Dam Methylation Sites Research Articles

Trans- and Cis-acting elements for the replication of P1 miniplasmids

Replication-deficient mutants of the unit-copy miniplasmid λ-P1:5R were isolated after hydroxylamine mutagenesis. Complementation tests showed that the majority of these mutants are defective in the production of the repA protein product. Two of these mutants have suppressible nonsense (amber) mutations. The DNA sequence of one of these, repA103, has been determined. The lesion lies within the repA open reading frame, showing that the repA product is essential for plasmid replication. Complementation of deletion mutants of λ-P1:5R by repA protein showed that the origin of replication lies to the left of repA and that this 300-base-pair origin region is the only portion of the DNA essential for plasmid replication if repA protein is supplied in trans. Six of the 21 hydroxylamine-induced mutants were not complemented by repA. Replication of three of these could be restored by introduction into the plasmid of a wildtype origin region, suggesting that they were origin-defective. The DNA sequence of two mutants was determined. Mutant rep-11 has a 43-base-pair deletion within the incC sequence ( incC is a series of five direct repeats of a 19-base-pair sequence known to be involved in the regulation of plasmid replication). The deletion appears to have been generated by homologous recombination between two repeats. Mutant rep-30 has a single base substitution in a region just to the left of incC that destroys one of five G-A-T-C ( dam methylation) sites in this region. As λ-P1:5R is unable to establish itself as a plasmid in a methylase-defective ( dam − ) strain, it seems probable that methylation of the G-A-T-C sequences is important for origin function. The incC region and the sequences to its left appear to constitute an essential part of the origin of replication.

Analysis of the methylation-regulated Mu mom transcript.

The Mu mom gene is transcribed only when DNA is methylated by Dam; this methylation dependence is determined by a region upstream of the gene containing three Dam-methylation sites. The mom operon contains two open reading frames, one of which codes for Mom. Experiments were done to find a function for the other reading frame; it is shown that it does not affect phage immunity or the regulation of mom expression. Deletion mapping and S1 nuclease mapping experiments identify the +1 of the mom mRNA: it is 45 bp downstream of the three Dam-methylation sites. Possible models for the short-distance transcription regulation by methylation are discussed. The mom promoter is surrounded by sequences that are shown to be homologous to the oriC region of E. coli. This suggests a relation between replication and methylation-dependent mom expression. It is shown however, that replication is not essential for mom expression.

Transcription initiation of Mu mom depends on methylation of the promoter region and a phage-coded transactivator.

The product of the bacteriophage Mu gene mom modifies adenine residues of DNA within the consensus sequence CGAGCNPy, providing protection against various restriction endonucleases (ref. 1 and D. Kamp, personal communication cited in ref. 2). The mom gene is only expressed during lytic development of the phage. It is known that mom is nonfunctional in Escherichia coli host mutants in a gene (dam) which itself encodes an adenine methylation system. We show here that the E. coli dam gene is essential for transcription initiation of the mom gene, and that this dependence on dam seems to lie in a short segment preceding the mom coding region, which also contains the mom promoter. The sequence of this segment reveals the presence of dam methylation sites (GATC), and suggests a model for the regulation of mom gene expression based on DNA secondary structure, which may explain why mom is only expressed during phage lytic development. We also show that expression of phage-coded proteins (A, B and C) is needed for transactivation of mom transcription.

Mutational and evolutionary analysis of an archaebacterial gene

The bacteriorhodopsin precursor gene of H. halobium is shown to contain 71 potential sites of mutation to premature stop codons, encompassing all detectable transitions and transversions. This same gene contains 24 potential frameshift hot-spots, all of the G and/or C type, nine possible dam methylation sites, and a unique mec methylation site which may act as a deamination mutagenesis hot-spot. While the gene contains pre-dominantly GC-rich codons, there is appreciable usage of other types of codons for particular amino acids. The bacteriorhodopsin precursor gene does not contain an abnormally high proportion of RNY-type, primitive codons, suggesting that even if distinct from the eubacteria, and an ancient group, modern-day archaebacteria have, nevertheless, undergone extensive evolution.