Mapping of Point Mutations on the Physical Map of Coliphage Lambda: Absence of Clustering for Odd-Numbered Exchanges

The lambda ral function modulates the restriction and modification activities of the Escherichia coli K12 and B restriction enzymes (Zabeau et al., 1980). In order to further analyse this function, ral deficient mutants have been isolated, using a method which exploits the property of the strong mutagen N-methyl-N'-nitro-N-nitrosoguanidine (N.G.) to induce multiple closely linked mutations. Hence, mutagenized phages carrying mutations in one locus were frequently found to contain additional mutations in adjacent loci. This very efficient mutagenesis procedure enabled us to isolate 27 independent Ral deficient mutants. Seven mutants were found to affect the ral gene directly and were located between the genes N anc cIII. Detailed mapping of two of these mutants showed that the lambda ral gene is located at position 70.6-70.9% on the physical map. The isolation and characterization of these mutants further supports the conclusion that ral is a gene different from the N gene, and demonstrates that the ral gene product is responsible for both counteracting restriction and enhancing modification.

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The ral gene of phage λ

Debrouwere

Zabeau

Montagu

et al. 1980

Molec. Gen. Genet.

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Specific mismatch correction in bacteriophage lambda crosses by very short patch repair

Lieb

1983

Molec Gen Genet

122

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In crosses under rec+, red+, gam+ conditions, mutation am6 in the cI (repressor) gene of bacteriophage lambda recombines with other cI mutations much more frequently than predicted by the physical distances involved. In four-factor crosses of am6 with mutations located 22-60 base pairs to the left, cI+ recombinants that are expected to require three crossovers (triple recombinants) are more frequent than recombinants that require only one crossover. However, when am6 is crossed with large insertions in cI, which may be expected to interfere with the formation of heteroduplexes by branch migration, the frequency of cI+ triple recombinants is very low. In addition, cI+ recombinants in crosses between am6 and adjacent mutations have a high probability of retaining the flanking markers of the am6 parent. These findings suggest that am6 is particularly susceptible to mismatch repair in heteroduplexes spanning cI. A large fraction of such heteroduplexes are presumed to be the result of branch migration from crossovers occurring at some distance from am6. The absence of co-repair when am6 is crossed with adjacent cI mutations indicates that most repair tracts extend no farther than about 20 bp to either side of the mismatch. The am6 mutation arose in the glutamine codon in a CCAGG sequence, in which the central cytosines are methylated in K12 strains. Their location in methylated sequences may make certain amber mutations susceptible to a specific very short patch (VSP) repair.

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Asymmetric Effects of Deletions and Substitutions on High Negative Interference in Coliphage Lambda

1982

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Experiments have been performed to help clarify the role of nonhomologies in phage λ recombination. Three-factor crosses were carried out, and the frequencies of single and double recombinants in the two adjoining intervals were compared when the central marker was either a double point mutation (v1v3) or deletion (rex-cI deletion) or nonhomologous substitution (imm434). In all cases the lefthand marker was a bio substitution (Fec- phenotype, which does not permit plating on recA -), and the righthand marker was an amber mutation in gene O. Experiments were performed in all four possible arrangements of the central and rightward markers, while selecting for the Fec+ phenotype on the recA - host. As anticipated, high negative interference (HNI) was observed with point mutations, but when the central marker was a substitution nonhomology, HNI was reduced about tenfold. Surprisingly, when the central marker was a simple deletion, a dramatic asymmetry in results was observed, with HNI being exhibited only when the central deletion marker was acquired by the double recombinant. These results indicate that under normal conditions (red +, gam +, rec +) and with noninhibited DNA replication, recombination in coliphage λ entails a highly asymmetric step that could be at the level of strand transfer or mismatch repair.

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Mapping of Point Mutations on the Physical Map of Coliphage Lambda: Absence of Clustering for Odd-Numbered Exchanges

Cited by 4 publications

References 13 publications

The ral gene of phage λ

The ral gene of phage λ

Specific mismatch correction in bacteriophage lambda crosses by very short patch repair

Asymmetric Effects of Deletions and Substitutions on High Negative Interference in Coliphage Lambda

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