Cloning, sequencing, and expression of the gene encoding a large S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 in Escherichia coli

The gene (xynA) encoding a surface-exposed, S-layer-associated endoxylanase from Thermoanaerobacterium sp. strain JW/SL-YS 485 was cloned and expressed in Escherichia coli. A 3.8-kb fragment was amplified from chromosomal DNA by using primers directed against conserved sequences of endoxylanases isolated from other thermophilic bacteria. This PCR product was used as a probe in Southern hybridizations to identify a 4.6-kb EcoRI fragment containing the complete xynA gene. This fragment was cloned into E. coli, and recombinant clones expressed significant levels of xylanase activity. The purified recombinant protein had an estimated molecular mass (150 kDa), temperature maximum (80 degrees C), pH optimum (pH 6.3), and isoelectric point (pH 4.5) that were similar to those of the endoxylanase isolated from strain JW/SL-YS 485. The entire insert was sequenced and analysis revealed a 4,044-bp open reading frame encoding a protein containing 1,348 amino acid residues (estimated molecular mass of 148 kDa).xynA was preceded by a putative promoter at -35 (TTAAT) and -10 (TATATT) and a potential ribosome binding site (AGGGAG) and was expressed constitutively in E. coli. The deduced amino acid sequence showed 30 to 96% similarity to sequences of family F beta-glycanases. A putative 32-amino-acid signal peptide was identified, and the C-terminal end of the protein contained three repeating sequences 59, 64, and 57 amino acids) that showed 46 to 68% similarity to repeating sequences at the N-terminal end of S-layer and S-layer-associated proteins from other gram-positive bacteria. These repeats could permit an interaction of the enzyme with the S-layer and tether it to the cell surface.     

Adaptation of pharmacomechanical coupling of vascular smooth muscle to chronic hypoxia

The Stoffel DNA fragment, shortened by 12 bp from 5' end, coding for Stoffel DNA polymerase (missing 4 amino acids at N-terminus of Stoffel amino-acids sequence) from the thermophilic Thermus aquaticus (strain YT-1) was amplified, cloned and expressed in Escherichia coli. The recombinant Stoffel fragment contained a polyhistidine tag at the N-terminus (21 additional amino acids) that allowed its single-step isolation by Ni2+ affinity chromatography. The enzyme was characterized and displayed high DNA polymerase activity and thermostability evidently higher than the native Taq DNA polymerase.     

Molecular cloning, characterization, and potential roles of cytosolic and mitochondrial aldehyde dehydrogenases in ethanol metabolism in Saccharomyces cerevisiae

The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption delta ald1 and delta ald2 strains on ethanol was marginally slower than that of the parent strain. The delta ald1 delta ald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.     

Ischemic preconditioning in isolated perfused mouse heart: reduction in infarct size without improvement of post-ischemic ventricular function

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.     

Identification of bifidobacteria from dairy products and evaluation of a microplate hybridization method

Sixteen strains of Bifidobacterium isolated from 15 dairy products such as yogurt, cultured milk, butter and cheese were characterized on the basis of phenotypic characteristics and DNA similarities were examined by a microplate hybridization method. Three of the strains were identified as Bifidodobacterium longum, one strain was identified as Bifidobacterium bifidm, and one strain was assigned to the species Bifidobacterium breve on the basis of phenotypic characteristics, and this identification was confirmed by the analysis of DNA similarities. The remaining 11 strains could not be identified by examining their phenotypic characteristics and, contrary to the product label information, these strains were identified as Bifudidobacterium animalis on the basis of DNA similarities. The applicability of the colorimetric hybridization method in micro dilution wells to genetic identification of Bifidobacterium species was also studied.     

Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Several different genomic fingerprints can be obtained from various commercially-important species of Bifidobacterium using pulsed-field gel electrophoresis (PFGE) following digestion of DNA with XbaI and SpeI. Four different genomic finger printings were discernible for reference strains of Bifidobacterium animalis, five for B. bifidum, three for B. breve, five for B. infantis and three for B. longum. Standard commercially-available industrial strains of B. animalis are identical to the reference strain ATCC 27536, previously isolated from chicken feces. There was more genomic heterogeneity among industrial strains of B. longum, in that only one gave profiles similar to the type strain of this species (ATCC 15707). The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. The PFGE method indicated that only five distinct strains of B. longum and one strain of B. animalis are used in commercial preparations. Additionally, the use of polymerase chain reaction amplification of portions of 16S rDNA provides a highly specific technique to discriminate between the species B. breve, B. infantis and B. longum.     

