The simplest method for attaching lambda to the host chromosome would be to insert it directly into the DNA. The simplest ideas are not always correct, however, and in the mid-60s when the question was being considered, chromosomal DNA seemed sacrosanct. Experiments were designed to determine whether lambda did integrate into the chromosome or whether it merely was stuck onto a special place on the chromosome. The genetic demonstration of lambda’s insertion into the host chromosome utilized deletions. The conclusion would be that lambda is integrated into the chromosome if a deletion of host genes extends into the lambda and removes some, but not all, lambda genes.

Often the identification of deletions is difficult. In this case, however, their identification and isolation were easy. The nitrate reductase com plex permits E. coli to use nitrate as an electron sink in the absence of oxygen. This enzyme complex is not entirely specific, and it will reduce chlorate as well. The product, chlorite, however, is toxic. Such a situation is a geneticist’s dream, for then mutants that are not killed by the chlorate can easily be isolated. These mutants have lost the ability to reduce chlorate, and many have been deleted of part or all of the nitrate reductase genes. Although it would be appropriate to name the genes involved after nitrate, the genetic locus is frequently called chl for chlorate resistance.

The deletions in the various chl loci frequently extend into adjacent genes. If chlorate-resistant mutants are isolated in a lambda lysogen, a few are found to be deletions and some of these are missing some but not all lambda genes. The pattern of lambda genes remaining or deleted is always consistent with the lambda genome being linearly incorporated into the chromosome in a specific orientation with respect to the flanking genes (Fig. 2). If some of the lambda genes have been deleted, how does one show that some lambda genes remain? The cell certainly cannot produce infective lambda. A method called marker rescue answers the question. Suppose we wish to test whether a lysogen with a deletion still has an intact J gene. A lawn of these cells is poured in soft agar on a plate and a small volume of a λimm⁴³⁴susJ (nonsense mutation in J) stock is spotted onto the lawn. Being heteroimmune, the superinfecting phage is not repressed even if the deleted lysogen still possesses immunity. A few of these infecting phage exchange their defective J gene by recombination for the good one from what remains of the partially deleted lysogenic lambda. Sufficient phage make this replacement, that cycles of growth, lysis, and infection of surrounding cells produces a turbid spot on the opaque lawn. If no J gene is present on the partially deleted chromosome, the infecting λimm⁴³⁴susJ cannot proceed through multiple growth cycles, and the lawn is unperturbed. In some cases, a gene on the partially deleted lysogen will be able to complement the superinfecting phage as well as recombine with it, but the mapping results are unaltered. Only if the gene is present in the host cells can the superinfecting phage grow.

Fig1. Deletions from chlD into lambda genes that are consistent with lambda being directly integrated into the host chromosome.

Marker rescue experiments on chl deletions revealed many deletions that extended only part way into the lambda genes. Certainly the simplest explanation for this result is that the DNA of lambda is contiguous with the host DNA.

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مواضيع ذات صلة

Amplification Techniques in Molecular Biology

Characteristics of Nucleic Acids

solation of Integration-Defective Mutants

Gene Order Permutation and the Campbell Model

Mapping Integrated Lambda

Expression Patterns of Developmental Genes

Enhancer Traps for Detecting and Cloning Developmental Genes

Cloning Developmental Genes

General Considerations on Signaling

DNA Strand Inheritance and Switching in Fission Yeast

DNA Cleavage at the MAT Locus

Glucose Metabolism