Role of DNA minor groove interactions in substrate recognition by the M.SinI and M.EcoRII DNA (cytosine-5) methyltransferases.

The SinI and EcoRII DNA methyltransferases recognize sequences (GG(A)/(T)CC and CC(A)/(T)GG, respectively), which are characterized by an (A)/(T) ambiguity. Recognition of the A.T and T.A base pair was studied by in vitro methyltransferase assays using oligonucleotide substrates containing a hypoxanthine.C base pair in the central position of the recognition sequence. Both enzymes methylated the substituted oligonucleotide with an efficiency that was comparable to methylation of the canonical substrate. These observations indicate that M.SinI and M.EcoRII discriminate between their canonical recognition site and the site containing a G.C or a C.G base pair in the center of the recognition sequence (GG(G)/(C)CC and CC(G)/(C)GG, respectively) by interaction(s) in the DNA minor groove. M.SinI mutants displaying a decreased capacity to discriminate between the GG(A)/(T)CC and GG(G)/(C)CC sequences were isolated by random mutagenesis and selection for the relaxed specificity phenotype. These mutations led to amino acid substitutions outside the variable region, previously thought to be the sole determinant of sequence specificity. These observations indicate that (A)/(T) versus (G)/(C) discrimination is mediated by interactions between the large domain of the methyltransferase and the minor groove surface of the DNA.

No mitochondrial haplotype was found to increase risk for Alzheimer's disease.

BACKGROUND: Seventy Alzheimer's disease (AD) patients and 80 age- and sex-matched controls were analyzed for mitochondrial mutations T4336C and A3397G, reported to be associated with AD, and for mutations T4216C/G13708A characteristic for a normal human haplotype associated with increased frequency of occurrence of some hereditary diseases. The distribution of apolipoprotein E (apoE) alleles was also analyzed. METHODS: Mitochondrial DNA was amplified by polymerase chain reaction, and the presence of mutations was detected by digestion with approximately chosen restriction endonucleases (restriction fragment length polymorphism). RESULTS: One patient and 2 controls were found to belong to the T4336C/T1630C haplotype. No A3397G mutant was detected. The T4216C/G13708A haplotype occurred at 5/70 and 5/80 frequency in the two groups. Prevalence of the apoE4 allele was significantly higher in AD patients (25%) than in the control group (8.1%). CONCLUSIONS: The T4336C/T16304C mutations were not found to associated with AD, and no predisposing mitochondrial haplotypes were found.

Mitochondrial mutation as a probable causative factor in familial progressive tubulointerstitial nephritis.

Renal biopsy of two children and a maternal relative, diagnosed with severe progressive tubulointerstitial nephritis, has shown the presence of distorted mitochondria. Mitochondrial DNA from the blood of these patients was analysed. No major deletions were found, but an A to G mutation was detected in position 5656. It is proposed that this mutation might play a causative role in the renal disease of the patients.

Purification of the KpnI DNA methyltransferase and photolabeling of the enzyme with S-adenosyl-L-methionine.

An Escherichia coli strain overproducing the KpnI DNA methyltransferase (M.KpnI) was constructed by cloning the kpnIM gene downstream from the inducible T7 phage luminal diameter 10 promoter. A method involving three chromatographic steps has been developed to purify M.KpnI to homogeneity. The purified enzyme has a pH optimum around 7.3 and is inhibited by salts. M.KpnI can be photolabeled by UV-irradiation of the enzyme in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet). Photolabeling results from a specific interaction between M.KpnI and AdoMet, as indicated by the dependence of photolabeling on native enzyme conformation and by the inhibitory effect of the AdoMet analogs, sinefungin and S-adenosyl-L-homocysteine (AdoHcy).

Self-methylation of the M.BspRI methyltransferase.

In the absence of DNA substrate, the DNA methyltransferase (MTase) M.BspRI can methylate itself using the methyl donor S-adenosyl-L-methionine (AdoMet). The methyl group is transferred to two Cys residues of the MTase.

Self-methylation of BspRI DNA-methyltransferase.

