Cloning and sequence analysis of the genes coding for Eco57I type IV restriction-modification enzymes.

A 6.3 kb fragment of E.coli RFL57 DNA coding for the type IV restriction-modification system Eco57I was cloned and expressed in E.coli RR1. A 5775 bp region of the cloned fragment was sequenced which contains three open reading frames (ORF). The methylase gene is 1623 bp long, corresponding to a protein of 543 amino acids (62 kDa); the endonuclease gene is 2991 bp in length (997 amino acids, 117 kDa). The two genes are transcribed convergently from different strands with their 3'-ends separated by 69 bp. The third short open reading frame (186 bp, 62 amino acids) has been identified, that precedes and overlaps by 7 nucleotides the ORF encoding the methylase. Comparison of the deduced Eco57I endonuclease and methylase amino acid sequences revealed three regions of significant similarity. Two of them resemble the conserved sequence motifs characteristic of the DNA[adenine-N6] methylases. The third one shares similarity with corresponding regions of the PaeR7I, TaqI, CviBIII, PstI, BamHI and HincII methylases. Homologs of this sequence are also found within the sequences of the PaeR7I, PstI and BamHI restriction endonucleases. This is the first example of a family of cognate restriction endonucleases and methylases sharing homologous regions. Analysis of the structural relationship suggests that the type IV enzymes represent an intermediate in the evolutionary pathway between the type III and type II enzymes.     

Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase

The genes for FokI, a type-IIS restriction-modification system from Flavobacterium okeanokoites (asymmetric recognition sequence: 5'-GGATG/3'-CCTAC), were cloned into Escherichia coli. Recombinants carrying the fokIR and fokIM genes were found to modify their DNA completely, and to restrict lambdoid phages weakly. The nt sequences of the genes were determined, and the probable start codons were confirmed by aa sequencing. The FokI endonuclease (R · FokI) and methyltransferase (M · FokI are encoded by single, adjacent genes, aligned in the same orientation, in the order M then R. The genes are large by the standards of type-II systems, 1.9 kb for the M gene, and 1.7 kb for the R gene. Preceding each gene is a pair of FokI recognition sites; it is conceivable that interactions between the sites and the FokI proteins could regulate expression of the genes. The aa sequences of the N- and C-terminal halves of M · FokI are similar to one another, and to certain other DNA-adenine methyltransferases, suggesting that the enzyme has a ‘tandem’ structure, such as could have arisen by the fusion of a pair of adjacent, ancestral M genes. Truncated derivatives of M · FokI were constructed by deleting the 5'- or 3' -ends of the fokIM gene. Deleting most of the C-terminus of M · FokI produced derivatives that methylated only the top (GGATG) strand of the recognition sequence. Conversely, deleting most of the N-terminus produced derivatives that methylated only the bottom (CATCC) strand of the recognition sequence. These results indicate that the domains in M · FokI for methylating the two strands of the recognition sequence are largely separate.     

Cloning and analysis of the four genes coding for Bpu10I restriction-modification enzymes.

The Bpu 10I R-M system from Bacillus pumilus 10, which recognizes the asymmetric 5'-CCTNAGC sequence, has been cloned, sequenced and expressed in Escherichia coli . The system comprises four adjacent, similarly oriented genes encoding two m5C MTases and two subunits of Bpu 10I ENase (34.5 and 34 kDa). Both bpu10IR genes either in cis or trans are needed for the manifestation of R. Bpu 10I activity. Subunits of R. Bpu 10I, purified to apparent homogeneity, are both required for cleavage activity. This heterosubunit structure distinguishes the Bpu 10I restriction endonuclease from all other type II restriction enzymes described previously. The subunits reveal 25% amino acid identity. Significant similarity was also identified between a 43 amino acid region of R. Dde I and one of the regions of higher identity shared between the Bpu 10I subunits, a region that could possibly include the catalytic/Mg2+binding center. The similarity between Bpu 10I and Dde I MTases is not limited to the conserved motifs (CM) typical for m5C MTases. It extends into the variable region that lies between CMs VIII and IX. Duplication of a progenitor gene, encoding an enzyme recognizing a symmetric nucleotide sequence, followed by concerted divergent evolution, may provide a possible scenario leading to the emergence of the Bpu 10I ENase, which recognizes an overall asymmetric sequence and cleaves within it symmetrically.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5′-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3′ end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Cloning and sequence comparison ofAvaI andBsoBI restriction-modification systems

