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B E Slatko

Publications and source records attributed to B E Slatko.

32 records · Page 2Linked 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↗
PubMed1990

Nucleotide sequence of the phage lambda gt11 SacI-KpnI lacZ region.

The nucleotide sequence of the lambda gt11 SacI-KpnI region, surrounding the unique EcoRI cloning site, was directly determined. This sequence previously had to be compiled from several diverse sources. The direct sequence confirms the sequence predicted from the compilation and pinpoints other unique restriction enzyme targets in the region for use in subcloning.

Bacteriophage lambda↗
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

M.FokI methylates adenine in both strands of its asymmetric recognition sequence.

M.FokI, a type-IIS modification enzyme from Flavobacterium okeanokoites, was purified, and its activity was characterized in vitro. The enzyme was found to be a DNA-adenine methyltransferase and to methylate both strands of the asymmetric FokI recognition sequence: (formula; see text) M.FokI does not methylate single-stranded DNA, nor does it methylate double-stranded DNA at sequences other than FokI sites.

Adenine↗
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

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

ATP-Binding Cassette Transporters↗
PubMed1988

Cloning and analysis of the HaeIII and HaeII methyltransferase genes.

The HaeIII methyltransferase (MTase) gene from Haemophilus aegyptius (recognition sequence: 5'-GGCC-3') was cloned into Escherichia coli in the plasmid vector pBR322. The gene was isolated on a single EcoRI fragment and on a single HindIII fragment. Clones carrying additional adjacent fragments were found to code also for the HaeII restriction endonuclease and HaeII modification MTase (recognition sequence: 5'-PuGCGCPy-3'). The sequence of the HaeIII modification gene was determined. The inferred amino acid sequence of the protein was found to share extensive similarity with other sequenced m5C-MTases. The central 'non-conserved' region of the M.HaeIII MTase, thought to form the nucleotide sequence-specificity domain, is almost identical to that of the M.BsuRI, M.BspRI and M.NgoPII MTases, which also recognize the sequence 5'-GGCC-3'.

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↗
PubMed1975

Elements causing male crossing over in Drosophila melanogaster.

A second chromosome line of Drosophila melanogaster (Symbol: T-007) has previously been shown to be responsible for the induction of male recombination. In the present investigation, the genetic elements responsible for this phenomenon have been partially identified and mapped. A major element (Symbol: Mr, for Male recombination) locates on the second chromosome between the pr (2L-54.4) and c (2R-75.5) loci and is responsible for the large majority of male recombination. In addition, there appear to be "secondary elements" present which have the ability to induce male recombination in much reduced frequencies and which are diluted out through successive back-cross generations when Mr is removed by recombination. The possible nature of these "secondary elements" is discussed.

Animals↗
PubMed1973

Mutation induction in the male recombination strains of Drosophila melanogaster.

One group of the second chromosome lines isolated from a southern Texas population of Drosophila melanogaster, which has been known to show relatively high frequencies of male recombinations, was found to increase the frequency of sex-linked recessive lethal mutations from a control frequency of 0.18% to 1.63%. The second group, which showed a very much reduced frequency of male recombinations, was found to cause a slight increase to 0.48%, although it was not statistically significant. The first group was also tested for the recessive lethal mutation frequency in the second chromosome; the frequency increased from a control frequency of 0.28% to 2.82%. Mapping of a portion of the sex-linked lethals indicated a distribution along the entire X chromosome, although there was a tendency of clustering towards the tip of the X chromosome. One sex-linked lethal line so far tested was found to be associated with an inversion (approximate breakpoints, 14A-18A). It was suggested that the element causing male recombination might be similar to the hi mutator gene studied earlier by Ives (1950).

Alleles↗