The DNA (cytosine-5)-methyltransferase (m5C-MTase) M.BspRI is able to accept the methyl group from the methyl donor S-adenosyl-L-methionine (AdoMet) in the absence of DNA. Transfer of the methyl group to the enzyme is a slow reaction relative to DNA methylation. Self-methylation is dependent on the native conformation of the enzyme and is inhibited by S-adenosyl-L-homocysteine, DNA and sulfhydryl reagents. Amino acid sequencing of proteolytic peptides obtained from M.BspRI, which had been methylated with [methyl-3H]AdoMet, and thin layer chromatography of the modified amino acid identified two cysteines, Cys156 and Cys181 that bind the methyl group in form of S-methylcysteine. One of the acceptor residues, Cys156 is the highly conserved cysteine which plays the role of the catalytic nucleophile of m5C-MTases.

Kinetic characterization of the EcaI methyltransferase.

A kinetic analysis of the EcaI adenine-N6-specific methyltransferase (MTase) is presented. The enzyme catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the adenine of the GGTNACC sequence with a random rapid-equilibrium mechanism. Experiments with a synthetic, 14-bp DNA substrate suggest that recognition of the specific site of DNA occurs after the binding of AdoMet. Proton concentration does not affect the dissociation constant of AdoMet while Vm and the dissociation constant of DNA show a maximum around pH 8. Increasing the amount of S-adenosyl-L-homocysteine decreases the inhibitory effect of methylated DNA which proves the active role of AdoMet in site recognition. Experiments with hemimethylated DNA show that the methylase binds the double-stranded DNA asymmetrically.

An Escherichia coli gene in search of a function: phenotypic effects of the gene recently identified as murI.

Earlier we reported that an open reading frame located at 89.5 min of the Escherichia coli map (ORFI) codes for a protein of unknown function that could be overexpressed and purified to homogeneity (G. Balikó, A. Raukas, I. Boros, and P. Venetianer, Mol. Gen. Genet. 211:326-331, 1988). In the work described here, we attempted to learn the function of this protein by inactivating the chromosomal gene and providing it or its deletion derivatives on temperature-sensitive plasmids. We found that the presence of the functional ORFI gene is essential; cells are not viable at the nonpermissive temperature or when the region coding for the C-terminal 50 amino acids of the protein is deleted. At intermediate temperatures or when the gene is overexpressed, characteristic changes occur in cell morphology, nucleoid separation during cell division, and supercoiling of plasmids. The possible mechanisms of these effects are discussed in view of the fact that Doublet et al. (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992) recently identified the ORFI gene as murI, involved in D-glutamic acid biosynthesis.

Purification and biochemical characterization of the EcaI DNA methyltransferase.

The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25 degrees C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The Km was 170 nM for DNA and 1.8 microM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 microM). The S-adenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.

Analysis of the complex transcription termination region of the Escherichia coli rrnB gene.

The complex terminator region of the Escherichia coli rrnB gene was analyzed by subcloning the terminators T1 and T2 and the inverted repeats IR1 and IR2 individually, or in various combinations, in a normal or inverted orientation into a terminator probe vector. The in vivo terminating efficiency was assayed by measuring the galactokinase activity encoded by the downstream galK gene. Termination efficiencies of all fragments were compared in two constructs, differing in the presence or absence of readthrough translation over the investigated terminator signal. The following main conclusions were drawn. (a) T1 and T2 are both efficient terminators in isolated forms. (b) IR1 and IR2 have some terminating effect (much lower than the proper terminators), especially in the inverted orientation. Their presence modifies the effect of the proper terminators in a quite unpredictable way, especially if these regions are translated. (c) The terminators are not symmetrical; in the inverted orientation T1 is practically inactive and T2 termination is reduced. (d) Translation radically decreases the efficiency of the terminators. (e) Several sequences in the rrnB gene, upstream of the terminator region (one in the 16S RNA and one in the 5S RNA coding region), are very efficient in vivo terminators in the inverted orientation.

Complementation by detached parts of GGCC-specific DNA methyltransferases.

Individually inactive N- and C-terminal fragments of the m5C-methyltransferase M.BspRI can complement each other resulting in specific, in vivo methylation of the DNA. This was shown by cloning the coding regions for N- and C-terminal parts of the enzyme in compatible plasmids and co-transforming them into E.coli cells. The enzyme could be detached at several different sites, producing either non-overlapping or partially overlapping fragments capable of complementation. Reconstitution of the active methyltransferase from inactive fragments was demonstrated in vitro, as well. Another GGCC-specific methyltransferase, M.BsuRI, showed a similar complementation phenomenon. Moreover, interspecies complementation was observed between appropriate fragments of the two closely related enzymes M.BspRI and M.BsuRI. Fragments of structurally and functionally more different methyltransferases were unable to complement each other.

