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J S Benner

Publications and source records attributed to J S Benner.

10 recordsLinked to original sources
PubMed1991

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.

Amino Acid Sequence↗
PubMed1991

Characterization and expression of the Escherichia coli Mrr restriction system.

The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified. The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E. coli strains that are Mrr+. A 2-kb EcoRI fragment from the plasmid pBg3 (B. Sain and N. E. Murray, Mol. Gen. Genet. 180:35-46, 1980) was cloned. The resulting plasmid restores Mrr function to mrr strains of E. coli. The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa. The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR. By using Southern blot analysis, E. coli RR1 and HB101 were found to lack the region containing mrr. The acceptance of various cloned methylases in E. coli containing the cloned mrr gene was tested. Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted. Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted. No simple consensus sequence causing restriction has been determined. The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined. The use of mrr strains of E. coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1988

The amino acid sequence of the eukaryotic DNA [N6-adenine]methyltransferase, M.CviBIII, has regions of similarity with the prokaryotic isoschizomer M.TaqI and other DNA [N6-adenine] methyltransferases.

The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) [Narva et al., Nucleic Acids Res. 15 (1987) 9807-9823] and M.TaqI (from the bacterium Thermus aquaticus) [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] have been determined recently. Both enzymes methylate adenine in the sequence TCGA. We have compared the predicted amino acid sequences of these two methyltransferases (MTases), with each other and with ten other N6 A-MTases and find regions of similarity. M.CviBIII and M.TaqI were most closely related followed by M.PaeR7, whose recognition sequence (CTCGAG) contains the M.TaqI/M.CviBIII recognition sequence TCGA, and M.PstI, whose recognition sequence is CTGCAG. All of the N6-MTases contain the sequence Asp/Asn-Pro-Pro-Tyr (B-P-P-Y) referred to by Hattman et al. [J. Bacteriol. 164 (1985) 932-937] as region IV. The predicted secondary structure of this region forms a finger-like structure ('beta finger') containing a beta-pleated sheet (...XXXB), two beta-turns (P-P) followed by another beta-pleated sheet [Y/FXXX...].

Amino Acid Sequence↗
PubMed1987

Cloning, sequencing and expression of the Taq I restriction-modification system.

The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.

Amino Acid Sequence↗
PubMed1986

Cloning the DdeI restriction-modification system using a two-step method.

DdeI, a Type II restriction-modification system from the gram-negative anaerobic bacterium Desulfovibrio desulfuricans, recognizes the sequence CTNAG. The system has been cloned into E. coli in two steps. First the methylase gene was cloned into pBR322 and a derivative expressing higher levels was constructed. Then the endonuclease gene was located by Southern blot analyses; BamHI fragments large enough to contain the gene were cloned into pACYC184, introduced into a host containing the methylase gene, and screened for endonuclease activity. Both genes are stably maintained in E. coli on separate but compatible plasmids. The DdeI methylase is shown to be a cytosine methylase. DdeI methylase clones decrease in viability as methylation activity increases in E. coli RR1 (our original cloning strain). Therefore the DdeI system has been cloned and maintained in ER1467, a new E. coli cloning strain engineered to accept cytosine methylases. Finally, it has been demonstrated that a very high level of methylation was necessary in the DdeI system for successful introduction of the active endonuclease gene into E. coli.

Bacteriophage lambda↗
PubMed1985

Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products.

Bal31 deletion experiments on clones of the PaeR7 restriction-modification system from Pseudomonas aeruginosa demonstrate that it is arranged as an operon, with the methylase gene preceding the endonuclease gene. The DNA sequence of this operon agrees with in vitro transcription-translation assays which predict proteins of 532 amino acids, Mr = 59,260 daltons, and 246 amino acids, Mr = 27,280 daltons, coincident with the methylase and endonuclease genes, respectively. These predicted values coincide with the measured molecular weights of the purified, denatured PaeR7 endonuclease and methylase proteins. The first twenty amino acids from the amino-terminus of the purified endonuclease exactly match those predicted from the DNA sequence. Finally, potential regulatory mechanisms for the expression of phage restriction are described based on the properties of several PaeR7 subclones.

Amino Acid Sequence↗