7.03 Genetics: Solutions to Problem Set 4 (1997)

cotransduction of 235/(235+100)=78%. This value is not completely accurate, because it fails to take into account the events which cotransduce pyrF" and argE +, but do not include the hisB + allele. (c) would If Arg + transductants had been selected (instead of His+), what fraction be His+? What fraction would be Pyr+? of these This problem relies on the cotransductional distances calculated in part (c). If the cotransductionat freqency between hisB and argE is 50%, then thefrequency of Arg + transductants that are also His + will be 50%. The accurate answer for the frequency of Arg + transductants that are also Pyr + is <50% (since the pyrF- allele is being cotransduced with the argE+ allele at afrequency >50%). Using our estimated value for the cotransductional distance between argE and pyrF, (78%) we can generate an estimated value for afrequency of Arg + transductants that are also Pyr+ = 22% 2, (a) Consider a portion of the E. coil chromosome that contains three genetic markers in the following order: metD -- proA -- lacZ. Starting with an F+, metD +, proA +, lacZ +, Str s strain, you isolate an Hfr that transfers metD + early and efficiently but transfers the proA + and LacZ + markers very inefficiently. Draw a map of the relevant portion of the E. coil chromosome in the Hfr showing the integrated F (indicate both the site of integration and the orientation of the origin of transfer). orIT 1 f,roA {at...-2 t_,-_e,u_earzf la_retd. (b) If the P1 cotransductional distance between metD and proA is 70% in a wild-type E. coil strain, what will the cotransduction frequency between metD and proA be in the Hfr strain isolated in part (a). The size of the F plasmid inserted benveen metD and proA is l0 s base pairs. The cotransduction frequency between metD and proA would go down to 0% in the isolated Hfr since they would be seperated by more than 105 base pairs. (c) You isolate an F' from the Hfr described in part (a) by mating to an F-, metD-, proA-, lacZ-, Str r strain and selecting for lacZ÷ and str r after a brief period of mating. This F' strain tois produce proA + and Describe the positions of the sequences that recombined the metD-. F'. .The recombination event that produces the isolated F' is as diagrammed below.. ....................... _ 4,, £'4 4- ..................... 3, (a) Phenyl p,D-galactoside inducer of the lac operon. (PG) is a substrate Isopropyl _,D-thiogatactoside operon, but not a substrate for _-Galactosidase. containing PG? IPTG? for p-Galactosidase, PG+IPTG? .-7 TT": ...... : but is not an (IPTG) is an inducer of the lac Can wild-type ceils grow on media Explain. The cells cannot grow on media containing PG alone, since they will not be able to activate tile lac operon. On IPTG, the cells will not grow because they cannot use IPTG as a substrate for flgalactosidase. On media containing both PG and IPTG, the cells will be able to grow, since IPTG can activate the lac operon, and PG can act as a subtrate for _-galactosidase. Following is a positional map of the lac operon, I POZY where P represents the promoter region necessary for transcription of the Z (_Galactosidase), and Y (Permease) cistrons. One can isolate mutations in this promoter 4 region (P-), which prevent the promoter from interacting with the RNA polymerase holoenzyme, thus blocking transcription. (b) Given what you know about the P- mutation and other mutations within the lac operon. give the induction state of the following Justify your answers. strains under inducing and non-inducing conditions. LacZ induction EXAMPLE GLUCOSE . I+ P+ O+ Z+ Y+ I+ P- 0 +z + Y+/F' I+ P+.O + Z+ y+ I+ P" 0 +z + Y+/F' I+ P+ 0 +z" IPTG + + Y" I+ P" 0 cZ + Y+/F' I+ P+ 0 + Z + Y+ " + I+ P" 0 +Z + Y+/F' l+ P+ 0 cZ + Y+ + + (c) You isolate an unidentified mutant (X'), that fails to induce LacZ expression on IPTG plates. You generate the following strains, and assay them for induction of LacZ under inducing and noninducing conditions. LacZinduction GLUCOSE IPTG X- - - X-/ F'I + P+ 0 + Z + Y+ " + X-/F' I"d P+ 0 + Z + Y+ + + X-/F' I+ P+ (:pc Z- Y+ - + X'/ F'I + P" 0 + Z + Y+ Where in the lac operon does the mutation X- likely reside? Explain how each of the strains above support your conclusion. The X- m_ttation leads to an uninducible phenotype. Thus _C is cannot be ]-_ 0 cor 1. The second strain, X'/F' I + P+ 0 + Z+ Y+, shows that the X- mutation is recessive. 22 cannot be Is, which is dominant. Finally, the X'/F' I + P+ 0 c Z- Y+ strain shows that X" complements a Z mutation, thus _ cannot be either a P or a Z mutation. Therefore, by elimination, the )C tnutation is likely to be a]r mutation in the permease gene. The other strains are consistent with the hypothesis that X" represents a mutation in the permease gene.