AvaI andBsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5′CYCGRG3′ and cleave between the first C and second Y to generate a four-base 5′ extension. TheAvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned intoEscherichia coli by the methylase selection method. TheBsoBI restriction endonuclease gene (bsoBIR) and part of theBsoBI methylase gene (bsoBIM) were cloned by the “endo-blue” method (SOS induction assay), and the remainder ofbsoBIM was cloned by inverse PCR. The nucleotide sequences of the two restriction-modification (RM) systems were determined. Comparisons of the predicted amino acid sequences indicated thatAvaI andBsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity. Although the two systems show similarity in protein sequence, their gene organization differs. TheavaIM gene precedesavaIR in theAvaI RM system, while thebsoBIR gene is located upstream ofbsoBIM in theBsoBI RM system. BothAvaI andBsoBI methylases contain motifs conserved among the N⁴ cytosine methylases.     

Cloning and sequence of UK bovine rotavirus gene segment 7: marked sequence homology with simian rotavirus gene segment 8.

The genome of the UK bovine rotavirus, which consists of eleven segments of dsRNA was polyadenylated and reverse-transcribed into cDNA. Complementary cDNA strands were annealed and the termini of the duplexes completed using DNA polymerase I. Full-length DNA copies of RNA segments 7, 8 and 9 were cloned into the Pst I site of pBR322 and a clone containing the entire gene 7 was identified and sequenced. Gene 7 is 1059 nucleotides in length and contains a single long open reading frame capable of coding for a protein of 317 amino-acids. The known gene product of segment 7 is a protein with an estimated molecular weight of 33,000 daltons. When the UK bovine rotavirus gene 7 sequence was compared with the published data for the homologous gene (segment 8) of the simian rotavirus SA11, it was found to be identical to it in size and the arrangement of the proposed coding and non-coding regions, and very similar in nucleotide sequence (88% homology). Most of the base changes are silent and the predicted amino-acid sequences are almost identical (96% homology).     

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.     

Cloning and sequence analysis of the gene encoding Lactococcus lactis malolactic enzyme: Relationships with malic enzymes

Abstract Malolactic enzyme is the key enzyme in the degradation of L-malic acid by lactic acid bacteria. Using degenerated primers designed from the first 20 N-terminal amino acid sequence of lactococcal malolactic enzyme, a 60-bp DNA fragment containing part of the mleS gene was amplified from Lactococcus lactis in a polymerase chain reaction. This specific probe was used to isolate two contiguous fragments covering the gene as a whole. The 1.9-kb region sequenced contains an open reading frame of 1623 bp, coding a putative protein of 540 amino acids. The deduced amino acid sequence reveals that lactococcal putative protein (Mlep) is highly homologous to the malic enzyme of other organisms. Expression of the mleS gene in Escherichia coli results in malolactic activity.     

Cloning and sequence analysis of the gene encoding L-lactate dehydrogenase from Lactococcus lactis: evolutionary relationships between 21 different LDH enzymes.

Lactate dehydrogenase (LDH; EC1.1.1.27) is a key enzyme in the fermentation of milk by lactic acid bacteria used in the dairy industry. An 800-bp DNA fragment containing part of the gene (ldh) encoding LDH was amplified from Lactococcus lactis in a polymerase chain reaction using primers designed from the partial amino acid sequence of a lactococcal LDH. This fragment was radioactively labelled and used to probe a phage lambda library of Lc. lactis genomic DNA. Fragments containing ldh were subcloned from lambda to pUC13 and pUC18 and a 1.2-kb region was sequenced. The deduced aa sequence reveals that the lactococcal LDH is highly homologous to the LDHs of other organisms. The active site and several other domains of unknown function are highly conserved between all LDH enzymes (prokaryotic and eukaryotic). An evolutionary study of LDH sequences clearly divides the prokaryotic from the eukaryotic enzymes except for the Bifidobacterium longum LDH which anomalously groups with the eukaryotic enzymes. The LDHs from Gram-positive bacteria form a separate group from the enzymes from the Gram-negative organisms. The lactococcal LDH is phylogenetically closest to the streptococcal LDH.     

Cloning and sequence analysis of gyrA gene of Klebsiella pneumoniae.

The gene gyrA encoding the DNA gyrase A subunit of Klebsiella pneumoniae has been cloned in the plasmid pBR322. Bases of about 3.5 Kb DNA have been sequenced to locate the gyrA gene. An open reading frame of 2628 nucleotides coding for a 97 KD protein has been identified. Homology to the extent of about 85% was detected at the nucleotide level and about 90% at the amino acid level, when the sequences were compared with that of Escherichia coli gyrA. Some very interesting differences have, however, been found in the promoter region.     

