Lederberg and Tatum discovered that some bacterial strains can transfer DNA to recipient cells, that is, they can mate. This discovery opened the doors for two types of research. First, genetic manipulations could be used to assist other types of studies involving bacteria, and throughout the book we shall see many examples of the assistance genetics provides to biochemical, physiological, and physical studies. Second, the mechanism of bacterial mating itself was interesting and could be investigated. In this section we shall review the actual mechanism of bacterial mating.

Maleness is conferred upon cells when they contain a mating module called the F-factor. F stands for fertility. This module is a circular DNA molecule containing about 25 genes involved with conjugation. One of the F genes codes for the major protein of the F-pilus. This is an appendage essential for DNA transfer. Other genes code for additional parts of the F-pilus, its membrane attachment, and DNA replication of F-factor DNA. The system also contains at least one regulatory gene.

F-pilus contact with a suitable female activates the mating module. As a result, a break is made within the F sequences of DNA and a single strand of DNA synthesized via the rolling circle replication mode is transferred into the female (Fig.1). Immediately upon entry of the single strand into the female, the complementary strand is synthesized. If no breakage occurs during transfer, all the F-factor DNA, including the portion initially left behind at the break, can be transferred into the female cell. The F-factor thus codes for transfer into female cells of itself and also any DNA to which it is connected. If the F-factor is integrated within the chromosome, then its mobilization in conjugation transfers the chromosome as well as itself. Cells which transfer their chromosomal genes to recipient cells are called Hfr for high frequency of recombination, whereas the cells which contain F-factors separate from the chromosome are called F or F’, and the females are called F-.

Fig1. Transfer of one strand of the double-stranded F factor from male cells into female cells.

A little over 100 minutes are required for transfer of the entire Escherichia coli chromosome to a female. Genes located anywhere on the chromosome can be mapped by determining the timing of their transfer to female cells. To do this mating is initiated by mixing male and female cells. Genetic markers on one side of the F-factor will be transferred to the female cells with only a few minutes of mating, while markers on the opposite side of the chromosome will not be transferred for over half an hour.

Random breakage of the DNA during mating from an Hfr to an F often interrupts the transfer so that only infrequently can the complete chromosome be transferred. This is a blessing in disguise however, for in addition to the time of transfer, the frequency of a marker’s transfer also indicates its chromosomal position with respect to the integrated F-factor. More than one integration site of F has been found on the chromosome, so that a variety of origins of transfer are available and the entire chromosome can be easily mapped. Once mating has been started and been allowed to proceed for a period of time, any further transfer of markers can be stopped merely by vigorously shaking the culture to separate the mating couples.

A mating experiment might be performed in the following way. Male Arg⁺ Gal⁺ Leu⁺ Sm^s (streptomycin-sensitive) and female Arg^- Gal^- Leu Sm^r cells would be grown to densities of about 1 × 10⁸ per ml and mixed together in equal portions. The cells would then be shaken very gently. At intervals, a sample of cells would be taken, vigorously shaken so as to separate mating couples and dilutions spread on streptomycin-containing medium so as to select for Arg⁺ or Gal⁺ or Leu⁺ Sm^r recombinants (Fig. 2).

Fig2. (a) The number of recombinants of the types indicated as a function of the duration of mating. (b) The approximate chromosomal locations of the markers used in the mating.

F-factors facilitate genetic study with Escherichia coli in a second way. They need not be associated with the chromosome. Instead, they can be autonomous DNA elements existing alongside the chromosome. The transfer properties remain the same for an F-factor in this position, but the genetics are slightly altered in two ways. First, the extra-chromosomal F-factor can have some chromosomal genes associated with it, in which case it is called an F’-factor. Presumably these genes were picked up at an earlier time by an excision of the integrated F-factor from the chromosome of an Hfr cell. The second alteration of an extrachromosomal F-factor is that often the entire mating module and attached genes are transferred to a female. As a result, the female acquires a functional F-factor with its associated genes and becomes diploid for a portion of the chromosome as well as becoming capable of transferring copies of the same F’ on to other female cells.

مواضيع ذات صلة

Fate Mapping and Study of Tissue-Specific Gene Expression

Isolating Mutations in Muscle or Nerve in Drosophila

Elements of Drosophila Genetics

Elements of Yeast Genetics

Crossover

Mapping with Generalized Transducing Phage

Genetic Selections

Growing Cells for Genetics Experiments

Genetic Systems

Elements of Recombination in E. coli, RecA, RecBCD, and Chi

Branch Migration and Isomerization

Heteroduplexes and Genetic Recombination