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Mapping By Bacterial Conjugation |
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Mapping By Bacterial Conjugation
A. Early genetic crosses (or mapping by uninterrupted conjugation)- Many aspects of the bacterial sexuality were worked out in the 1950s by W. Hayes, P. Jacob, J. and E. Lederberg and E. Wollman well before the molecular details of the process were known. Their studies revealed that recombination would occur when Hfr cells of one genotype were crossed with F- cells of another genotype. Jacob and Wollman (1958), for example, used an Hfr strain of E. coli with the following genotype-thr+ leu+ azi-s T1-s lac+ gal+ str-s(Hfr). It is capable of synthesizing the amino acids threonine (thr+) and leucine (leu+), it is sensitive to the metabolic inhibitor sodium azide (azi-s), and it is sensitive to the phage T1 (T1- s). It can ferment lactose (lac+) and galactose (gal+) as well as glucose and is sensitive to the antibiotic streptomycin (str-s).
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The F- strain of E. coli has the complementary genotype, that is, thr-, leu-, azi-r T1-r lac-gal-str-r (F-), where resistance is denoted by -r. A cross is made by mixing the two cell types and letting them remain together for 60 minutes. The cells are then plated to various media and allowed to grow.Jacob and Wollman began by plating cells to a minimal medium containing streptomycin. On such a medium all unmated Hfr cells being streptomycin sensitive were killed, and any cell that required threonine or leucine, including unmated F- cells, were unable to grow. This step therefore allowed only F- cells with the recombinant genotype thr+ leu+ str-, to form colonies, Such recombinants emerged with very high frequency-some 10 per cent of the total cells plated-a result that suggests that thr and leu gene loci are quite loosely linked to the str locus. In other words, genetic exchange occurred frequently between the thr+ leu+ markers contributed by Hfr donor and str-r marker contributed by the F- recipient.
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Further, for the determination of rest of the genotype of the thr+ leu+ str-r recombinants, Jacob and Wollman replica-plated the recombinant colonies to a minimal medium that contained either sodium azide or bacteriophage T1 and determined the distribution of sensitivity and resistance to these agents among the colonies. They also replica-plated to the special indicator media to determine whether the various strains were able to ferment lactose or galactose. Typically, they found that among the thr+ leu+ str-r, recombinants 90 per cent carried the azi-s marker of the Hfr donor 80 per cent, the T1-s marker; 40 per cent, tbe lac+ marker, and 25 per cent the gal+ marker.
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Moreover, if it was also estimated that 1 minute of conjugation is equivalent to 20 recombination units in E. coli, and the entire chromosome is transferred in 100 minutes, then the total map length is 2000 recombination units. If 107 nucleotide pairs exist in the chromosome, then 1 recombination unit represents 107/2000=5000 nucleotide pairs.
B. Mapping by interrupted mating- When Hfr and F- bacterial cell cultures are mixed, conjugation can be stopped at any desired time by subjecting the mixture to the shearing forces of Waring Blender which artificially disrupts the conjugation bridge. Samples are allowed to mate for 5 minutes, 10 minutes, and so on and then each sample is diluted immediately, plated to a streptomycin-containing minimal medium to select recombinant F- cells that are thr+ leu+ str-r. Thus, a Hfr strain carrying selected marker and a distal auxotrophic or sensitivity marker (e.g., str+ or str-r) which prevents the growth of Hfr cells on the selective medium and thereby allowing only recombinant cells (e.g., thr+ leu+ str-r cells) to appear.
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The order of the genes in the Hfr donor strain is b+-c+a+; b is less than 5 time-units from the origin; c is less than 10 time-units from b; a is less than 10 time-units from c. |
This technique is called contraselection (Stansfield, 1969). Because of the polarity with which the Hfr chromosome is transferred, the time at which various genetic markers appear in the recipient indicates their linear organization in the donor chromosome. At a given temperature, the transfer of the first half of Hfr chromosome proceed at a relatively uniform rate. Therefore the time of entry of different markers into a recipient (F-) cell is a function of the physical distance between them.
Example: An Hfr strain of E. coli carrying the prototrophic markers a+, b+, c+ is mixed with a F- strain carrying the auxotrophic alleles a, b, c. Conjugation was interrupted at 5 minute intervals and plated on media which revealed the presence of recombinants.
Time(minutes) |
Recombinants |
5 |
ab+c |
10 |
ab+c+ |
15 |
a+b+c+ |
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