The FokI restriction-modification system. II. Presence of two domains in FokI methylase responsible for modification of different DNA strands

Based on the previous findings that the FokI methylase (MFokI) consists of 647 amino acid residues and contains two copies of the segment specific for adenine methylase, Asp-Pro-Pro-Tyr, at amino acid positions 218-221 and 548-551, the role of these copies in the methylation reaction was investigated by introduction of a mutation into each segment. The MFokI gene was inserted into M13 vectors, and the Asp residues in the two segments were converted to Gly and Ala by oligonucleotide-directed mutagenesis. The wild-type and mutant genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the FokI 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 FokI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by the introduction of mutations into one of the two segments, and that the segments at the N-terminal and C-terminal moieties participated in modification of the strands carrying 5'-GGATG-3' and 3'-CCTAC-5', respectively. We concluded that MFokI contained two functional domains each of which was responsible for modification of different strands in the target DNA.     

Cloning and expression of the HpaI restriction-modification genes.

The genes from Haemophilus parainfluenzae encoding the HpaI restriction-modification system were cloned and expressed in Escherichia coli. From the DNA sequence, we predicted the HpaI endonuclease (R.HpaI) to have 254 amino acid residues (Mr 29,630) and the HpaI methyltransferase (M.HpaI) to have 314 amino acid residues (37,390). The R.HpaI and M.HpaI genes overlapped by 16 base pairs on the chromosomal DNA. The genes had the same orientation. The clone, named E. coli HB101-HPA2, overproduced R.HpaI. R.HpaI activity from the clone was 100-fold that from H. parainfluenzae. The amino acid sequence of M.HpaI was compared with those of other type II methyltransferases.     

Cloning and expression of the BalI restriction-modification system.

BalI, a type II restriction-modification (R-M) system from the bacterium, Brevibacterium albidum, recognizes the DNA sequence 5'-TGGCCA-3'. We cloned the genes encoding the BalI restriction endonuclease and methyltransferase and expressed them in Escherichia coli. The two genes were aligned tail-to-tail and their termination codons overlapped. BalI restriction endonuclease and methyltransferase comprise 260 and 280 amino acids, respectively, and have molecular weights of 29 043 and 31 999 Da. The amino acid sequence of BalI methyltransferase is similar to that of other m6A MTases, although it has been categorized as a m5C methyltransferase. A high expression system for the BalI restriction endonuclease was constructed in E. coli for the production of large quantities of enzyme.     

Evolution of sequence recognition by restriction-modification enzymes: selective pressure for specificity decrease

Several type II restriction-modification (RM) gene complexes kill host bacterial cells that have lost them, through attack on the chromosomal recognition sites of these cells. Two RM gene complexes recognizing the same sequence cannot simultaneously enjoy such stabilization through postsegregational host killing, because one will defend chromosomal sites from attack by the other. In the present work, we analyzed intrahost competition between two RM gene complexes when the recognition sequence of one was included in that of the other. When the EcoRII gene complex, recognizing 5'-CCWGG (W = A, T), is lost from the host, the SsoII gene complex, which recognizes 5'-CCNGG (N = A, T, G, C), will prevent host death by protecting CCWGG sites on the chromosome. However, when the SsoII (CCNGG) gene complex is lost, the EcoRII (CCWGG) gene complex will be unable to prevent host death through attack by SsoII on 5'-CCSGG (S = C, G) sites. These predictions were verified in our experiments, in which we analyzed plasmid maintenance, cell growth, cell shape, and chromosomal DNA. Our results demonstrate the presence of selective pressure for decrease in the specificity of recognition sequence of RM systems in the absence of invading DNA.     

Cloning and sequence analysis of the LPD-glc structural gene of Pseudomonas putida.

Pseudomonas putida is able to produce three lipoamide dehydrogenases: (i) LPD-glc, which is the E3 component of the pyruvate and 2-ketoglutarate dehydrogenase complexes and the L-factor for the glycine oxidation system; (ii) LPD-val, which is the specific E3 component of the branched-chain keto acid dehydrogenase complex and is induced by growth on leucine, isoleucine, or valine; and (iii) LPD-3, which was discovered in a lpdG mutant and whose role is unknown. Southern hybridization with an oligonucleotide probe encoding the highly conserved redox-active site produced three bands corresponding to the genes encoding these three lipoamide dehydrogenases. The complete structural gene for LPD-glc, lpdG, was isolated, and its nucleotide sequence was determined. The latter consists of 476 codons plus a stop codon, TAA. The structural gene for LPD-glc is preceded by a partial open reading frame with strong similarity to the E2 component of 2-ketoglutarate dehydrogenase of Escherichia coli. This suggests that lpdG is part of the 2-ketoglutarate dehydrogenase operon. LPD-glc was expressed in Pseudomonas putida JS348 from pHP4 which contains a partial open reading frame corresponding to the E2 component, 94 bases of noncoding DNA, and the structural gene for lpdG. This result indicates that lpdG can be expressed separately from the other genes of the operon.     

