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S Chai

Publications and source records attributed to S Chai.

33 records · Page 2Linked to original sources
PubMed1994

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M. phi 3TI and M. rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M. phi 3TII and M. rho 11sII. These enzymes modify the C of TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M. phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M. phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M. phi 3TII does not show pronounced similarity to M. phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M. phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.TaqI--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.

Amino Acid Sequence↗
PubMed1994

M.phi 3TII: a new monospecific DNA (cytosine-C5) methyltransferase with pronounced amino acid sequence similarity to a family of adenine-N6-DNA-methyltransferases.

The temperate B.subtilis phages phi 3T and rho 11s code, in addition to the multispecific DNA (cytosine-C5) methyltransferases (C5-MTases) M.phi 3TI and M.rho 11sI, which were previously characterized, for the identical monospecific C5-MTases M.phi 3TII and M.rho 11sII. These enzymes modify the C to TCGA sites, a novel target specificity among C5-MTases. The primary sequence of M.phi 3TII (326 amino acids) shows all conserved motifs typical of the building plan of C5-MTases. The degree of relatedness between M.phi 3TII and all other mono- or multispecific C5-MTases ranges from 30-40% amino acid identity. Particularly M.phi 3TII does not show pronounced similarity to M.phi 3TI indicating that both MTase genes were not generated from one another but were acquired independently by the phage. The amino terminal part of the M.phi 3TII (preceding the variable region 'V'), which predominantly constitutes the catalytic domain of the enzyme, exhibits pronounced sequence similarity to the amino termini of a family of A-N6-MTases, which--like M.Taql--recognize the general sequence TNNA. This suggests that recently described similarities in the general three dimensional organization of C5- and A-N6-MTases imply divergent evolution of these enzymes originating from a common molecular ancestor.

Amino Acid Sequence↗
PubMed1994

Analysis of the Bacillus subtilis bacteriophages SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging.

Gene 1 product (G1P) of the related Bacillus subtilis bacteriophages SPP1, SF6, and rho 15 is essential for DNA maturation and packaging. A DNA segment containing gene 1 of phage SF6 or rho 15 origin was cloned and sequenced. SF6 and rho 15 G1P (both with predicted molecular mass of 16.7 kDa) share 71% identity with G1P of SPP1. The G1P of all three phages contains three conserved segments (I, II, and III). Within segments I and II helix-turn-helix DNA binding and nucleotide binding motifs were predicted. G1P of both SPP1 and SF6 origin was purified. SPP1 G1P protein (20.7 kDa), purified from cells overexpressing the cloned gene, purifies together with another polypeptide, having a molecular mass of about 13 kDa. The 13-kDa polypeptide results from a translation start signal within gene 1, and hence was termed SPP1 G1P*. G1P of both SPP1 and SF6 binds specifically to a pac-containing DNA fragment, whereas G1P*, which lacks segment I, does not. Chimeric G1P proteins were obtained by domain swapping between gene 1 of SPP1 and SF6. The results presented here suggest that the G1P DNA binding motif lies in segment I and the major determinant for G1P::G1P interaction might lie in segment II. Segment III and the extended C-terminal part of SPP1 G1P are dispensable. The G1P::G2P interacting region remains uncharacterized.

Adenosine Triphosphatases↗
PubMed1994

Nucleotide sequence and complementation studies of the gene 35 region of the Bacillus subtilis bacteriophage SPP1.

Genetic analysis of the Bacillus subtilis bacteriophage SPP1 defective in gene 35 shows that the gene 35 product (G35P) is essential for SPP1 growth. The defect in growth of SPP1tsl17 and SPP1tsl20F at nonpermissive temperature is overcome by wild-type gene 35 expressed from a plasmid. The region where gene 35 maps was cloned and sequenced. Analysis of the nucleotide sequence (5884-bp) around gene 35 revealed 13 open reading frames (orfs). We have assigned the term gene to three of these orfs; gene 35, gene 36, the product of which shares homology with SSB proteins, and gene 38, given the gene order orf 34-orf 34.1-orf 34.2-orf34.3-orf34.4-gene 35-gene 36-orf 36.1-orf 37-orf 37.1-orf 37.2-orf 37.3-gene 38. Gene 35 encodes a protein of 32.0 kDa. By using the T7 promoter-expression system for gene 35 a radioactive band of the expected molecular mass was detected.

Bacillus Phages↗
PubMed1994

Analysis of cis and trans acting elements required for the initiation of DNA replication in the Bacillus subtilis bacteriophage SPP1.

