33. A cross is mode between an Hfr strain that is met⁺ thi⁺ pur⁺ and an F^– that is met^– thi^– pur^–. The recombinants are selected on medium that contains supplements that satisfy only the requirements for pur and thi and not for met. These recombinants are tested for the presence of the thi⁺ and pur⁺ alleles. The following number of colonies are found for each genotype:

met⁺ thi⁺ pure⁺        200
met⁺ thi⁺ pur^–          0
met⁺ thi^– pur⁺          7
met⁺ thi^– pur^–           60

What is the order of genes in Hfr strain?
(1) met⁺ pur⁺ thi⁺           (2) met⁺ thi⁺ pur⁺
(3) thi⁺ met⁺ pur⁺       (4) pur⁺ met⁺ thi⁺

Introduction: In bacterial conjugation experiments with Hfr strains, gene order determination relies on recombinant frequencies from selective media, crucial for CSIR NET Life Sciences preparation on microbial genetics. This Hfr met+ thi+ pur+ cross with F- met- thi- pur- reveals precise mapping through met+ selection on pur/thi-supplemented media lacking methionine.

Experimental Setup Analysis

The Hfr donor transfers chromosomal genes linearly from the origin of transfer (oriT), with proximal genes entering first and distal genes later, often interrupted before full transfer. Recombinants are met+ (requiring integration of met+), tested for unselected thi+ and pur+ markers on minimal media. Total met+ colonies: 200 (thi+ pur+) + 0 (thi+ pur-) + 7 (thi- pur+) + 60 (thi- pur-) = 267. Rare double crossovers explain low frequencies for certain genotypes.

Gene Order Determination

Frequencies indicate proximity: highest met+ thi+ pur+ (200/267 ≈ 75%) shows thi+ and pur+ often co-inherited with met+, placing both proximal to met+. Few met+ thi- pur+ (7/267 ≈ 2.6%) versus many met+ thi- pur- (60/267 ≈ 22%) reveal thi+ lies between met+ and pur+, requiring rare double crossover for thi- pur+ but single for thi- pur-. Thus, Hfr order is thi+ → met+ → pur+, with thi+ first, met+ intermediate, pur+ last.

Option-by-Option Analysis

• (1) met+ pur+ thi+: met+ first would yield high met+ thi- pur- (pur+ distal), contradicting observed high met+ thi+ pur+ and low met+ thi- pur+. Incorrect.

• (2) met+ thi+ pur+: met+ first, thi+ next, pur+ last predicts few met+ thi- pur+ (double crossover) matching data, but high met+ thi+ pur- (0 observed) expected if pur+ distal—contradicts. Incorrect.

• (3) thi+ met+ pur+: thi+ first (frequent), met+ next (selected), pur+ last (requires extra crossover for exclusion, yielding thi- pur-). Matches 200:0:7:60 perfectly. Correct.

• (4) pur+ met+ thi+: pur+ first yields high met+ thi- pur+, but observed only 7; contradicts. Incorrect.

This thi+ met+ pur+ order aligns with Hfr conjugation principles where gradient transfer and even double crossover frequencies determine mapping.