Working with Molecular Genetics (Hardison)

Gene mapping by conjugal transfer

Conjugal transfer can also be used for genetic mapping. By using many different hfr strains, each with the F factor integrated at a different part of the E. colichromosome, the positions of many genes were mapped. These studies showed that the genetic map of the E. coli chromosome is circular. During conjugal transfer, genes closer to the site of F integration are transferred first. By disrupting the mating at different times, one can determine which genes are closer to the integration site. Thus on the E. colichromosome, genes are mapped in terms of minutes (i.e., the time it takes to transfer to recipient).

For example, for an hfr strain with the F factor integrated at 0 min on the E. colimap, conjugal transfer to a female recipient would transfer

• leuACBD at 1.7 min
• pyrH at 4.6 min
• proAB at 5.9 min
• bioABFCD at 17.5 min.

Bacteriophage

Bacteriophageare viruses that infect bacteria. Because of their very large number of progeny and ability to recombine in mixed infections (more than one strain of bacteria in an infection), they have been used extensively in high-resolution definition of genes. Much of what we know about genetic fine structure, prior to the advent of techniques for isolating and sequencing genes, derive form studies in bacteriophage.

Bacteriophage have been a powerful model genetic system, because they have small genomes, have a short life cycle, and produce many progeny from an infected cell. They provide a very efficient means for transfer of DNA into or between cells. The large number of progeny makes it possible to measure very rare recombination events.

Lytic bacteriophage form plaqueson lawns of bacteria; these are regions of clearing where infected bacteria have lysed. Early work focused on mutants with different plaque morphology, e.g. T2 r, which shows rapid lysis and generates larger plaques, or on mutants with different host range, e.g. T2 h, which will kill both host strains B and B/2.

A cis-trans complementation test defines a cistron, which is a gene

Seymour Benzer used the rIIlocus of phage T4 to define genes by virtue of their behavior in a complementation test, and also to provide fundamental insight into the structure of genes (in particular, the arrangement of mutable sites – see the next section). The difference in plaque morphology between rand r+phage is easy to see (large versus small, respectively), and Benzer isolated many r mutants of phage T4. The wild type, but not any rIImutants, will grow on E. colistrain K12(l), whereas both wild type and mutant phage grow equally well on E. colistrain B. Thus the wild phenotype is readily detected by its ability to grow in strain K12 (l).

If E. colistrain K12 (l) is co-infected with 2 phage carrying mutations at different positions in rIIA, you get no multiplication of the phage (except the extremely rare wild type recombinants, which occur at about 1 in 106 progeny). In the diagram below, each line represents the chromosome from one of the parental phage.