The development of SPP1 has been studied in several B. subtilis mutants conditionally defective in initiation of DNA replication. Initiation of SPP1 replication is independent of the host DnaA (replisome organizer), DnaB, DnaC and DnaI products, but requires the DnaG (DNA primase) and the DNA gyrase. Furthermore, SPP1 replication is independent of the DnaK (heat shock) protein. The phage-encoded products required for initiation of SPP1 replication have been genetically characterized. Analysis of the nucleotide sequence (3.292 kilobases) of the region where SPP1 initiation replication mutants map, revealed five open reading frames (orf). We have assigned genes 38, 39 and 40 to three of these orfs, which have the successive order gene 38-gene 39-orf39,1-gene 40-orf41. The direction of transcription of the reading frames, the lengths of the mRNAs as well as the transcription start point, upstream of gene 38 (PE2), were identified. Proteins of 29.9, 14.6 and 46.6 kDa were anticipated from translation of gene 38, gene 39 and gene 40, respectively. The purified G38P and G39P have estimated molecular masses of 31 and 15 kDa. G38P and G39P do not share significant identity with primary protein sequences currently available in protein databases, whereas G40P shares substantial homology with a family of DNA primase-associated DNA helicases. G38P binds specifically to two discrete SPP1 DNA restriction fragments (EcoRI-4 and EcoRI-3). The G38P binding site on EcoRI-4 was localized on a 393 bp DNA segment, which lies within the coding sequence of gene 38. The putative binding site on EcoRI-3 was inferred by DNA sequence homology, it maps in a non-coding segment. G39P, which does not bind to DNA, is able to form a complex with G38P. The organization of the SPP1 genes in the gene 38 to gene 40 interval resembles that one found in the replication origin regions of different Escherichia coli double-stranded DNA phages (lambda, phi 80 and P22). We propose that the conserved gene organization is representative of the replication origin region of a primordial phage.

Amino Acid Sequence↗
PubMed1994

[Cloning and sequencing of promoter and signal sequence coding regions from Bacillus subtilis].

Promoter and signal sequence coding regions from B. subtilis were cloned in E. coli using a bifunctional and signal sequence selection plasmid pGPB14 as a vector. The Sau3A digested chromosome DNA was ligated with BamHI digested pGPB14. The ligated mixture was used to transform E. coli C600. Ampicillin and erythromycin resistant clones were selected. Recombinant plasmids were isolated from double resistant transformants. Restriction analysis showed that inserts of various length had been cloned and these inserts rendered the E. coli cells resistant to different concentrations of ampicillin. The recombinant plasmids were transformed and showed the same secretion function in B. subtilis. The amount and localization of beta-lactamase were determined in transformed E. coli and B. subtilis. The results indicate that the beta-lactamase activities of E. coli mainly in periplasm while the enzyme produced by B. subtilis secreted extracellularly. 10 of the cloned fragments were sequenced by Sanger's dideoxy chain termination method. The sequencing data show that all fragments contain promoter, ribosome binding site and signal sequence coding region.

Amino Acid Sequence↗
PubMed1993

Analysis of the stabilization system of pSM19035-derived plasmid pBT233 in Bacillus subtilis.

The low-copy-number, 9.0-kb pSM19035-derived plasmid pBT233, is stably inherited in Bacillus subtilis. The complete nucleotide (nt) sequence of pBT233 has been determined. Analysis of the nt sequence revealed nine major open reading frames (orfs). The repS, erm1 and erm2 genes have been assigned to three of these orfs, and given the gene order, repS-orf alpha-orf beta-orf gamma-orf delta-orf epsilon-orf zeta-erm2-erm1. The organization of genes of the repS-orf gamma region resembles the organization of genes in the repE-orfI region of pAM beta 1. Messenger RNA species of molecular weights corresponding to repS, orf alpha + orf beta, orf gamma, orf delta and orf epsilon + orf zeta were detected by Northern blotting. Proteins of 23.8, 81.3, 34.4, 10.7 and 32.4 kDa correspond to Orfs beta, gamma, delta, epsilon and zeta, respectively. Bands of radioactive proteins of 25, 81, 34, 10 and 32 kDa were detected using the T7 promoter-expression system. The orf beta and orf gamma encode proteins that share homology to site-specific recombinases and type-I topoisomerases, respectively. The orfs, delta, epsilon and zeta, encode proteins with unknown activity. Deletion of a 1.5-kb segment (nt 2999-4552) with coding capacity for orf beta, orf gamma and orf delta does not seem to affect plasmid maintenance. Removal of a 3.0-kb fragment (nt 4598-7689) with coding capacity for orf epsilon and orf zeta reduced plasmid segregational stability, but deletion of a 5.2-kb DNA segment (nt 2546-7826) abolished it.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Sequence analysis of the left end of the Bacillus subtilis bacteriophage SPP1 genome.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.

Amino Acid Sequence↗
PubMed1993

Characterization of the effectors required for stable inheritance of Streptococcus pyogenes pSM19035-derived plasmids in Bacillus subtilis.

The low-copy-number and broad-host-range pSM19035-derived plasmid pBT233 is stably inherited in Bacillus subtilis cells. Two distinct regions, segA and segB, enhance the segregational stability of the plasmid. Both regions function in a replicon-independent manner. The maximization of random plasmid segregation is accomplished by the recombination proficiency of the host or the presence of the pBT233 segA region. The segA region contains two open reading frames (orf) [alpha and beta]. Inactivation or deletion of orf beta results in SegA- plasmids. Better than random segregation requires an active segB region. The segB region contains two orfs (orf epsilon and orf zeta). Inactivation of either of the orfs does not lead to an increase in cell death, but orf zeta- plasmids are randomly segregated. These results suggest that pBT233 stabilization relies on a complex system involving resolution of plasmid oligomers (segA) and on the function(s) encoded by the segB region.

