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.)

What is the purpose of rifampicin in the experiment?

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1. Rifampicin is an antibiotic that inhibits bacterial RNA polymerase, preventing transcription and thus halting bacterial growth. In this experiment, it is used to ensure that only the recipient F⁻ cells, which are rifampicin-resistant (rif^T), can grow on the plates.
2. The Hfr donor cells are rifampicin-sensitive (rif), so they cannot grow in the presence of rifampicin. This ensures that any colonies observed on the plates are derived from the recipient F⁻ cells that have successfully received genetic material from the Hfr donor during conjugation.
3. By including rifampicin in the medium, the experiment eliminates the possibility of donor cells contributing to the observed growth, allowing the researchers to focus solely on the transfer of genetic material to the recipient cells.
4. This selective pressure ensures that the experiment accurately reflects the transfer of the a⁺, b⁺, and c⁺ alleles from the Hfr donor to the F⁻ recipient, as only the recipient cells can survive and form colonies.
5. In summary, rifampicin serves as a selective agent to distinguish between donor and recipient cells, ensuring the validity of the interrupted mating experiment results.

Key Concepts

Hfr Strains and Gene Mapping

Hfr (high frequency of recombination) strains are bacterial strains that facilitate the transfer of chromosomal genes during conjugation. In gene mapping experiments, Hfr strains are used to determine the order and distance between genes based on the timing of their transfer. By interrupting the mating process at various time intervals, researchers can analyze which genes are transferred first, providing insights into their relative positions on the chromosome.

Auxotrophy and Nutritional Requirements

Auxotrophy refers to the inability of an organism to synthesize a particular compound required for its growth, necessitating the addition of that compound to the growth medium. In this experiment, the F⁻ strain is auxotrophic for nutrients A, B, and C, meaning it cannot grow without these nutrients. The presence of specific nutrients in the medium allows researchers to determine which genes have been successfully transferred from the Hfr strain, as only those with the corresponding functional alleles can grow.

Role of Rifampicin

Rifampicin is an antibiotic that inhibits bacterial RNA synthesis by targeting RNA polymerase. In this experiment, rifampicin is used to prevent the growth of the Hfr donor strain after conjugation, ensuring that only the recipient F⁻ strain can grow on the selective media. This allows for the accurate assessment of gene transfer and the identification of successful recombinants, as only those that have received the necessary genes from the Hfr strain will be able to grow in the presence of rifampicin.