bacterial host range temperature range (T4: 25-42 C) By early 1950s, Alfred Hershy had isolated a number of phage T4 mutants and constructed a (circular) genetic map based on (1) complementation (to group the mutants into genes) and (2) recombination to organize those genes on a map. …and along came physicist Seymour Benzer P.N.A.S.-US v41p344 (1955) The young S. Benzer: (1950s) phage T4 as his genetic workhorse • reconciled the segregrational and functional definitions of the gene •set up the experimental system used in the 2nd most elegant genetics paper in history An older Benzer: (1967-present) fruit fly (D.melanogaster) as his genetic workhorse •founded many of the most interesting areas of modern behavioral genetics (clocks & learning, etc.) •set up the experimental system most effective for studying fly development: the compound eye 4 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) (1) rII vs. rII+ easily distinguished based on plaque morphology Among Hershey’s T4 collection were rapid lysis (r) mutants --- produced distinctively large plaques. (2) extremely rare (<10-7) rII+ recombinants easy to recover 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. Fig. 7.20 p.229 Fig. 7.20 (1) Characterize mutants: fell into two complementation groups, rIIA & rIIB very close on a genetic map. infect 1:1 mixture at “multiplicity of infection” of ~3 phage:1 bacterium (still) NON-permissive host mutants 1&2 in same “gene” alternatives mutants 1&2 in different “genes” Complementation test: phenotype of the hybrid Question: did he test by plaque morphology or ability to growth on K(λ)? No growth growth (AND no recombination!) BUT WHAT did Benzer do FIRST?! determine that mutants are recessive 5 (for complementation test) (1) Characterize mutants: fell into two complementation groups, rIIA & rIIB very close on the genetic map. Yes, but extremely tedious (almost never do) p.229 Fig. 7.20 (still) 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 YES …or NO: segregational alleles, so only… rIIA1 & rIIA2 (“unchanged in the hybrid”) (can be either a quantitative test or a qualitative test) no progeny lyse K(λ) 6