Bacillus subtilis↗
PubMed1993

Potassium channel antagonists and vascular reactivity in stroke-prone spontaneously hypertensive rats.

The goal of this study was to characterize differences in contractile responsiveness to several potassium channel antagonists in vascular smooth muscle from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY). Helically-cut strips of carotid arteries (endothelium removed) from SHRSP and WKY were mounted in muscle baths for measurement of isometric force generation. Contractile responses to tetraethylammonium (10(-4) to 3 x 10(-2) mol/L) and barium (3 x 10(-5) mol/L), blockers of the voltage-dependent and large conductance, calcium activated potassium channels, were greater in carotid arteries from SHRSP than in those from WKY. In contrast, contractile responses to the voltage-dependent potassium channel blockers 3,4-diamino-pyridine (10(-6) to 3 x 10(-3) mol/L) and sparteine (10(-6) to 3 x 10(-2) mol/L) in arteries from SHRSP did not differ from WKY values. Carotid arteries from SHRSP and WKY did not contract to apamin (10(-9) to 10(-6) mol/L), an antagonist of the small conductance, calcium activated potassium channel. Furthermore, relaxation responses to diazoxide (3 x 10(-4) mol/L), an activator of the ATP-sensitive potassium channel, and subsequent contractions to the ATP-sensitive potassium channel blocker glyburide (10(-8) to 3 x 10(-6) mol/L) in arteries from SHRSP did not differ from WKY values. Carotid artery segments from SHRSP were more sensitive to the contractile effects of elevated potassium than those from WKY. We conclude that altered activity of the large conductance, calcium activated potassium channel may play a role in the increased responsiveness observed in arteries from SHRSP.

4-Aminopyridine↗
PubMed1992

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Amino Acid Sequence↗
PubMed1992

Extracellular calcium, contractile activity and membrane potential in tail arteries from genetically hypertensive rats.

OBJECTIVE AND DESIGN: This study compares the effect of extracellular calcium on contractile responsiveness and membrane potential (E(m)) in arteries from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive (WKY) rats. METHODS: Isometric force and E(m) were measured in isolated tail artery strips using standard muscle bath and microelectrode techniques, respectively. RESULTS: The resting contractile force in SHRSP and WKY arteries was not influenced by the extracellular calcium concentration. However, the rate of force development in response to norepinephrine (3 x 10(-8) mol/l) was slowed when calcium was elevated and increased when calcium was reduced. Compared with WKY rats, this stabilizing action of calcium on contractions to norepinephrine was reduced in SHRSP. In 1.6 mmol/l calcium, resting E(m) in SHRSP did not differ from that in WKY rats. Calcium-free buffer caused depolarization in SHRSP and WKY rats. Reductions in calcium below physiological levels resulted in depolarization, whereas elevations in calcium caused hyperpolarization. Regardless of the calcium concentration, E(m) values in SHRSP did not differ from those in WKY rats. Norepinephrine (3 x 10(-8) mol/l) caused a depolarization in WKY rat and SHRSP arteries, and the magnitude of this depolarization was not influenced by calcium. Endothelium removal did not alter the stabilizing effects of calcium on the membrane potential or contractile activity in WKY rats or SHRSP. CONCLUSIONS: The reduced stabilizing effect of calcium on the contractile activity in SHRSP arteries is not due to an alteration in the general effect of the cation on the membrane potential.

Animals↗
PubMed1990

Protective anti-sporozoite antibodies induced by a chemically defined synthetic vaccine.

Chemically defined synthetic polymers, known as multiple antigen peptide systems (MAPs) represent an effective and novel approach for engineering peptide-based vaccines. Ten different mono and diepitope MAP models, containing different arrangements and stoichiometry of functional B and/or T helper epitopes from the circumsporozoite protein of Plasmodium berghei were used to immunize mice. High titers of antibody and protective immunity against sporozoite challenge were elicited by MAPs containing T and B epitopes arranged in tandem and in equimolar amounts. These results indicate that MAPs may serve as a basis for developing subunit vaccines to induce high levels of antibodies against sporozoites.

Amino Acid Sequence↗
PubMed1990

Basal body temperature and endometriosis.

This investigation examined the association between pelvic endometriosis and altered basal body temperature (BBT). The study population consisted of infertile women who have been diagnosed as having endometriosis. A significant association was found between the presence of pelvic endometriosis (without previous treatment) and the appearance of a late decline in BBT during the early follicular phase of the menstrual cycle. A temperature greater than 97.8 degrees F on the first 3 days of menses is associated with pelvic endometriosis. The findings of this study support the early clinical diagnosis of endometriosis in infertile women. Basal body temperature chart analysis may be useful as a clinical adjunct when endometriosis is suspected.

Adult↗