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Problem Set 2
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©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
VI C2. GENERALIZED TRANSDUCTION
Imagine what would happen if a phage incorporates host DNA instead of phage DNA inside a fraction of the produced phage heads. The cell would still lyse, and these novel particles, termed transducing particles, could still recognize an appropriate bacteria, attach, and inject their DNA (sometimes called a transducing fragment). The result would be the transfer of DNA from one host to another, since no phage DNA would be found in transducing particles of this type. This DNA transfer mechanism has been enormously useful for E. coli, S. typhimurium and B. subtilis, but such phage have not been identified and developed for most bacteria.
How is this host DNA "mispackaged"? The most commonly used generalized transducing phages, P22 (infecting S. typhimurium) and P1 (infecting E. coli), package by "headfull packaging", so that they fill the head with a little more than one phage length of DNA. They typically recognize their own DNA as appropriate for packaging because of special sequences termed pac sites. Occasionally, the phage make an error and begin to package a host "concatamer", presumably because a site on the host DNA is reminiscent of the phage pac site. It is also conceivable that insertion sequences in the host might cause a transposition of the host DNA into the phage concatamer, thus allowing packaging of host DNA even when the packaging scheme started correctly on phage concatamers.
How often does this "mispackaging" occur? For both P22 and P1, about one phage virion in l,000 actually contains host DNA. It is possible to obtain mutants which make errors more frequently. An example of this is the mutant termed P22HT which has completely lost the ability to recognize its pac sites so that it packages DNA randomly. Since an infected cell contains about half host and half phage DNA, the P22HT variant causes the generation of a phage lysate in which half of the phage heads contain host DNA. In the normal case, to the extent that mispackaging is due to pseudo-pac sites in the host DNA, one would expect that not all regions of the host DNA would be transduced at equal frequency. Such seems to be the case with wild-type P22 and this differential transduction is eliminated in mutants like P22HT.
How is the transduced DNA stably inherited? As always, for the transferred DNA to become stably inherited, it must become associated with a replicon in the recipient cell. Since it is a linear molecule, this requires two homologous recombination events with the net result that a portion of the chromosome in the recipient is replaced by a portion of the incoming DNA. Thus, in contrast with specialized transduction, generalized transduction requires that the recipient possess both a functional rec system and DNA homologous to the transferred fragment. It has been observed in generalized transduction that there is an approximately l0% chance of the incoming chromosomal DNA being recombined into the recipient's chromosome. A recipient cell whose genotype and phenotype have been changed by a transduction event is called a transductant.
What are the frequencies at which phage can generate transductants? The two phage referred to above carry l-2% of the host DNA and normally package chromosomal DNA at a frequency of 10-3. Thus, about one in l05 phage particles carries a given region of the host DNA. Since a given gene injected into a recipient cell by a transducing particle has a l0% chance of being recombined into the chromosome then one might expect to get approximately one transductant for a given region per l06 phage particles. In the case of the P22HT derivative, since approximately half of the phage particles carry chromosomal DNA, one can achieve the extraordinary frequency of one transductant per l000 phage particles. There is also a remarkable phage in B. subtilis that carries l0% of the host chromosome, though apparently its frequency of mispackaging is sufficiently low that the number of transductants per phage particle is around one in l07. In general, the virtue of generalized transduction is not its efficiency, but its ease of use. For strain construction, it is also an advantage that only a portion of the chromosome is transferred, in contrast to Hfr's.
[See sample problem 14]
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