Cloning and structural characterization of the mcrA locus of Escherichia coli.

Escherichia coli has DNA restriction systems which are able to recognize and attack modified cytosine residues in the DNA of incoming bacteriophages and plasmids. The locus for the McrA/RglA system of modified cytosine restriction was located near the pin gene of the defective element, e14. Hence, loss of the e14 element through abortive induction after UV irradiation caused a permanent loss of McrA restriction activity. e14 DNA encoding McrA restriction was cloned and sequenced to reveal a single open reading frame of 831 bp with a predicted gene product of 31 kDa. Clones expressing the complete open reading frame conferred both McrA and RglA phenotypes; however, a deletion derivative was found which complemented RglA restriction against nonglucosylated T6gt phage but did not complement for McrA restriction of methylated plasmid DNA. Possible explanations for this activity and a comparison with the different organization of the McrB/RglB restriction system are discussed.     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.     

Highly efficient positive selection of recombinant plasmids using a novel rglB-based Escherichia coli K-12 vector system

We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.     

Effects of mcr restriction of methylated CpG islands of the L1 transposons during packaging and plating stages of mammalian genomic library construction.

The use of optimally methylation-tolerant mcrA- mcrB- strains has been shown to produce an over tenfold increase in the plating efficiencies of mammalian genomic libraries, compared to a superior conventional phage host strain LE392 which is mcrB+. However, there is an even more significant effect of mcr restriction. Amongst the recombinants recovered with an mcrB+ host, we have found that there is an additional 30-fold reduction in the frequencies of clones containing the heavily methylated 5'-CpG island sequences of both the human and rat L1 repetitive elements. The mcrA product was also found to restrict clones of these methylated genomic segments, but not as strongly as mcrB. However, the use of packaging extracts made from mcrA+ lysogens did not result in convincing reductions in the recoveries of these dispersed methylated elements. The magnitude of mcr restriction during plating due to methylated dispersed elements is sufficient to make a significant proportion of mammalian genomes unclonable from genomic libraries constructed previously using conventional mcr+ hosts.     

Physical mapping and cloning of bacteriophage T4 anti-restriction endonuclease gene.

We have proposed that the ability of T4 to produce non-glucosylated progeny after a single cycle of growth on a galU rglA rglB+ mutant of Escherichia coli is due to the initiation of the rglB+ function by a phage-coded, anti-restriction endonuclease protein. Based on this hypothesis, we screened T4 deletion mutants for failure to give a burst in this host. The absence of an arn gene in phage mutants lacking the 55.5- to 58.4-kilobase region is verified by their inability to protect secondary infecting non-glucosylated phage from rglB-controlled cleavage. A functional arn gene was cloned on plasmid pBR325, and the 0.8-kilobase insert DNA was shown to be homologous to the DNA missing in the arn deletion phage.     

Isolation of temperature-sensitive McrA and McrB mutations and complementation analysis of the McrBC region of Escherichia coli K-12.

We isolated temperature-sensitive mcrA and mcrBC mutants of Escherichia coli. At 42 degrees C, they were unable to restrict the T-even bacteriophages T6gt and T4gt or plasmids encoding cloned DNA methylase genes whose specificities confer sensitivity to the McrA and McrBC nucleases. Complementation analysis of the McrBC region (mcrB251) with the complete cloned McrBC system or a derivative with mcrB alone indicated that the mutation shows an absolute defect for the restriction of DNA containing hydroxymethylcytosine and a thermosensitive defect for the restriction of DNA containing methylcytosine. The properties of the McrA temperature-sensitive mutants suggest that some of these mutations can also influence the restriction of DNA containing hydroxymethylcytosine or methylcytosine residues.     

Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.     

Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains.

Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli. We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues. This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E. coli cells for subsequent infection and propagation. This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E. coli locus responsible for this difference in efficiency. It was determined that the E. coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity. (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E. coli genome at one copy per chromosome. A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome. The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences. Removal of this DNA region including the mrr gene from E. coli K-12 strains allows high rescue efficiencies equal to those of E. coli C strains. These modified E. coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.     

Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage

The diversity of methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen-specific fingerprinting assay based on terminal restriction fragment length polymorphism (T-RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group-specific Sau96I restriction sites. Even genes for the isoenzyme methyl-coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T-RF). Both cloning and T-RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T-RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50 degrees C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T-RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC-I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T-RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC-I. Thus, we could demonstrate the genotype and phenotype of RC-I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.     

Characterization of the mcrBC region of Escherichia coli K-12 wild-type and mutant strains.

We have carried out an analysis of the Escherichia coli K-12 mcrBC locus in order to (1) elucidate its genetic organization, (2) to identify the proteins encoded by this region, and (3) to characterize their involvement in the restriction of DNA containing methylated cytosine residues. In vitro expression of recombinant plasmids carrying all or portions of the mcrBC region revealed that the mcrB and mcrC genes are organized as an operon. The mcrBC operon specifies five proteins, as evident from parallel in vitro and in in vivo expression studies. Three proteins of 53, 35 and 34 kDa originate from mcrB expression, while two proteins of 37 and 16 kDa arise from mcrC expression. Products of both the mcrB and mcrC genes are required to restrict the methylated substrate DNA used in this study. We also determined the nature of mutant mcrBC loci in comparison to the E. coli K-12 wild-type mcrBC locus. A major goal of these studies was to clarify the nature of the mcrB-1 mutation, which is carried by some strains employed in previous analyses of the E. coli K-12 McrBC system. Based on our analyses the mutant strains investigated could be divided into different complementation groups. The mcrB-1 mutation is a nonsense or frameshift mutation located within mcrB. It causes premature termination of mcrB gene product synthesis and reduces the level of mcrC gene expression. This finding helps to understand an existing conflict in the literature. We also describe temperature-sensitive McrA activity in some of the strains analysed and its relationship to the previously defined differences in the tolerance levels of E. coli K-12 mcrBC mutants to cytosine methylation.     

Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.     

Identification of a second polypeptide required for McrB restriction of 5-methylcytosine-containing DNA in Escherichia coli K12

Summary The McrB restriction system in Escherichia coli K12 causes sequence-specific recognition and inactivation of DNA containing 5-methylcytosine residues. We have previously located the mcrB gene near hsdS at 99 min on the E. coli chromosome and demonstrated that is encodes a 51 kDa polypeptide required for restriction of M.AluI methylated (A-G-5mC-T) DNA. We show here, by analysis of maxicell protein synthesis of various cloned fragments from the mcrB region, that a second protein of approximately 39 kDa is also required for McrB-directed restriction. The new gene, designated mcrC, is adjacent to mcrB and located distally to hsdS. The McrB phenotype has been correlated previously with restriction of 5-hydroxy-methylcytosine (HMC)-containing T-even phage DNA that lacks the normal glucose modification of HMC, formally designated RglB (for restriction of glucoseless phage). This report reveals a difference between the previously correlated McrB and RglB restriction systems: while both require the mcrB gene product only the McrB system requires the newly identified mcrC-encoded 39-kDa polypeptide.     

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Cloning the KpnI restriction-modification system in Escherichia coli

The genes encoding the KpnI restriction and modification (R-M) system from Klebsiella pneumoniae, recognizing the sequence, 5'-GGTAC decreases C-3', were cloned and expressed in Escherichia coli. Although the restriction endonuclease (ENase)- and methyltransferase (MTase)-encoding genes were closely linked, initial attempts to clone both genes as a single DNA fragment in a plasmid vector resulted in deletions spanning all or part of the gene coding for the ENase. Initial protection of the E. coli host with MTase expressed on a plasmid was required to stabilize a compatible plasmid carrying both the ENase- and the MTase-encoding genes on a single DNA fragment. However, once established, the MTase activity can be supplied in cis to the kpnIR gene, without an extra copy of kpnIM. A chromosomal map was generated localizing the kpnIR and kpnIM genes on 1.7-kb and 3.5-kb fragments, respectively. A final E. coli strain was constructed, AH29, which contained two compatible plasmids: an inducible plasmid carrying the kpnIR gene which amplifies copy number at elevated temperatures and a pBR322 derivative expressing M.KpnI. This strain produces approx. 10 million units of R.KpnI/g of wet-weight cells, which is several 1000-fold higher than the level of R.KpnI produced by K. pneumoniae. In addition, DNA methylated with M.KpnI in vivo does not appear to be restricted by the mcrA, mcrB or mrr systems of E. coli.     