Cloning and sequence analysis of ornithine decarboxylase gene fragments from the Ascomycota.

Ornithine decarboxylase (ODC; EC 4.1.1.17) catalyzes the initial step in the biosynthesis of polyamines, the conversion of ornithine to putrescine. Based on the most conserved regions of fungal ODCs, we designed and synthesized oligonucleotides to amplify homologous fragments of three important plant pathogenic Pyrenomycete fungi (Ascomycota), Magnaporthe grisea, Colletotrichum lindemuthianum and Fusarium solani, and one insect pathogenic fungus Metarhizium anisopliae. Cloning and sequencing of the amplified fragments revealed homologies of between 37 to 88% with other fungal ODCs. The predicted peptide sequences were compared by Clustal analysis and conserved sequences corresponding to the substrate and cofactor binding sites were identified. Comparative analyses of the ODC fragments isolated in this study, revealed high homology between them (68.3-81.1%) and also with other Pyrenomycetes such as Neurospora crassa (order Sordariales; 68.6-72.9%) and Fusarium graminearum (order Hypocreales; 70.8-88.1%). Data obtained in this work revealed that these fungi constitute a compact group separated from other eukaryotic ODCs.     

ISHAN: sequence homology analysis package

Sequence based homology studies play an important role in evolutionary tracing and classification of proteins. Various methods are available to analyze biological sequence information. However, with the advent of proteomics era, there is a growing demand for analysis of huge amount of biological sequence information, and it has become necessary to have programs that would provide speedy analysis. ISHAN has been developed as a homology analysis package, built on various sequence analysis tools viz FASTA, ALIGN, CLUSTALW, PHYLIP and CODONW (for DNA sequences). This JAVA application offers the user choice of analysis tools. For testing, ISHAN was applied to perform phylogenetic analysis for sets of Caspase 3 DNA sequences and NF-kappaB p105 amino acid sequences. By integrating several tools it has made analysis much faster and reduced manual intervention.     

Cloning and nucleotide sequence analysis of the human beta-microseminoprotein gene

Beta-microseminoprotein (MSP) is a small protein (94 amino acids) synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. Restriction endonuclease mapping of human genomic DNA with a human MSP cDNA probe identified a 19 kilobase (Kb) hybridizing band in both EcoRI and BamHI digestions. Subsequently, the 19 Kb EcoRI fragment of human genomic DNA containing the MSP gene was isolated and cloned into an EMBL4 phage vector. Screening of the recombinant phages resulted in the isolation of one clone containing the MSP gene. Restriction endonuclease mapping and sequence analysis of this clone revealed the human MSP gene of approximately 15 Kb in length. The gene contains four exons and three large introns of approximately 6, 1, and 7 Kb.     

Cloning and sequence analysis of the muramidase-2 gene from Enterococcus hirae.

Extracellular muramidase-2 of Enterococcus hirae ATCC 9790 was purified to homogeneity by substrate binding, guanidine-HCl extraction, and reversed-phase chromatography. A monoclonal antibody, 2F8, which specifically recognizes muramidase-2, was used to screen a genomic library of E. hirae ATCC 9790 DNA in bacteriophage lambda gt11. A positive phage clone containing a 4.5-kb DNA insert was isolated and analyzed. The EcoRI-digested 4.5-kb fragment was cut into 2.3-, 1.0-, and 1.5-kb pieces by using restriction enzymes KpnI, Sau3AI, and PstI, and each fragment was subcloned into plasmid pJDC9 or pUC19. The nucleotide sequence of each subclone was determined. The sequence data indicated an open reading frame encoding a polypeptide of 666 amino acid residues, with a calculated molecular mass of 70,678 Da. The first 24 N-terminal amino acids of purified extracellular muramidase-2 were in very good agreement with the deduced amino acid sequence after a 49-amino-acid putative signal sequence. Analysis of the deduced amino acid sequence showed the presence at the C-terminal region of the protein of six highly homologous repeat units separated by nonhomologous intervening sequences that are highly enriched in serine and threonine. The overall sequence showed a high degree of homology with a recently cloned Streptococcus faecalis autolysin.