Purification and characterization of a β-galactosidase from Sclerotinia sclerotiorum

The DNA coding for the eight structural genes and uncI of the sodium dependent ATPase of Propionigenium modestum has been cloned and sequenced. Based on sequence homology, the genes were determined to appear in the order uncBEFHAGDC as in several other bacterial species. Minicell experiments revealed that plasmids containing the P. modestum DNA expressed those ATPase polypeptides in Escherichia coli. These were very similar in molecular mass to those obtained from the purified ATPase of P. modestum. No membrane-bound ATPase activity was observed in E. coli unc deletion strains containing the P. modestum ATPase genes. Amino acid alignments which were done with the Fo subunits revealed only a few conservative changes in the highly conserved regions of the polypeptides.     

Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase

A resident-plasmid cloning system developed for Bacillus subtilis has been used to isolate recombinant plasmids carrying DNA from Bacillus licheniformis which confer alpha-amylase activity on alpha-amylase-negative mutants of B. subtilis. These plasmids contain a 3550-bp insert at the EcoRI site of the plasmid pBD64. Subcloning various lengths of the B. licheniformis DNA has localised the gene to a 2550-bp BclI fragment. We present evidence that the cloned fragment codes for a B. licheniformis heat-stable alpha-amylase with a temperature optimum of 93 degrees C. The foreign gene is expressed efficiently in B. subtilis and is stably maintained.     

Mapping of the gene for endo-β-1,3-1,4-glucanase of Bacillus subtilis

Abstract An integrating plasmid has been used to mutagenise the gene coding for endo-β-1,3-1,4-glucanase of Bacillus subtilis . The gene, named bgl , has been mapped by PBS-1 transduction to the sacA-pureA region of the B. subtilis chromosome and is closely linked to the hutP 1 locus. The order of markers in this region is sacA 321- thiC 5- bgl - hutP 1- purA 16.     

DNA shuttling between plasmid vectors and a genome vector: systematic conversion and preservation of DNA libraries using the Bacillus subtilis genome (BGM) vector

The combined use of the contemporary vector systems, the bacterial artificial chromosome (BAC) vector and the Bacillus subtilis genome (BGM) vector, makes possible the handling of giant-length DNA (above 100 kb). Our newly constructed BGM vector efficiently integrated DNA prepared in the BAC vector. A BAC library comprised of 18 independent clones prepared from mitochondrial DNA (mtDNA) of Arabidopsis thaliana was converted to a parallel BGM library using the new BGM vector. The effectiveness of the combined use of the vector systems was confirmed by the stable recovery of all 18 DNAs as BAC clones from the respective BGM clones. We show that DNA in BGM was stably preserved at room temperature after spore formation of the host B.subtilis. Rapid and stable shuttling between Escherichiacoli and the B. subtilis host, combined with spore-mediated DNA storage, may facilitate the long-term and low-cost preservation and the transportation of DNA resources.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Construction of yeast artificial chromosome (YAC) clone banks covering three genome equivalents and isolation of YACs containing the human gene encoding tumor necrosis factor receptor β

Yeast artificial chromosome (YAC) banks covering in total about three haploid genome equivalents were constructed using a human Epstein-Barr-virus-transformed B lymphocytic cell line. Two clone banks were made: 20 000 clones with average inserts of 350 kb in the pYAC4 vector and 9850 clones with average inserts of 180 kb using vectors pJS89 and pJS91. Direct comparison of pYAC4 with pJS89 and pJS91 showed pYAC4 to be the most suitable cloning vector. Two partial banks with average insert sizes of 220 kb for human endothelial cell DNA and epithelial HEp2 cell DNA were also constructed, each covering 10% of the haploid genome. A rapid, three-step PCR screening procedure for isolation of individual YAC clones was developed and used to identify two clones encoding TNF-Rβ. These clones cover about 200 kb and have 170 kb in common. TNF-Rβ is 9.3 kb long and contains two introns within the protein-coding sequence.     

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.     

Sequence of the gene for a high-alkaline mannanase from an alkaliphilic Bacillus sp. strain JAMB-750, its expression in Bacillus subtilis and characterization of the recombinant enzyme

A novel alkaline mannanase Man26A has been found in the culture of an alkaliphilic Bacillus sp. strain JAMB-750 and the optimal pH for the mannanase activity of the enzyme was around pH 10 (J Biol Macromol 4: 67–74, 2004). This optimal pH is the highest among those of the mannanases reported to date. The gene man26A coding the enzyme was cloned from the genomic DNA of strain JAMB-750 and sequenced. It encodes a protein of 997 amino acids including a signal peptide. The N-terminal half (Glu27–Val486) of the enzyme exhibited moderate similarities to other mannanases belonging to glycoside hydrolase family 26, such as the enzymes from Cellvibrio japonicus (37% identity), Cellulomonas fimi (33% identity), and Bacillus sp. strain AM-001 (28% identity). The C-terminal half was found to contain four domains. The first, second, third, and fourth domains exhibited similarities to the carbohydrate-binding module, the mannan-binding module, the Homo sapiens collagen type IX alpha I chain, and the membrane anchor region of Gram-positive surface proteins, respectively. Its recombinant mannanase was produced extracellularly using Bacillus subtilis as the host. The optimal pH for the mannanase activity of the recombinant enzyme was around pH 10. The enzyme was very resistant to surfactants, for example, SDS up to 2.0% (w/v).     