Different mechanisms for SOS induced alleviation of DNA restriction in Escherichia coli.

The alleviation of DNA restriction during the SOS response in Escherichia coli has been further investigated. With the EcoK DNA restriction system UV irradiated wild-type cells show a 10(4)-fold increase in ability to plate non-modified lambda phage and a 3-4 fold increase in transformation by non-modified plasmid DNA. A role for the umuDC genes of E coli in the process of SOS-induced restriction alleviation was identified by showing that a umuC122::Tn5 mutant could alleviate EcoK restriction to only 5% that of wild-type levels. Although umuDC are better characterized for their pivotal role in SOS induced mutagenesis, it is demonstrated here that umu-dependent alleviation of EcoK restriction is a transient process in which umu-dependent mutagenesis plays little part. A second form of SOS induced alleviation of DNA restriction is described in this paper involving the McrA restriction system. The mcrA gene is shown to be encoded within a defective prophage called e14 situated at the 25 min region on the Escherichia coli genetic map. e14 is known to abortively excise from the chromosome after SOS induction and it is demonstrated in this report that mcrA is lost from the genome after SOS induction as part of e14. This results in co-ordinate decrease in the level of McrA restriction within a population of cells.     

Albinism in the domestic cat (Felis catus) is associated with a tyrosinase (TYR) mutation

Albino phenotypes are documented in a variety of species including the domestic cat. As albino phenotypes in other species are associated with tyrosinase (TYR) mutations, TYR was proposed as a candidate gene for albinism in cats. An Oriental and Colourpoint Shorthair cat pedigree segregating for albinism was analysed for association with TYR by linkage and sequence analyses. Microsatellite FCA931, which is closely linked to TYR and TYR sequence variants were tested for segregation with the albinism phenotype. Sequence analysis of genomic DNA from wild-type and albino cats identified a cytosine deletion in TYR at position 975 in exon 2, which causes a frame shift resulting in a premature stop codon nine residues downstream from the mutation. The deletion mutation in TYR and an allele of FCA931 segregated concordantly with the albino phenotype. Taken together, our results suggest that the TYR gene corresponds to the colour locus in cats and its alleles, from dominant to recessive, are as follows: C (full colour) > c(b) (burmese) > or = c(s) (siamese) > c (albino).     

Isolation of mutants in a DNA methyltransferase through mcrB-mediated restriction

A procedure has been developed that permits the positive selection of mutants in a DNA methyltransferase (MTase) gene. The stringency of this selection can be varied so as to yield null mutants only, or a mixture of null and partially defective mutants. The procedure was developed with the PvuII MTase gene (pvuIIM), which was subcloned into a bacteriophage lambda vector. Growth of this lambda pvuIIM construct on an mcrB+ host selected for non-methylating mutants, and the stringency of selection was proportional to the number of consecutive lytic cycles. Many cytosine MTases have been found to generate substrates for mcrB-mediated restriction, and this procedure should be applicable to a number of cytosine MTase genes.     

Development of a SCAR (sequence characterised amplified region) marker for molecular tagging of the dwarf BREIZH (Bzh) gene in Brassica napus L.

 A SCAR (sequence characterised amplified region) has been developed for optimal tagging of the dwarf Bzh gene in Brassica napus. A RAPD marker named OPMO7-730 was previously found closely linked to the dwarf locus at 0.8±0.7 cM. The DNA band corresponding to this marker was cloned and sequenced, and specific PCR primers were designed. The PCR test allowed us to amplify the locus corresponding to OPM07-730. With a restriction endonuclease digest and optimal electrophoresis conditions, three allelic forms of this marker have been recovered on the 40 B. napus accessions tested. The usefullness of this marker in breeding dwarf rapeseed lines is discussed. Received: 20 April 1998 / Accepted: 29 April 1998