Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.     

Cloning and complete nucleotide sequences of the type II restriction-modification genes of Salmonella infantis.

The complete type II restriction-modification system of Salmonella infantis was cloned in Escherichia coli as an R . Sau3AI fragment of 3,430 base pairs. The clone was shown to express the restriction endonuclease as well as the modification methylase. The nucleotide sequence of the above fragment showed two open reading frames of 461 and 230 codons in tail-to-tail orientation. These were shown to represent the modification methylase M . SinI and the restriction endonuclease R . SinI, respectively. The methylase M . SinI amino acid sequence revealed a considerable similarity to those of other deoxycytidylate methylases. In contrast, endonuclease R . SinI did not exhibit such a similarity to other restriction enzymes.     

The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

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.     

Sse9I restriction-modification system: Organization of genes and structural comparison of proteins

The nucleotide sequence was established for the operon of the Sse9I type II restriction-modification system of Sporosarcina species 9D. The enzymes of the Sse9I system recognize the 5′-AATT-3′ tetranucleotide. The operon includes three genes, sse9IC-sse9IR-sse9IM, which are transcribed unidirectionally and code, respectively, for the controller protein (C.Sse9I), restriction endonuclease (R.Sse9I), and DNA methyltransferase (M.Sse9I). The region immediately upstream of sse9IC was found to contain a conserved nucleotide sequence (C box) providing a binding site for C. Sse9I. The amino acid sequences of C.Sse9I and R.Sse9I were compared with those of related proteins. In the case of R.Sse9I, the highest homology was observed with the R.MunI (5′-CAATTG-3′) and R.EcoRI (5′-GAATTC-3′) regions that harbor the amino acid residues involved in recognizing the AATT inner tetranucleotide. The sse9IR gene was cloned in an expression vector, and recombinant R.Sse 9I was isolated.     

The corrected nucleotide sequences of the TaqI restriction and modification enzymes reveal a thirteen-codon overlap.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.     

The EcoDXX1 restriction and modification system: Cloning the genes and homology to type I restriction and modification systems

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and Ecob. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.     

The nucleotide sequence recognised by the Escherichia coli D type I restriction and modification enzyme.

A type I restriction endonuclease from a new isolate of Escherichia coli (E. coli E166) has been purified and characterised. The enzyme, EcoD, has a recognition sequence similar in overall structure to the previously determined type I enzyme sequences, an exception being that it is degenerate. The sequence is 5'-T-T-A-N-N-N-N-N-N-N-G-T-C-Y-3' 3'-A-A-T-N-N-N-N-N-N-N-C-A-G-R-5' where Y is a pyrimidine, R is a purine and N can be any nucleotide. The enzyme methylates adenosyl residues in both strands of the DNA that are separated by ten base pairs, suggesting that the enzyme interacts with DNA along one face of the helix making contacts in two successive major grooves.     

Cloning and characterization of the genes encoding the MspI restriction modification system.

The genes encoding the MspI restriction modification system, which recognizes the sequence 5' CCGG, have been cloned into pUC9. Selection was based on expression of the cloned methylase gene which renders plasmid DNA insensitive to MspI cleavage in vitro. Initially, an insert of 15 kb was obtained which, upon subcloning, yielded a 3 kb EcoRI to HindIII insert, carrying the genes for both the methylase and the restriction enzyme. This insert has been sequenced. Based upon the sequence, together with appropriate subclones, it is shown that the two genes are transcribed divergently with the methylase gene encoding a polypeptide of 418 amino acids, while the restriction enzyme is composed of 262 amino acids. Comparison of the sequence of the MspI methylase with other cytosine methylases shows a striking degree of similarity. Especially noteworthy is the high degree of similarity with the HhaI and EcoRII methylases.     

Cloning and characterization of genes for the PvuI restriction and modification system.

The genes encoding the endonuclease and the methylase of the PvuI restriction and modification system were cloned in E.coli and characterized. The genes were adjacent in tandem orientation spanning a distance of 2200 bases. The PvuI endonuclease was a single polypeptide with a calculated molecular weight of 27,950 daltons. The endonuclease was easily detectable when the gene was expressed from its endogenous promotor and present on a low copy plasmid, but expression was considerably enhanced when the endonuclease gene was placed under the control of a strong promoter on a high copy plasmid. The methylase did not completely protect plasmid DNA from R.PvuI digestion until the methylase gene was placed under lac promotor control in a multicopy plasmid. In the absence of the M.PvuI methylase, expression of the R.PvuI endonuclease from the lac promotor on a multicopy plasmid was not lethal to wild type E.coli, but was lethal in a temperature-sensitive ligase mutant at the non-permissive temperature. Moreover, induction of the R.PvuI endonuclease under lambda pL promotor control resulted in complete digestion of the E.coli chromosome by R.PvuI.     

The HgaI restriction-modification system contains two cytosine methylase genes responsible for modification of different DNA strands.