Transfusion virology: progress and challenges

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3'-5' exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction.     

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.     

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.     

Molecular cloning, sequencing, and expression of the genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca

The pdd genes encoding adenosylcobalamin-dependent diol dehydrase of Klebsiella oxytoca were cloned by using a synthetic oligodeoxyribonucleotide as a hybridization probe followed by measuring the enzyme activity of each clone. Five clones of Escherichia coli exhibited diol dehydrase activity. At least one of them was shown to express diol dehydrase genes under control of their own promoter. Sequence analysis of the DNA fragments found in common in the inserts of these five clones and the flanking regions revealed four open reading frames separated by 10-18 base pairs. The sequential three open reading frames from the second to the fourth (pddA, pddB, and pddC genes) encoded polypeptides of 554, 224, and 173 amino acid residues with predicted molecular weights of 60,348 (alpha), 24,113 (beta), and 19,173 (gamma), respectively. Overexpression of these three genes in E. coli produced more than 50-fold higher level of functional apodiol dehydrase than that in K. oxytoca. The recombinant enzyme was indistinguishable from the wild-type one of K. oxytoca by the criteria of polyacrylamide gel electrophoretic and immunochemical properties. It was thus concluded that these three gene products are the subunits of functional diol dehydrase. Comparisons of the deduced amino acid sequences of the three subunits with other proteins failed to reveal any apparent homology.     

Dopamine-stimulated sexual behavior is testosterone dependent in mice

The ingestion of viable bacteria is thought to be required to modify intestinal microflora. In the present study, the effects on fecal flora of consumption of cell-free concentrated whey from milk that had been fermented with Bifidobacterium breve C50 was tested using 10 healthy human volunteers. Results were compared with effects of a commercial milk formula that had been fermented with Streptococcus thermophilus and B. breve C50 and given to 10 control subjects. Nitroreductase and beta-glucuronidase activities were assessed as risk indexes for colon carcinogenesis, and beta-galactosidase was measured as an indicator of the fermentation capacity of the colonic flora. Fecal excretion of Bacteroides fragilis, Clostridium perfringens, and clostridial spores decreased after 7 d of consumption of either preparation; however, counts of bifidobacteria only increased after intake of B. breve whey. Fecal pH was reduced from 7.1 +/- 0.2 to 6.6 +/- 0.3 after intake of whey that had been fermented with Bif. breve. Fecal nitroreductase and beta-glucuronidase significantly decreased, and beta-galactosidase activity increased, after consumption of either preparation. The results indicate that ingestion of viable bifidobacteria was not required to modify intestinal flora of humans. Repression of B. fragilis and clostridia seems to be independent of colonic bifidobacterial overgrowth in humans.     

Digoxigenin-labeled deoxyribonucleic acid probes for the enumeration of bifidobacteria in fecal samples

The numbers of bifidobacteria in fecal samples were specifically determined by colony hybridization with the mixture of digoxigenin-labeled DNA probes that were prepared from whole chromosomal DNA of Bifidobacterium longum 6001 and Bifidobacterium adolescentis 6003. These DNA probes strongly hybridized with DNA of B. longum, B. adolescentis, Bifidobacterium breve, Bifidobacterium suis, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium angulatum, and Bifidobacterium animalis. Detectable positive signals with DNA of Bifidobacterium pseudolongum ssp. pseudolongum, Bifidobacterium catenulatum, and Bifidobacterium thermophilum were also found after hybridization. When dot-blot hybridization was performed with whole cells of 47 reference strains containing 11 species (16 strains) of bifidobacteria, all of the bifidobacteria tested could be specifically detected by using these DNA probes; Lactobacillus fermentum JCM 1173, however, showed a slight nonspecific signal. The counts of bifidobacteria by colony hybridization in the fecal samples of four of the five subjects were the same as the counts that were obtained by the conventional method using BL agar medium. Furthermore, no significant difference existed in the number of bifidobacteria that were determined by either method.     

Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.     

