Isolation of F-primes
F-primes are formed from Hfr's by illegitimate recombination (i.e. recombination between sequences with little sequence homology). Compared to homologous recombination events that result in precise excision of the F-plasmid from an Hfr, such illegitimate recombination events are very rare -- typically about a million-fold less frequent. If the illegitimate recombination events occurs with the chromosome sequences that flank the Hfr, the aberrant excision transfers the adjacent chromosomal sequence to the plasmid, leaving behind a deletion of these sequences on the chromosome. The steps of F-prime formation are shown in the cartoon below.
F-primes can include chromosomal sequences from either side of the integrated Hfr. For example, if an Hfr is inserted in the region between the proBA and proC genes, aberrant excision on one side of the Hfr will produce F' proC plasmids while aberrant excision on the other side will produce F' proBA plasmids. The events leading to each of these types of F-prime are shown in the cartoon below.
The illegitimate recombination events that lead to F-primes are rare, but chromosomal genes can be readily transferred to a recipient via an Hfr. Therefore, a selection is required to isolate F-primes. The selection must demand transfer of a chromosomal marker from the donor to the recipient, while excluding transfer from an Hfr. Two approaches can be used to select for F-primes:
Recall that, as with all matings, transfer of an F-prime requires a counterselection against donor cells.
- Selection for transfer of a distal marker following a short mating. This approach is based upon the requirement of 100 minutes to transfer the entire E. coli chromosome from an Hfr donor into a recipient. If the time allowed for mating is limited to 30 minutes, the distal genes cannot be transferred via the Hfr. However, F-primes are much plasmids that only require a few minutes for transfer into a recipient (the time required depends upon the size of the chromosomal DNA fragment on the F-prime). Thus, the chromosomal genes carried on an F-prime can be readily transferred to the recipient during a short mating.
- Selection for transfer of a chromosomal marker into a recA-recipient. Because the DNA brought in on an Hfr must recombine with the recipient chromosome via homologous recombination, the recA mutation prevents inheritance of donor markers from an Hfr. In contrast, because F-primes circularize after the complete plasmid sequence has been transferred into the recipient and because F-primes are plasmids that can replicate independant of the chromosome, F-primes can be readily transferred to a recA recipient. In a recA recipient the donor markers are maintained on the F-prime and complement the chromosomal copy in trans.
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Last modified October 17, 2000