Increased mutation rate in bacteria overexpressing a cloned foreign gene.

Escherichia coli cells growing slowly as a result of the overexpression of a cloned foreign gene were shown to exhibit an increased mutation rate in the foreign gene as well as in several non-selected markers. This phenomenon is discussed in terms of the model proposed by Hall (1990).

A family of expression vectors based on the rrnB P2 promoter of Escherichia coli.

We describe here the construction of a family of expression vectors, based on the P2 promoter of the Escherichia coli rrnB gene by removing regulatory sequences downstream of the Pribnow-box and replacing them with the lac operator. These vectors allow cloning of foreign genes in such a way that their products are synthesized either in the form of fusion proteins of different length, or without fusion partners, with or without the original translational initiation signals. One of the vectors contains a synthetic oligothreonine-coding sequence that helps to stabilize the product of the cloned gene. These vectors allow high-level regulated expression of foreign genes, even if their products are relatively short peptides.

Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes.

The genes coding for the GGNCC specific Sau96I restriction and modification enzymes were cloned and expressed in E. coli. The DNA sequence predicts a 430 amino acid protein (Mr: 49,252) for the methyltransferase and a 261 amino acid protein (Mr: 30,486) for the endonuclease. No protein sequence similarity was detected between the Sau96I methyltransferase and endonuclease. The methyltransferase contains the sequence elements characteristic for m5C-methyltransferases. In addition to this, M.Sau96I shows similarity, also in the variable region, with one m5C-methyltransferase (M.SinI) which has closely related recognition specificity (GGA/TCC). M.Sau96I methylates the internal cytosine within the GGNCC recognition sequence. The Sau96I endonuclease appears to act as a monomer.

Regulatory elements downstream of the promoter of an rRNA gene of E. coli.

Previously we have shown that plasmid constructs carrying a reporter gene fused to the P2 promoter of the E. coli rrnB gene exhibited a strange two-phase kinetics of expression depending on the physiological conditions of the cell if a short DNA region downstream of the promoter was present between the promoter and the reporter gene (Lukacsovich et al. (1987) J. Bacteriol. 169, 272-277). Insertion of a synthetic oligonucleotide corresponding to the first half of this region into constructs where the reporter directly follows the promoter, leads to a complete block of expression in vivo, while in vitro--in a purified system--transcription is not inhibited. Band-shift experiments indicate that the putative regulatory region downstream of the promoter specifically binds protein(s) present in total bacterial extracts.

New approaches to increase the expression and stability of cloned foreign genes in Escherichia coli.

A family of expression plasmid vectors were constructed by fusing the strong P2 promoter of the rrnB gene of Escherichia coli (coding for ribosomal RNA) to the lac operator, thereby eliminating regulatory sequences from the rrnB gene and placing the expression under lac repressor control. This promoter proved to be stronger in vivo than the well-known consensus tac promoter, and its strength could be further increased by converting the sequence to consensus. The stability of the recombinant proteins could be increased by fusion to various lengths of the N-terminal end of beta-galactosidase, or by inserting a synthetic oligonucleotide, coding for heptathreonine. A new method was developed for the stabilization of recombinant plasmids without antibiotic selection, based on the presence of an essential gene on the plasmid and its absence from the chromosome. The application of this method is illustrated by the example of a plasmid expressing human proinsulin.

Cloning and nucleotide sequence of the gene encoding the Ecal DNA methyltransferase.

The gene coding for the GGTNACC specific Ecal DNA methyltransferase (M.Ecal) has been cloned in E. coli from Enterobacter cloacae and its nucleotide sequence has been determined. The ecalM gene codes for a protein of 452 amino acids (Mr: 51,111). It was determined that M.Ecal is an adenine methyltransferase. M.Ecal shows limited amino acid sequence similarity to other adenine methyltransferases. A clone that expresses Ecal methyltransferase at high level was constructed.

A new procedure for the targeted inactivation of essential bacterial genes.

A new method is described for the exchange of a plasmid encoded mutant gene with its chromosomal counterpart. The method is based on positive selection and is applicable for the exchange of essential genes. The main features of the method are: (1) cloning of an antibiotic resistance marker (without its promoter) downstream of the cloned target gene, thus forming an artificial operon; (2) inactivating the target gene (and consequently also the antibiotic resistance gene) by inserting a strong transcriptional termination signal into it; and (3) selection for double recombinants by means of the antibiotic resistance gene.

The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli.

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.