Insertion of eukaryotic DNA into the Bacillus subtilis genome by means of a temperature-sensitive plasmid vector

A hybrid temperature-sensitive plasmid capable of integration into the Bacillus subtilis genome was constructed. By using this vector, we inserted a 3.2-kb fragment of eukaryotic DNA (wheat 'Chinese Spring') into the bacterial genome. The fragment of wheat DNA was stably retained and replicated as a part of the bacterial genome. The position of the integrated plasmid in the B. subtilis genome was mapped, as was the site in wheat DNA insert on plasmid at which the integration occurred.     

Construction and application of a promoter-trapping vector with methyl parathion hydrolase gene mpd as the reporter

A facilitative and efficient promoter-trapping vector, pUC-mpd, was constructed with the promoterless methyl parathion hydrolase gene as the reporter. This reporter gene is easily used to clone promoters with different promoting strength on selective plates. Promoter regions of the ytkA and ywoF genes with strong promoting and signal peptide functions were cloned from the Bacillus subtilis 168 genomic promoter library with this vector.     

Purification of a β-galactosidase from rice shoots and its involvement in hydrolysis of the natural substrate in cell walls

Several glycosidase and glycanase activities have been detected in homogenates of rice ( Oryza sativa L. cv. Nipponbare) shoots after successive extraction with K-phosphate (pH 7. 0) and buffer containing 3 M LiCl. The major β-D-galactosidase (EC 3. 2. 1. 23) present in the buffer-soluble protein fraction was purified to electrophoretic homogeneity by a combination of chromatographic techniques including DEAE-Sepharose CL-6B, Sephacryl S-200HR and p -aminophcnyl-β-D-thiogalactopyranoside–Sepharose 4B. Analysis by denaturing gel electrophoresis revealed a single polypeptide chain with an apparent molecular mass of 42 kDa. Similar to the value of 40 kDa estimated for the native protein by gel-permeation. The isoelectric point was pH 6. 0. The K_m and V_max values for p -nitrophenyl (PNP)-β-D-galactopyra-noside were 0. 63 m M and 0. 32 mmol (mg protein)⁻¹ h⁻¹, respectively. Maximum activity in McIlvaine buffer occurred at pH 3. 4, and the activity was inhibited by Ag²⁺, Cu²⁺. Hg²⁺, p -chloromercuribenzoate (PCMB) and D-galactono-l,4-lactone. The enzyme hydrolyzed larchwood arabinogalactan in an exo-fashion, and acted weakly on arabinosyl and galactosyl residue-rich polymer of pectic polysaccharides and cell walls from rice shoots.     

Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum β-lactamase VEB-1 in Pseudomonas aeruginosa

A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.     

Cloning and genetic organization of the gene cluster encoding F71 fimbriae of a uropathogenic Escherichia coli and comparison with the F72 gene cluster

Abstract The gene cluster coding for expression of F7₁ fimbriae of the uropathogenic Escherichia coli strain AD110 has been cloned by a cosmid-cloning procedure. A positive clone was further subcloned to a plasmid of 17.5 kilobases (kb), pPIL110-75. Analysis of pPIL110-75 showed that at least six genes are present encoding proteins with apparent M _rs of 75 000, 36 000, 23 000, 20 000, 17 000 and 14 000. The 20-kDa protein, encoding the F7₁ fimbrial subunit is dispensable for expression of the MRHA phenotype. Complementation experiments of mutants in the F7₂ gene cluster by gene products of the F7₁ gene cluster show that the two gene clusters are related.     

Sequence and chromosomal localisation of the gene encoding ovine latent transforming growth factor-β1

Transforming growth factor-β1 (TGF-β1) plays important roles in pathologic processes. To further investigate the actions of this cytokine in sheep, the entire 1170-bp ovine TGF-βl pro-protein-encoding sequence has been determined by the cloning and sequencing of specific polymerase-chain-reaction amplification products of TGF-β1 cDN A sequences. In addition, these sequences have been used to estimate the length of the TGF-β1 mRNA as 1.5-1.7 kb by Northern blot hybridization and determine that the ovine TGF-β1 gene occupies a single locus in the sheep genome by chromosomal in situ hybridization.     

Nucleotide and deduced amino acid sequence of the gene for a novel protein with a possible regulatory function encoded in the β operon of Staphylococcus aureus

Abstract Although considerable homology exists between the translation products of the rplL, rpoB and rpoC genes of the β operons of the Gram-negative organism Escherichia coli and the Gram-positive Staphylococcus aureus the region between the rplL and rpoB genes is quite different in the two bacterial species. In E. coli the 324 bp has three centres of dyad symmetry in the first half of the sequence and multiple nonsense codons in all three reading frames. By contrast, the corresponding region in S. aureus consists of 1000 bp capable of forming a similar arrangement of stem-loop structures but with an open reading frame, sited 177 bp downstream of the end of rplL and 217 bp upstream of the beginning of the rpoB gene, with consensus initiation and termination signals, which if translated would generate a 22,665 Da protein with 202 amino acids. In view of the inability to find any significant homology with other proteins in the data bank and because the evidence suggests, as in E. coli , that the rplL-rpoB intergenic sequence is involved in regulation it is proposed that the expression product of orf202 may be a further element of control in the S. aureus β operon.