32. A cross was made between Hfr met⁺ arg⁺ leu⁺ str^s X F^– met^– arg^– leu^– str^r in which leu⁺ exconjugants are selected. If the linear organization of the genes are leu⁺ arg⁺ met⁺, which one of the following genotypes is expected to occur in the lowest frequency?
(1) leu⁺ arg^– met^–          (2) leu⁺ arg⁺ met^–
(3) leu⁺ arg⁺ met^–      (4) leu⁺ arg^– met⁺

In Hfr conjugation, the donor Hfr met+ arg+ leu+ strs transfers genes linearly starting from the origin of transfer (oriT), with leu+ entering first, followed by arg+, then met+ in the recipient F- met- arg- leu- strr. Leu+ exconjugants are selected on streptomycin media, requiring recombination of the leu+ donor fragment into the recipient chromosome. Due to the gradient of transfer and breakage during conjugation, distal markers like met+ recombine less frequently than proximal ones like arg+.​

Gene Transfer Mechanism

Genes transfer sequentially: oriT → leu+ → arg+ → met+. Selection for leu+ favors early transfers, but complete transfer of all three genes is rare because conjugation bridges break randomly. Recombinants form via double crossovers (even number minimum) between homologous donor and recipient DNA. Frequency decreases for genotypes needing more distal donor DNA or complex crossovers.​

Option Analysis

• (1) leu+ arg- met-: Retains recipient arg- met-; single leu+ integration via crossovers flanking leu. Highest frequency as it requires minimal donor DNA.​

• (2) leu+ arg+ met-: Needs leu+ arg+ from donor; crossovers before leu and between arg-met. Moderate frequency since arg+ is proximal to leu+.​

• (3) leu+ arg+ met-: Duplicate of option (2); same moderate frequency requiring leu+ arg+ transfer.​

• (4) leu+ arg- met+: Needs leu+ and met+ but not arg+; requires four crossovers (flanking leu, around arg region, flanking met) due to skipping middle arg+. Lowest frequency as it demands longest donor fragment and extra recombination events.​

The correct answer is option (4) leu+ arg- met+, occurring at the lowest frequency.