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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
VI C. TRANSDUCTION
VI C1. MOLECULAR BIOLOGY OF PHAGE
Transduction is defined as the transfer of genetic information between cells through the mediation of a virus (phage) particle. It therefore does not require cell to cell contact and is DNase resistant. In order to understand transduction it is necessary to understand a bit of the biology of bacteriophage. There are a wide variety of bacteriophages, but we will concentrate on those that can mediate genetic transfer and therefore only on those bacteriophage that contain double stranded DNA. These phage can be broken down into two classes, both of which are useful for genetic analysis, and these are termed lytic and temperate (or lysogenic).
When a lytic phage injects its DNA into a host cell, it begins an ordered sequence of gene expression that involves the generation of many copies of the phage DNA, typically in the form of long concatamers (a linear sequence of covalently attached phage genomes). The phage also direct the generation of proteinaceous "heads" and then pack their DNA into these heads by some mechanism. When "mature" phage are assembled, they cause the cell to lyse and release approximately l02 to l03 progeny. Another class of phage, termed temperate, have a choice of "lifestyles" upon injecting their DNA into the host. Under some conditions, they follow a route similar to that chosen by lytic phage sketched above. However, they also possess the ability to repress their lytic functions and take up semi-stable residency in the host. This residency typically takes the form of an integration of the phage DNA into the host chromosome, usually by a site-specific recombination event with the resulting integrated phage termed a prophage. An example of such an integration is shown below.
This integration event is reversible and is driven largely by phage factors in both directions. If a prophage decides to lyse the cell, it excises itself from the chromosome, begins expression of DNA synthesis and other lytic functions, packages its DNA appropriately, and lyses the cell.
There is another class of E. coli phage that possess a number of qualities useful for molecular genetics, but cannot mediate transduction. These are the single-stranded DNA phages (M13 is the best known example) that are filamentous and produce progeny without lysing the host cell. The first feature has the effect that there are not strong size constraints on inserts into these phage, since they do not utilize a "headfull packaging" scheme. The latter property allows very high titres of phage to accumulate in the medium without significant amounts of cell debris. Their single-strandedness is also useful in sequencing strategies and as a substrate for site-directed mutagenesis in vitro. The phage can also be used as "helpers" to cause single-stranded replication and extrusion of plasmids that have been engineered to contain the phage origin of replication.
The production and quantitation of phage stocks requires the exposure of phage to actively growing bacteria. The optimum production of a high-titre lysate (greater than l09 phage/ml) requires inoculation of a bacterial culture with phage under conditions where the phage just lyses the culture as the cell concentration reaches saturation. Optimal levels of bacterial and phage inocula are a function of the growth rate of the bacteria, the latent period (the time from phage infection until cell lysis), and the burst size of the phage. If purified phage (free of cells and debris) are desired, this is usually accomplished by a centrifugation step where the phage position themselves in a salt gradient according to their buoyant density. Phage concentrations can be titred (counted) by plating by various dilutions of the phage stock onto a soft agar lawn containing susceptible cells. The dilution that yields a countable number of plaques, each arising from a single phage, is used to calculate the concentration of the original phage stock.
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