Wht do we men (wht hve we ment) y " gene": Reding for lectures 15-17 (We F27, Fr F29, We M5) Chp 8: from 258 (Nonoverlpping...) to 261 ( Crcking) & from 285 (8.6) to 293 (end of "essentil concepts) Chp 14: Using deletions to locte genes (496-498) Chp 20: ll except (for the time eing) 724-25 (cloning) & RNA Interference (726-728). How do we recognize if A given heritle difference in (ppernce) is cused y the simplest possile difference in genotype (reeding ehvior) SIMPLEST DIFFERENCE:. difference with respect to one gene.different lleles (forms) of one gene. "monogenic trite" A definition of wht we men y "gene": the thing tht exists in two different forms (lleles) Two different opertionl tests for llelism: Mendel's "segregtionl test" The gene s the unit of segregtion -- reeding ehvior of the hyrid (1:2:1) (=genotype of the hyrid)) Recll tht conflict rose Green's/Lewis'/Benzers' "complementtion test" The gene s the cis-cting unit of function --- of the hyrid recessive lozenge-shped-fly-eye "lleles" in trns: lz(g) recessive lozenge-shped-fly-eye "lleles" in trns: recomintion lz(g) wt lz(g) lz(bs) hyrid looks mutnt (sme defective unit of function = functionl lleles) lz(bs) hyrid looks mutnt ( - function) lz(bs) wt nd yet: nd yet: wt rre wildtype gmetes hence cn't e segregtionl lleles (reeding ehvior of hyrid) wt wt wt rre wildtype gmetes (lso: lz(bs) lz(g) ) hence cn't e segregtionl lleles (reeding ehvior of hyrid) 1
Are nd lleles (i.e. genetic lterntives) Do we go with Mendel or (Green/Lewis)/Benzer high-resolution segregtion test for recomintion + + reeding ehvior Are & lterntive units of segregtion during meiosis i.e. re they segregtionl lleles complementtion test + + Are & lterntive cis-cting units of function during development i.e. re they functionl lleles The complementtion test is generlly fvored: Genes s defined s cis-cting units of function hve multiple prts tht re seprle y recomintion Different lleles of the sme gene my e defective for different resons Segregtion test is useful preliminry test (especilly when we cn't mke the hyrids we would like) If: lleles with similr phenotypic effects mp to pproximtely the sme point Then: those mutnts re likely to e lleles of the sme gene Are nd lleles (i.e. genetic lterntives) --- could we ever e misled y the pprent filure of these recessive mutnts to complement in trns complementtion test + + wildtype mutnt (complement) (don t complement) not lleles lleles Are nd lleles (i.e. genetic lterntives) could mutnts e recessive individully nd in different cis-cting units of genetic function (i.e. not e functionl lleles), ut interct in comintion to pper dominnt together) + + + + wildtype wildtype mutnt complementtion test + + wildtype mutnt (complement) (don t complement) not lleles lleles Are nd lleles (i.e. genetic lterntives) When lleles FAIL TO COMPLEMENT we my need the cis control: + + BECAUSE: If the two mutnts re individully recessive lleles of different genes, ut dominnt in comintion the cis control will lso e mutnt! complementtion test + + wildtype mutnt (complement) (don t complement) not lleles lleles Are nd lleles (i.e. genetic lterntives) AND sme recessive mutnts in CIS + + wildtype then we cn trust the trns result IF: recessive mutnts in TRANS mutnt don t pper to complement complementtion test trns cis, hence & re functionl lleles of the sme gene orienttion of the genetic informtion mtters for genes 2
Are nd lleles (i.e. genetic lterntives) sme recessive mutnts in CIS + + wildtype Phenotype we cn trust the trns result recessive mutnts in TRANS mutnt don t pper to complement CIS/TRANS test complementtion test But noody others with the cis control for recessive mutnts when doing complementtion tests despite wht ll the textooks sy (not even Benzer did it, s we will see) Are nd lleles (i.e. genetic lterntives) CIS/TRANS test complementtion test Cis test is too hrd in most cses, nd in most cses unnecessry. then why m I wsting your time with this Are nd lleles (i.e. genetic lterntives) CIS/TRANS test for functionl llelism The complete cis/trns test will llow us to determine llelism even if one or oth of the mutnts re not recessive! Rememer: the complementtion test per se is limited to recessive mutnts. Most mutnts re recessive, ut some of the most useful & interesting re not. CIS TRANS, hence functionl lleles CIS = TRANS, hence NOT functionl lleles nd ll tht mtters is whether the cis vs. trns s re the sme or different, not wht the s of ech ctully re! complementtion test lleles s lterntive cis-cting units of function Intuitively, etter opertionl definition of lterntive forms of genes Rises questions: Conflict fvored high-resolution segregtion test for recomintion: lleles s lterntive units of segregtion (recomintion) If one cn get recomintion etween functionl lleles (lterntive forms of gene), then how do the genetic mps one cn therefore construct within single genes compre to the genetic mps tht cn e (hd een) constructed etween genes --- WHAT IS THE NATURE OF GENETIC FINE STRUCTURE nd wht is the sic (miniml) unit of recomintion (i.e. wht re true segregtionl lleles) Fig. 7.20 p228 Phge kill cteri for living (nd replicte in the process). geneticists count centers of klling (plques) 3
(centers of infection) multiplicity of infection Must hve phenotypic difference etween pure-reeding lines to do genetics Phge s (differences): sed on growth plque morphology lrge/smll (growth rte) cler/turid smooth edged/rough edged growth conditions cteril host rnge temperture rnge (T4: 25-42 C) By erly 1950s, Alfred Hershy hd isolted numer of phge T4 mutnts nd constructed (circulr) genetic mp sed on (1) complementtion (to group the mutnts into genes) nd (2) recomintion to orgnize those genes on mp. nd long cme physicist Seymour Benzer P.N.A.S.-US v41p344 (1955) The young S. Benzer: (1950s) phge T4 s his genetic workhorse reconciled the segregrtionl nd functionl definitions of the gene set up the experimentl system used in the 2nd most elegnt genetics pper in history An older Benzer: (1967-present) fruit fly (D.melnogster) s his genetic workhorse founded mny of the most interesting res of modern ehviorl genetics (clocks & lerning, etc.) set up the experimentl system most effective for studying fly development: the compound eye 4
Need selective genetic system (one with high resolving power for smll mp distnces) Rf - = NP pfu from hyrid / totl pfu from hyrid pfu = plque-forming units hyrid = mixed infection Phge re smll, ut plques re often lrger thn fruit flies! How do phge help with mesuring smll Rfs Use selective systems to esily mesure NP pfu concentrtion without compliction from the much lrger numer of P pfu Benzer s system mde mesuring 0.0001 cm (1x10-6 ) esy nd y the wy, s n dded onus for mpping, phge hppen to hve MUCH greter rte of recomintion per unit DNA thn fruit flies or grden pes In rii, smllest non-0 recomintion rte mesured ws 0.02 cm (mutnts 1 p prt) (2 NP/10,000 totl) In my first effort t fine-structure mpping in flies, I mesured 0.007 cm (one recominnt) for distnce (I only lter found out to e) 3,100 p (1 NP/14,286 totl) (1) rii vs. rii + esily distinguished sed on plque morphology Among Hershey s T4 collection were rpid lysis (r) mutnts --- produced distinctively lrge plques. (2) extremely rre (<10-7 ) rii + recominnts esy to recover They rose spontneously (1:10-4 ) nd fell into severl different complementtion groups tht mpped t different plces One complementtion group ws rii nd Benzer discovered something specil out it tht mde it perfect for studying genetic fine structure. Fig. 7.20 (1) Chrcterize mutnts: fell into two complementtion groups, riia & riib very close on genetic mp. p.229 Fig. 7.20 (still) mutnts 1&2 in sme gene lterntives infect 1:1 mixture t multiplicity of infection of ~3 phge:1 cterium NON-permissive host mutnts 1&2 in different genes Complementtion test: of the hyrid Question: did he test y plque morphology or ility to growth on K(λ) No growth (AND no recomintion!) growth BUT WHAT did Benzer do FIRST! determine tht mutnts re recessive 5
(for complementtion test) Yes, ut extremely tedious (lmost never do) (1) Chrcterize mutnts: fell into two complementtion groups, riia & riib very close on the genetic mp. p.229 Fig. 7.20 (still) p.229 Fig. 7.20 (still) Mke recominnts y mixed infection of the permissive host Assy progeny pfu concentrtion on permissive (=totl) nd nonpermissive (=50% of nonprentl) hosts (cn e either quntittive test or qulittive test) YES or NO: segregtionl lleles, so only riia 1 & riia 2 ( unchnged in the hyrid ) no progeny lyse K(λ) 6