Molecular cloning and expression of GalNAc alpha 2,6-sialyltransferase

cDNA clones encoding GalNAc alpha 2,6-sialyltransferase (EC 2.4.99.3) have been isolated from chick embryo cDNA libraries using sequence information obtained from the conserved amino acid sequence of the previously cloned enzymes. The cDNA sequence included an open reading frame coding for 566 amino acids, and the deduced amino acid sequence showed 12% identity with that of Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase from chick embryo. The primary structure of this enzyme suggested a putative domain structure, like that in other glycosyltransferases, consisting of a short NH2-terminal cytoplasmic domain, a signal-membrane anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain. The identity of this enzyme was confirmed by the construction of a recombinant sialyltransferase in which the NH2-terminal part (232 amino acid residues) was replaced with the immunoglobulin signal sequence. The expression of this recombinant in COS-7 cells resulted in secretion of a catalytically active and soluble form of the enzyme into the medium. The expressed enzyme exhibited activity toward only asialomucin and (asialo)fetuin, no significant activity being detected toward the other glycoprotein and glycolipid substrates tested. 14C-Sialylated glycols obtained from asialomucin re-sialylated with this enzyme were identical to NeuAc alpha 2,6-GalNAc-ol and GlcNAc beta 1,3(NeuAc alpha 2,6) GalNAc-ol. Synthetic GalNAc-SerNAc also served as an acceptor for alpha 2,6-sialylation. These results clearly showed that the expressed enzyme is GalNAc alpha 2,6-sialyltransferase.     

Molecular cloning and nucleotide sequences of cDNA and gene encoding endo-inulinase from Penicillium purpurogenum

A cDNA and a gene encoding endo-inulinase from Penicillium purpurogenum were isolated, and were cloned for the first time. Two oligonucleotide probes, which were synthesized based on the partial amino acid sequences of the purified endo-inulinase, were used to screen a cDNA library. A 1.7-kb DNA fragment encoding endo-inulinase was isolated and analyzed. A single open reading frame, consisting of 1548-bp, was found to encode a polypeptide that comprised a 25-amino acid signal peptide and 490-amino acid mature protein. All the partial amino acid sequences of the purified enzyme were discovered in the deduced ones. The deduced amino acid sequences of endo-inulinase had similar sequences to those of fructan hydrolases. A 3.5-kb chromosomal DNA fragment encoding endo-inulinase was also isolated and analyzed. The same ORF with cDNA clone as identified. There were no introns in the endo-inulinase gene.     

3-Ketosteroid-delta1-dehydrogenase of Rhodococcus rhodochrous: sequencing of the genomic DNA and hyperexpression, purification, and characterization of the recombinant enzyme

The gene encoding 3-ketosteroid-Delta1-dehydrogenase from Rhodococcus rhodochrous was cloned and sequenced. The gene (ksdD) consists of 1,536 nucleotides and encodes an enzyme protein of 511 amino acid residues. The amino terminal methionine residue was deleted in the mature protein. The amino acids involved in the flavin binding site are conserved in the dehydrogenase sequence. The deduced amino acid sequence is highly homologous to that from Arthrobacter simplex but less so to that from Pseudomonas testosteroni. Upstream of the gene was located a heat shock protein gene, dnaJ, and downstream, a gene of a hypothetical protein. The enzyme gene was ligated with an expression vector to construct a plasmid pDEX-3 and introduced into Escherichia coli cells. The transformed cells hyperexpressed the 3-ketosteroid-Delta1-dehydrogenase as an active and soluble protein at more than 30 times the level of R. rhodochrous cells. Purification of the recombinant 3-ketosteroid-Delta1-dehydrogenase from the E. coli cells by a simplified procedure yielded about 13 mg of enzyme protein/liter of the bacterial culture. The purified recombinant dehydrogenase exhibited identical molecular and catalytic properties to the R. rhodochrous enzyme.     

Studies on almond emulsin beta-D-glucosidase. I. Isolation and characterization of a bifunctional isozyme

A beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) isozyme has been isolated from almond emulsin. The isolated enzyme is a glycoprotein and migrates as a single band on Sephadex G-200 filtration, CM 52 ion exchange chromatography, polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and isoelectric focussing. The glucosidase and galactosidase activities traverse together during Sephadex G-200 gel filtration. Polyacrylamide gels stained specifically for the 2 enzymes reveal that the two activities comigrate. The molecular weight of the isozyme has been found to be 135 180 +/- 770, and that of its protomers to be 65 150 +/- 650.