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To demonstrate that the transfer of genetic material from the donor to the recipient cell during conjugation is a linear event, F. Jacob and E. Wollman devised the technique of interrupted mating. In this technique, F and Hfr strains were mixed together in a food blender.
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Figure 7.15 Bacterial conjugation. (a) The F-pilus draws an Hfr
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Figure 7.14 Electron micrograph of conjugation between an
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F (upper right) and an F (lower left) cell with the F-pilus between them. Magni cation 3,700 . (Courtesy of Wayne
Rosenkrans and Dr. Sonia Guterman.)
and an F cell close together. (b) The Hfr chromosome then begins to pass into the F cell, beginning at the F region of the Hfr chromosome but in the direction away from the F factor. Only a single strand passes into the F cell; this strand and the single strand remaining in the Hfr cell are replicated. After the process is interrupted (c), two crossovers bring the a allele into the F a chromosome (d ).
Tamarin: Principles of Genetics, Seventh Edition
II. Mendelism and the Chromosomal Theory
7. Linkage and Mapping in Prokaryotes and Bacterial Viruses
The McGraw Hill Companies, 2001
Seven
Linkage and Mapping in Prokaryotes and Bacterial Viruses
Elie Wollman (1917 ).
(Courtesy of Dr. Elie Wollman and the Pasteur Institute.)
Frequency (percent)
After waiting a speci c amount of time, Jacob and Wollman turned the blender on. The spinning motion separated conjugating cells and thereby interrupted their mating. Then the researchers tested the F cells for various alleles originally in the Hfr cell. In an experiment like this, the Hfr strain is usually sensitive to an antibiotic such as streptomycin. After conjugation is interrupted, the cells are plated onto a medium containing the antibiotic, which kills all the Hfr cells. Then the genotypes of the F cells can be determined by replica-plating without fear of contamination by Hfr cells. The mating outlined in table 7.3 was carried out. In the food blender, an Hfr strain sensitive to streptomycin (str s ) but resistant to azide (azi r ), resistant to phage T1 (tonAr ), and prototrophic for the amino acid leucine (leu ) and the sugars galactose ( galB ) and lactose (lac ) was added to an F strain that was resistant to streptomycin (str r ), sensitive to azide (azi s ), sensitive to T1 (tonAs ), and auxotrophic for leucine, galactose, and lactose (leu , galB , and lac ). After a speci c number of minutes (ranging from zero to sixty), the food blender
was turned on.To kill all the Hfr cells, the cell suspension was plated on a medium containing streptomycin. The remaining cells were then plated on medium without leucine. The only colonies that resulted were F recombinants.They must have received the leu allele from the Hfr in order to grow on a medium lacking leucine. Hence, all colonies had been selected to be F recombinants. By replica-plating onto speci c media, investigators were able to determine the azi, tonA, lac, and galB alleles and the percentage of recombinant colonies that had the original Hfr allele (leu ). (Note that by trial and error, it was determined that leucine should be the locus to use to select for recombinants. As we will see, the leucine locus entered rst.) Figure 7.16 shows that as time of mating increases, two things happen. First, new alleles enter the F cells from the Hfr cells. The tonAr allele rst appears among recombinants after about ten minutes of mating, whereas galB rst enters the F cells after about twenty- ve minutes. This suggests a sequential entry of loci into the F cells from the Hfr ( g. 7.17). Second, as time proceeds, the percentage of recombinants with a given allele from the Hfr increases. At ten minutes, tonAr is rst found among recombinants. After fteen minutes, about 40% of recombinants have the tonAr allele from the Hfr; and after about twenty- ve minutes, about 80% of the recombinants have the tonAr allele. This limiting percent-
tonAr*
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