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Table of Contents
Why Do Genetics
More Basic Concepts
Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
Problem Set 2
F factor and other incompatibility groups
THE SPECIFIC CASE OF F FACTOR
F factor is the best studied of the incompatibility groups that have the property of conjugative ability. It was also at the core of most of the gene transfer systems that were used by bacterial geneticists in the '60's and '70's. In its extrachromosomal state the factor has a molecular weight of approximately 62 kb and encodes at least 20 tra genes. It also contains three copies of IS3, one copy of IS2, and one copy of a À sequence as well as genes for incompatibility and replication. The F factor can exist in three different states: "F+" refers to a factor in an autonomous, extrachromosomal state containing only the genetic information described above. The "Hfr" (which refers to "high frequency recombination") state describes the situation when the factor has integrated itself into the chromosome presumably due to its various insertion sequences. Finally, the "F'" or (F prime) state refers to the factor when it exists as an extrachromosomal element, but with the additional requirement that it contain some section of chromosomal DNA covalently attached to it. A strain containing no F factor is said to be "F-".
In mating an F+ with an F+ strain, one finds fast, efficient transfer of F+ (approximately 50% transfer in one hour), but chromosomal transfer is only at the level of l0-5 to l0-7 per donor cell. This is probably due to rare, spontaneous Hfr formation. As mentioned above, Hfr's arise by integration into the chromosome due to the insertion sequences on the plasmid. These seem to cause integration at preferred sites, so that one finds a variety of different Hfr's which differ with respect to their origins of transfer and direction of transfer. When one performs a cross between an Hfr strain and an F- strain, one sees transfer of chromosomal markers at high frequency (10-2 to 10-5). This transfer is both oriented and time dependent. Since transfer begins at the oriT site in the F factor, a portion of the F factor is transferred first followed by the remainder of the chromosome. If the entire chromosome is transferred, then the other portion of the F factor is transferred. The F factor itself does not integrate into the recipient as there is no homology for such integration but the chromosomal DNA which has been transferred can recombine in by homologous recombination. The transfer of the entire E. coli chromosome takes approximately l00 minutes but you very often get spontaneous breakage of the mating pair. Such breakage means that markers transferred late are often not transferred at all yielding a gradient of transfer which tends to be of the order of l03 (that is, early markers are transferred approximately l03times more frequently than the most distal markers). The net result is that one often fails to transfer the entire chromosome. In crosses between an F' and an F- strain, two possible donation events can occur, depending on the genotype of the donor. If the donor strain is Rec-, then the plasmid will remain as an extrachromosomal element in the donor and will be the only genetic information transferred in the conjugation event (true, you will get IS directed chromosome integration at approximately 10-5 to 10-7 in line with section IV E1. If, however, the donor cell is Rec+, then homologous recombination will cause some of the F's to integrate into the chromosome of the donor and thus act like Hfr's. Typically, for that reason, Rec- donors are used for such analyses.
The major "failing" of F factors is that their use is limited to E. coli and its close relatives. The bottom line is that, in those organisms, F's are large, low-copy, conjugative plasmids capable of chromosomal mobilization, but are too large to physically handle with any ease.
OTHER INCOMPATIBILITY GROUPS
Plasmids of a variety of other incompatibility groups, with a wide range of properties, have been useful in genetic analysis. Natural isolates vary in copy number (from approximately one per cell to hundreds), size (several kB to hundreds of kb), stability, conjugative ability, host range, and drug resistance. Moreover, because many of these properties are the product of one or a small set of genes, a vast array of plasmids have been engineered to have a specific set of useful properties, including a number of unique cloning sites for in vitro manipulation. Specialized manuals describe the general choices and new versions are constantly being described in the literature.
OTHER CONSIDERATIONS CONCERNING PLASMIDS AND CONJUGATION
The above sections are in no way meant to be an all-inclusive summary of plasmids in prokaryotic organisms. Clearly, molecular biology is only scratching the surface of the various forms and properties that these elements can possess. There are, however, several other classes of plasmids which deserve mention. Many bacteria have plasmids with no obvious function. These are often assigned the name "cryptic" plasmids and their existence is only noted because of their physical detection as mentioned at the beginning of this section. You may well ask, "if they aren't necessary, why aren't they lost?" There are two possibilities: (l) They are necessary but you have not found the growth conditions which select for the plasmid or (2) the plasmid transfers so well that, though they are segregated frequently, any plasmid-free cell soon receives another from a plasmid-containing cell. Some are extremely large and this property makes them even harder to detect physically. In general, if you isolate a new organism and it has an unstable phenotype, it would be prudent to consider the possibility that a plasmid is harbored in the strain.
So what are plasmids good for:
[See sample problems 12 and 13]
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