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8.17 Jacob and Wollman used interrupted conjugation to map bacterial genes.

and to infection by bacteriophage T1 {tort); auxotrophic for threonine (thr-) and leucine (leu-); and unable to breakdown lactose (lac-) and galactose (gat). Thus, the genotypes of the donor and recipient cells were:

Donor Hfr cells: Hfr strs thr+ leu+ azf torf lac+ gat

Recipient F- cells: F- stf thr- leu- azf torf lac- gal-

The two strains were mixed in nutrient medium and allowed to conjugate. After a few minutes, the medium was diluted to prevent any new pairings. At regular intervals, a sample of cells was removed and agitated vigorously in a kitchen blender to halt all conjugation and DNA transfer. The cells were plated on a selective medium that contained streptomycin and lacked leucine and threonine. The original donor cells were streptomycin sensitive (strs) and would not grow on this medium. The F- recipient cells were aux-otrophic for leucine and threonine and also failed to grow on this medium. Only cells that underwent conjugation and received at least the leu+ and thr+ genes from the Hfr donors could grow on the selective medium. All stf leu+ thr+ cells were then tested for the presence of other genes that might have been transferred from the donor Hfr strain.

All of the cells that grow on the selective medium are strr leu+ thr+; so we know that these genes were transferred. The percentage of strr leu+ thr+ exconjugates receiving specific alleles (azf, torf, lac+, and gat) from the Hfr strain are plotted against the duration of conjugation ( FIGURE 8.17b). What is the order and distances among the genes?

The first donor gene to appear in all of these exconjugates (at about 9 minutes) was azf. Gene torf appeared next (after about 10 minutes), followed by lac+ (at about 18 minutes) and by gat (after 25 minutes). These transfer times indicate the order and relative distances among the genes (< Figure 8.17b).

Time (min)

Direction of <"

transfer Gene lO

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