The preceding sections have described a little of what has been learned from genetic experiments about lambda’s ability to integrate and excise. One way to study the biochemistry of integration and excision is to construct an in vitro system that mimics the in vivo reaction. The first requirement for integration or excision is to bring the two participating molecules close together. In vivo, this requirement is partially met as a simple consequence of the fact that both DNA molecules are confined to the volume of the cell and therefore are held in close proximity. The in vitro integration or excision reaction should be greatly speeded if the two att regions similarly can be forced close to one another. One way to accomplish this is to place both att sites on the same DNA molecule. Then the concentration of one att site in the vicinity of the other is, of necessity, high.

This section describes the isolation and properties of such a double att phage, and the following sections describe its use and the use of a similar phage to study the integration and excision reactions more deeply both in vivo and in vitro.

Some distance to either side of an integrated lambda are two sites that the host recombination system will recombine at a reasonable frequency. This event excises a phage that has picked up host DNA from both sides of the phage integration site (Fig. 1). Ordinarily this event cannot be detected because the resulting DNA is too long to be packaged in a lambda coat, but if several deletions have first been put into lambda, this particular excision product will fit into the head, and viable phage are formed.

Fig1. Recombination between two points beyond the ends of an integrated lambda phage generated the λatt² phage containing attR and attL.

The structure of the phage produced by the recombination between two sites flanking lambda is most interesting. The phage contains two att regions, attL and attR, and is called att2. Such an att2 phage can lose the extra bacterial DNA by a reaction analogous to normal excision:

This process requires the Int and Xis proteins and produces a viable lambda phage genome and a minicircle (Fig. 2).

Fig2. An excision reaction between the two att regions on att2 generates a viable phage with a single att region and a minicircle.

Study of the excision reaction requires quantitating the input phage λatt² as well as the product phage. Fortunately, the two may be readily distinguished. The concentrations of λatt² and the λ generated by excision can be assayed in a mixture of the two by first separating the phage on the basis of density in equilibrium centrifugation. The λatt² phage is more dense than lambda as a result of its additional DNA.

A simpler assay of the two types of phage makes use of the sensitivity of lambda to heat and chelators of Mg⁺⁺. Removal of Mg⁺⁺ ion from the phage reduces the charge neutralization between phosphates of the DNA backbone, and as a result, the DNA expands and can burst the phage coat. Phage particles possessing less than the usual amount of DNA are more resistant to heating and removal of Mg⁺⁺. Combinations of Mg⁺⁺ chelators and elevated temperatures can be found in which wild-type lambda is killed by factors of 10³ to 10⁴, but the λb2 deletion mutant is unharmed (Fig. 3). The same principle can be used to titer λ in the presence of λatt2. If the mixture is titered on plates containing pyrophosphate, which chelates magnesium, only the λ can proceed through multiple infective cycles and generate plaques. By titering also on normal plates, the total number of both phage types present can be determined since both form plaques.

Fig3. Killing of λb2 and wild-type λ by heating in the presence of a Mg⁺⁺ chelator.

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مواضيع ذات صلة

Transducing Phage Carrying Genes Other than gal and bio

Incorrect Excision and gal and bio Transducing Phage

Isolation of Excision-Deficient Mutants

Amplification Techniques in Molecular Biology

Characteristics of Nucleic Acids

solation of Integration-Defective Mutants

Gene Order Permutation and the Campbell Model

Simultaneous Deletion of Chromosomal and Lambda DNA

Mapping Integrated Lambda

Expression Patterns of Developmental Genes

Enhancer Traps for Detecting and Cloning Developmental Genes

Cloning Developmental Genes