Concepts

Conjugation in E. coli is controlled by an episome called the F factor. Cells containing F (F+ cells) are donors during gene transfer; cells without F (F~ cells) are recipients. Hfr cells possess F integrated into the bacterial chromosome; they donate DNA to F~ cells at a high frequency. F' cells contain a copy of F with some bacterial genes.

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Mapping bacterial genes with interrupted conjugation

The transfer of DNA that takes place during conjugation between Hfr and F~ cells allows bacterial genes to be mapped. During conjugation, the chromosome of the Hfr cell is transferred to the F~ cell. Transfer of the entire E. coli chromosome requires about 100 minutes; if conjugation is interrupted before 100 minutes have elapsed, only part of the chromosome will pass into the F~ cell and have an opportunity to recombine with the recipient chromosome. Chromosome transfer always begins within the integrated F factor and proceeds in a continuous direction; so genes are transferred according to their sequence on the chromosome. The time required for individual genes to be transferred indicates their relative positions on the chromosome. In most genetic maps, distances are expressed as percent recombination; but, in bacterial maps constructed with interrupted conjugation, the basic unit of distance is a minute.

Worked Problem

To illustrate the method of mapping genes with interrupted conjugation, let's look at a cross analyzed by Fran├žois Jacob and Elie Wollman, who first developed this method of gene mapping ( FIGURE 8.17a). They used donor Hfr cells that were sensitive to the antibiotic streptomycin (genotype strs); resistant to sodium azide (azf) and infection by bacteriophage T1 (torf); prototrophic for threonine (hr+) and leucine (leu+); and able to break down lactose (lac+) and galactose (gal+). They used F- recipient cells that were resistant to streptomycin (strr); sensitive to sodium azide (azf)

J Experiment)-

Question: How can interrupted conjugation be used to map bacterial genes?

An Hfr cell with genotype str5 thr+ leu+ azf torf lac+ gal+.

Start ton r lac *

Jlthr a V

An Hfr cell with genotype str5 thr+ leu+ azf torf lac+ gal+.

Jlthr a V

.was mated with a F- cell with genotype strr thr~ leu-azP ton5 lac- gat.

Genes transferred: thr +, leu + , and str+ (first selected genes, defined as zero time)

.was mated with a F- cell with genotype strr thr~ leu-azP ton5 lac- gat.

Genes transferred: thr +, leu + , and str+ (first selected genes, defined as zero time)

^ Conjugation was interrupted at regular intervals.

8 min

Bacteria separate w w

Bacteria separate thr + leu + str+

-ailtf'

10 min

16 min

Bacteria separate

Bacteria

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