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Why Do Genetics
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Mutagenesis in vitro
Effects of Mutations
Two Factor Crosses
Other Mapping Methods
Problem Set 1
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©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison
VI C3. SPECIALIZED TRANSDUCTION
Specialized phage are derivatives of temperate phage where some portion of the phage genome has been replaced by a portion of other genetic material, typically the bacterial chromosome. In this way, all of the phage particles carry the same portion of the bacterial chromosome.
How are specialized phage generated? If one considers the structure of a prophage as indicated below, and asks what would happen if the prophage came out imprecisely, the result would be a specialized phage. This phage would have the property that it would be mutant in some way (although they may still possess all functions necessary for growth; if they fail to grow without helper phage, they are termed "defective") as some of the phage functions had been effectively deleted; and all progeny would carry the same host sequence. Typically one solves the difficulty of the defectiveness of the phage by supplying a normal wild-type version of the phage, termed a "helper" which grows along with a specialized phage and supplies whatever functions are necessary for generating phage particles. Since it would not be very interesting to pick up only those genes which happen to be located on either side of the phage att site, other genes can be put on specialized phage by several methods including (a) forcing the prophage to go in at alternate sites, (b) moving the gene of interest near a phage attachment site, (c) cloning the gene of interest onto the specialized phage. While the first two methods require little understanding of the physical structure of the phage, the last method requires a good deal of that information. One needs to have at least one restriction sequence that occurs only once in the phage (and in a non-essential region) to serve as a site for cloning. Fortunately, through the diligent efforts of molecular biologists, there are a plethora of phage that have all the desired properties, at least for E. coli. Since phage can only carry a "headfull" of DNA, a region of non-essential DNA needs to be removed from the phage genome to compensate for that inserted DNA. Alternatively, a helper phage can be used as noted above. Finally, it can often be useful to have a drug-resistance gene cloned into the phage to serve as an easy assay for the genetic presence of the phage.
How do these specialized phage integrate their "host" genes into the chromosome? Specialized phage have two possible ways of introducing genes into a recipient. The first and most efficient involves the normal phage integration system. By this means, the entire specialized phage DNA is integrated at the phage attachment site, possibly in conjunction with the wild-type helper phage. This generates a merodiploid (a strain partially diploid for a portion of its genome) for the region carried by the phage. The other possibility involves recombination between the chromosomal DNA carried by the phage and the chromosome itself.
As always, for a transferred gene to be "stably" inherited in the recipient, it needs to be replicated in that recipient. In the case of transduction, this means that the incoming DNA must (generally) integrate into a replicon in the recipient (either the chromosome or a plasmid). Transferred DNA in generalized transduction can only do this by homologous recombination while DNA associated with specialized phage also has the phage int system as a means of association with the replicon. Other "exceptions" to these rules are (i) transposons that can transpose into the chromosome of a Rec+ or Rec- cell from DNA of a specialized or generalized transducing phage; (ii) a specialized phage that has the ability to circularize and replicate as a plasmid in the recipient cell, thus not requiring any recombination into the chromosome; (iii) a plasmid that is moved by generalized transduction or by transformation and that has the ability to circularize and subsequently replicate in the recipient.
Advantages of each type of transduction (for organisms where the appropriate phage have been developed):
It is relatively easy.
It is rather efficient (10-3 per recipient with P22HT, 10-6 with P22 or P1), using the correct phage.
It moves only a small part of the chromosome which allows you to change part of a strain's genotype without affecting the rest of the chromosome (with chromosomal mobilization you cannot help but move in regions of the chromosome which you might not want, whenever you seek transfer of another region).
The high frequency of transfer and the small region transferred allows fine-structure mapping (see section VIII).
Very efficient transfer of a small region--can be useful for fine-structure mapping (see section VIII).
Excellent source of DNA for the chromosomal region carried by the phage, since every phage carries the same DNA.
Can often be used to select for deletions of some of the chromosomal genes carried on the phage (see the section VIIIC on deletion mapping).
Merodiploids generated using specialized phage can be quite useful in complementation analyses.
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