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Chapter: Genetics and Molecular Biology: Lambda Phage Genes and Regulatory Circuitry

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Genetic Structure of Lambda

A turbid or opaque lawn of cells is formed when 105 or more cells are spread on the agar surface in a petri plate and allowed to grow until limited by nutrient availability.

The Genetic Structure of Lambda

A turbid or opaque lawn of cells is formed when 105 or more cells are spread on the agar surface in a petri plate and allowed to grow until limited by nutrient availability. If a few of the original cells are infected with a virus, severalcycleof lysis of the infected cells and infection of adjacent cells during growth of the lawn leaves a clear hole in the bacterial lawn. These holes are called plaques. The first mutations isolated and mapped in phage lambda were those that changed the morphology of its plaques. Ordinarily, lambda plaques are turbid or even contain a minicolony of cells in the plaque center. Both result from growth of cells that have become immune to lambda infection. Conse-quently, lambda mutants that do not permit cells to become immune produce clear plaques. These may easily be identified amid many turbid plaques. Kaiser isolated and mapped such clear plaque mutants of lambda. These fell into three complementation groups, which Kaiser called CI, CII, and CIII, with the C standing for clear.

One particular mutation in the CI gene is especially useful. It is known as CI857, and the mutant CI product is temperature-sensitive. At tem-peratures below 37° the phage forms normal turbid plaques, but at temperatures above 37° the mutant forms clear plaques. Furthermore, lysogens of lambda CI857 can be induced to switch from lysogenic to lytic mode by shifting their temperature above 37°. They then excise from the chromosome and grow vegetatively.

Many additional lambda mutations were isolated and mapped by Campbell. These could be in essential genes, as he used conditional mutations. Such mutants are isolated by plating mutagenized phage on a nonsense-suppressing strain. Plaques deriving from phage containing nonsense mutations may be identified by their inability to grow after being spotted onto a nonsuppressing strain.

 

Phage with nonsense mutations in various genes may then be studied by first preparing phage stocks of the nonsense mutants on suppressing hosts. The phage can then be used in a variety of studies. For example, pairs of mutants can be crossed against one another and the frequency


Figure 14.2 The complete physi-cal map of lambda determined from its DNA sequence. The sizes of genes are indicated along with the functions of various classes of proteins. The genetic map is simi-lar except that the circle is opened between genes Rz and Nu1.

of generation of wild-type phage by recombination between the two mutations can be quantitated by plating on nonsuppressing strains. In this way a genetic map can be constructed. The nonsense mutants also facilitate study of phage gene function. Nonsuppressing cells infected with a nonsense mutant phage stock progress only partway through an infective cycle. The step of phage development and maturation that is blocked by the mutation can be determined with radioactive isotopes to quantitate protein, RNA, and DNA synthesis, or electron microscopy to determine which phage macromolecules or structures are synthesized.

 

Campbell named the genes he found and mapped A through R, left to right on the genetic map. The genes identified and mapped after his work are identified by the remaining letters of the alphabet, by three- letter names, and by other symbols.


 

The sequence of the entire lambda DNA molecule has been deter-mined. A few of the known genes could be identified in the sequence by the amino acid sequences of their products or by mutations that changed the DNA sequence. Many others could be identified with a high degree of confidence by the correspondence between genetic and physi-cal map location (Fig. 14.2). The identification of such open reading frames was greatly assisted by examination of codon usage. An open reading frame that is not translated into protein usually contains all sense codons at about the same frequency, whereas the reading frames that are translated into protein tend to use a subset of the codons. That is, many proteins use certain codons with a substantially higher fre-quency than other codons. Unexpectedly, applying these criteria to the DNA sequence of phage lambda revealed more than ten possible genes not previously identified genetically or biochemically. It remains to be shown how many of these actually play a role in phage growth and development.

The DNA sequencing revealed a second unexpected property of the lambda genes. Many are partially overlapped. The function of this overlap could be to conserve coding material, although it may also play


a role in translation. Since ribosomes drift phaselessly forward and backward after encountering a nonsense codon and before dissociating from the mRNA, this overlapping may help adjust the translation efficiency of the downstream gene product.

 

Genes of related function are clustered in the lambda genome. The genes A through F are required for head formation, and Z through J for tail formation. The genes in the b2 region are not essential for phage growth under normal laboratory conditions and may be deleted without material effect. Since such deletion phage still possess the normal protein coat but lack about 10% of their DNA, they are less dense than the wild-type phage; that is, they are buoyant density mutants. The genes int and xis code for proteins that are involved with integration andexcision and will be discussed in a later chapter. The genes exo, β, and γ are involved with recombination. The proteins from genesCIII,N,CI,cro, and CII are expressed early in the growth cycle and regulate theexpression of phage genes expressed early. The genes O and P code for proteins required for initiation of lambda DNA replication. The Q gene product regulates expression of late genes. These genes include S, R, and Rz, which are required for lysis of the cell as well as the genes that encode proteins comprising the head and tail of the phage particle.


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