A DNA fragment of about 3.4 kilobase pairs that expressed the HgaI modification activity was cloned from the chromosomal DNA of Haemophilus gallinarum, and its nucleotide sequence was determined. Two open reading frames (ORF) which could code for structurally similar proteins were identified in the upstream and middle regions and a truncated ORF in the downstream region in the same orientation. When the respective ORFs were separately cloned, the clones carrying the upstream and middle ORFs both expressed the modification activity, indicating that the two genes are involved in modification of the HgaI restriction-modification system. In order to determine the sites of modification precisely, the respective genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the HgaI recognition site was treated with each of these enzymes, and, after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to HgaI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by each gene product. Analysis of the species and positions of modified bases by the Maxam-Gilbert method further demonstrated that the gene products from the upstream and middle ORFs participated in methylation of the internal cytosine residues of the strands carrying 3'-CTGCG-5' and 5'-GACGC-3', respectively. We concluded that the HgaI modification system consisted of two cytosine methylase genes responsible for modification of different strands in the target DNA.     

Organization of restriction-modification systems.

The genes for over 100 restriction-modification systems have now been cloned, and approximately one-half have been sequenced. Despite their similar function, they are exceedingly heterogeneous. The heterogeneity is evident at three levels: in the gene arrangements; in the enzyme compositions; and in the protein sequences. This paper summarizes the main features of the R-M systems that have been cloned.     

Cloning, nucleotide sequence, and expression of the HincII restriction-modification system.

Two genes, coding for the HincII from Haemophilus influenzae Rc restriction-modification system, were cloned and expressed in Escherichia coli RR1. Their DNA sequences were determined. The HincII methylase (M.HincII) gene was 1,506 base pairs (bp) long, corresponding to a protein of 502 amino acid residues (Mr = 55,330). The HincII endonuclease (R.HincII) gene was 774 bp long, corresponding to a protein of 258 amino acid residues (Mr = 28,490). The amino acid residues predicted from the R.HincII and the N-terminal amino acid sequence of the enzyme found by analysis were identical. These methylase and endonuclease genes overlapped by 1 bp on the H. influenzae Rc chromosomal DNA. The clone, named E. coli RR1-Hinc, overproduced R.HincII. The R.HincII activity of this clone was 1,000-fold that from H. influenzae Rc. The amino acid sequence of M.HincII was compared with the sequences of four other adenine-specific type II methylases. Important homology was found between tne M.HincII and these other methylases.     

The nucleotide sequences of two leghemoglobin genes from soybean.

We present the complete nucleotide sequences of two leghemoglobin genes isolated from soybean DNA. Both genes contain three intervening sequences in identical positions. Comparison of the coding sequences with known amino-acid sequences of soybean leghemoglobins suggest that the two genes correspond to leghemoglobin C2 and leghemoglobin C3, respectively.     

Organization and nucleotide sequences of two lactococcal bacteriocin operons.

Two distinct regions of the Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6, each of which specified bacteriocin production as well as immunity, have been sequenced and analyzed by deletion and frameshift mutation analyses. On a 1.8-kb ScaI-ClaI fragment specifying low antagonistic activity, three open reading frames (ORFs) were present, which were organized in an operon. The first two ORFs, containing 69 and 77 codons, respectively, were involved in bacteriocin activity, whereas the third ORF, containing 154 codons, was essential for immunity. Primer extension analysis indicated the presence of a promoter upstream of the ORFs. Two ORFs were present on a 1.3-kb ScaI-HindII fragment specifying high antagonistic activity. The first ORF, containing 75 codons, specified bacteriocin activity. The second ORF, containing 98 codons, specified immunity. The nucleotide sequences of both fragments upstream of the first ORFs as well as the first 20 bp of the first ORF of both bacteriocin operons appeared to be identical.     

Cloning the BamHI restriction modification system.

BamHI, a Type II restriction modification system from Bacillus amyloliquefaciensH recognizes the sequence GGATCC. The methylase and endonuclease genes have been cloned into E. coli in separate steps; the clone is able to restrict unmodified phage. Although within the clone the methylase and endonuclease genes are present on the same pACYC184 vector, the system can be maintained in E. coli only with an additional copy of the methylase gene present on a separate vector. The initial selection for BamHI methylase activity also yielded a second BamHI methylase gene which is not homologous in DNA sequence and hybridizes to different genomic restriction fragments than does the endonuclease-linked methylase gene. Finally, the interaction of the BamHI system with the E. coli Dam and the Mcr A and B functions, have been studied and are reported here.     

Cloning type-II restriction and modification genes

We have cloned into Escherichia coli the genes for 38 type-II bacterial modification methyltransferases. The clones were isolated by selecting in vitro for protectively modified recombinants. Most of the clones modify their DNA fully but a substantial number modify only partially. In approximately one-half of the clones, the genes for the corresponding endonucleases are also present. Some of these clones restrict infecting phages and others do not. Clones carrying endonuclease genes but lacking methyltransferase genes have been found, in several instances, to be viable.