An Hfr strain is used to map three genes in an interrupted mating experiment. The cross is Hfr/a⁺b⁺c⁺ rif x F⁻/a⁻b⁻c⁻ rif^T (No map order is implied in the listing of the alleles; rif^T is resistance to the antibiotic rifampicin.) The a⁺ gene is required for the biosynthesis of nutrient A, the b⁺ gene for nutrient B, and the c⁺ gene for nutrient C. The minus alleles are auxotrophs for these nutrients. The cross is initiated at time = 0, and at various times, the mating mixture is plated on three types of medium. Each plate contains minimal medium (MM) plus rifampicin plus specific supplements that are indicated in the following table. (The results for each time interval are shown as the number of colonies growing on each plate.)

Based on these data, determine the approximate location on the chromosome of the a, b, and c genes relative to one another and to the F factor.

Verified step by step guidance

1. Step 1: Understand the experimental setup. An Hfr strain (donor) with genes a⁺, b⁺, and c⁺ is crossed with an F⁻ strain (recipient) that is auxotrophic (a⁻, b⁻, c⁻). The interrupted mating experiment measures the time it takes for different genes to be transferred from the Hfr to the F⁻ strain. The presence of rifampicin resistance (rif^T) allows selection for recipient cells that have received donor DNA.
2. Step 2: Analyze the data for each pair of nutrients at different time points. The number of colonies growing on minimal medium plus rifampicin and specific nutrient supplements indicates successful transfer and expression of those genes. The earliest time at which colonies appear for a gene pair suggests the order and relative position of those genes on the chromosome.
3. Step 3: Determine the order of gene transfer by comparing the times at which colonies first appear for each nutrient pair. For example, if colonies appear earlier for the A and C pair than for the A and B pair, gene C is transferred before gene B relative to gene A. Use the times when colonies start to appear (non-zero counts) to infer the sequence of gene transfer.
4. Step 4: Use the relative timing of gene transfer to map the genes on the chromosome. The gene transferred first is closest to the origin of transfer (near the F factor integration site), and subsequent genes are farther away. The difference in time between the appearance of colonies for each gene pair corresponds to the distance between those genes on the chromosome.
5. Step 5: Summarize the gene order and approximate distances based on the interruption times. Express the gene order as a linear sequence (e.g., F factor - gene A - gene C - gene B) and indicate the relative distances between genes proportional to the time intervals observed in the experiment.

Key Concepts

Hfr Strain and Interrupted Mating

An Hfr (high frequency recombination) strain contains the F factor integrated into its chromosome, allowing it to transfer chromosomal genes to an F- recipient during conjugation. Interrupted mating experiments stop conjugation at specific times, enabling mapping of gene order based on the time genes are transferred. Genes closer to the F factor transfer earlier.

Gene Mapping by Time of Entry

In interrupted mating, the order and relative distances of genes on the bacterial chromosome are determined by the time at which they enter the recipient cell. Genes transferred earlier are closer to the origin of transfer (F factor), and the time intervals between gene transfer reflect their physical distances on the chromosome.

Auxotrophic Markers and Selective Media

Auxotrophic mutations (a-, b-, c-) require specific nutrients for growth, while wild-type alleles (a+, b+, c+) enable biosynthesis of those nutrients. By plating on minimal media supplemented with specific nutrients and rifampicin, only recombinants receiving certain genes survive, allowing identification of gene transfer timing and mapping.