Question

An Hfr strain is used to map three genes in an interrupted mating experiment. The cross is \(H f r / a^{+} b^{+} c^{+} r i f \times F^{-} / a^{-} b^{-} c^{-}\) rif \(^{r} .\) (No map order is implied in the listing of the alleles; rif \(^{r}\) is resistance to the antibiotic rifampicin.) The \(a^{+}\) gene is required for the biosynthesis of nutrient \(\mathrm{A}\), the \(b^{+}\) gene for nutrient \(\mathrm{B}\), and \(c^{+}\) for nutrient \(\mathrm{C}\). 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 \((\mathrm{MM})\) 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. (a) What is the purpose of rifampicin in the experiment? (b) 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. (c) Can the location of the rif gene be determined in this experiment? If not, design an experiment to determine the location of rif relative to the \(\mathrm{F}\) factor and to gene \(b\)

Short answer

Answer: The purpose of the antibiotic rifampicin in the experiment is to select for recombinants that have acquired the rif^r gene (resistance to rifampicin), ensuring only the successful recombinants grow on the medium containing rifampicin. The position of the rif gene cannot be determined from this experiment. To determine its location, a new Hfr strain with the rif gene near gene b or integrated within it can be created, and another interrupted mating experiment can be conducted to analyze the time intervals for each gene transfer and establish the relative position of the rif gene.

Step-by-step solution

Purpose of rifampicin in the experiment

The antibiotic rifampicin is used in the experiment to select for recombinants that have acquired the rif^r gene (which confers resistance to rifampicin). This ensures that only the cells with successful recombination will grow on the medium containing rifampicin.

Determine the time intervals for each gene transfer

To determine the location of a, b, and c genes, we need to analyze the data and find when each gene is being transferred. This will give us an idea of the distance between the genes and the order in which they occur on the chromosome. Unfortunately, the mentioned table is not provided with the exercise, but the subsequent analysis is still possible. Once the time intervals for each gene's transfer would be identified, we can proceed with determining their relative positions on the chromosome.

Determine the relative order of genes a, b, and c

Based on the time intervals obtained in step 2, the relative order of genes can be determined. The gene that transfers first is closest to the F factor, and as time passes, genes progressively further away are transferred. This sequence provides us with the relative order of genes a, b, and c on the bacterial chromosome.

Location of rif gene

Based on the exercise information, it's not possible to determine the location of the rif gene. An experiment to determine its location relative to the F factor and gene b would be to introduce the rif gene in the Hfr strain as a selectable marker. By creating a new Hfr strain with the rif gene near gene b and conducting an interrupted mating experiment with the same setup, we could determine the relative position of rif with respect to gene b by analyzing the time intervals for each gene transfer. (c) The location of the rif gene cannot be determined in this experiment as the data does not allow for it. To determine the location of the rif gene relative to the F factor and gene b, we can construct a new Hfr strain with the rif gene adjacent to gene b or integrated within it. By performing an interrupted mating experiment with this new Hfr strain and analyzing the time intervals for each gene transfer, we could determine the relative position of rif with respect to the F factor and gene b.

Key concepts

Hfr strain

An Hfr (High-frequency recombination) strain plays a critical role in bacterial genetic studies. These strains possess a unique ability where the F factor, a plasmid that facilitates DNA transfer, integrates into their chromosome. This integration allows them to efficiently transfer genetic material during conjugation, a process where two bacterial cells connect and exchange fragments of DNA.
The Hfr strain acts as a donor in mating experiments, typically with an F- (lacking F factor) recipient strain. The presence of integrated F factor in Hfr strains leads to the transfer of chromosomal genes from the donor to the recipient in a sequential manner. This sequential transfer reflects the linear order of genes on the chromosome, which aids in mapping genes within a chromosome.
Hfr strains are invaluable for constructing genetic maps, as the time taken for a gene to be transferred during conjugation provides clues about its location. The closer a gene is to the integrated F factor, the quicker it is transferred to the recipient. Understanding this mechanism is essential for deciphering bacterial genetics and for experiments like the one highlighted, which aim to determine the chromosome map using gene transfer timings.

chromosome mapping

Chromosome mapping is a technique used to determine the specific physical locations of genes within a chromosome. This is achieved by examining the sequence in which genes are transferred from an Hfr strain to a recipient strain during bacterial conjugation. Conjugation is interrupted at various time intervals, and the sequence and timing of gene entry into the recipient is recorded.
Through chromosome mapping, we can establish the relative positions of genes. For example, in the interrupted mating experiment, if gene 'a' is transferred before gene 'b', and gene 'b' is transferred before gene 'c', it indicates that 'a' is positioned closest to the F factor origin, followed by 'b', and then 'c'.
Chromosome mapping is crucial because it helps describe genetic layout and linkage. By knowing which genes are close to one another on a chromosome, scientists can predict the likelihood of genetic recombination between them. This technique helps in understanding bacterial gene function, mutation effects, and can pave the way for genetic engineering applications.

genetic recombination

Genetic recombination is a natural process where DNA sequences are rearranged to create genetic diversity. In bacteria, this occurs during conjugation where DNA is exchanged between a donor (like the Hfr strain) and a recipient cell (like the F- strain). This is one of the primary methods of horizontal gene transfer among bacteria, aside from transformation and transduction.
During the process, a segment of the donor's chromosomal DNA is inserted into the recipient's genome. This exchange is facilitated by the F factor in Hfr strains, which acts as a key to unlock the DNA transfer process.
Through recombination, bacteria can acquire new traits, such as antibiotic resistance, which in the provided experiment is selected by using rifampicin resistance (rif^r). This process enriches bacterial adaptability, helping them endure and evolve in changing environments. Understanding genetic recombination is pivotal in microbiology as it has implications for the development of antibiotic resistance and is a prime focus in genetic manipulation for research and therapeutic purposes.