Epidemiological study of latent and recent infection by Toxoplasma gondii in pregnant women from a regional population in the U.K

The last two amino acids in the nascent peptide influence translation termination in E. coli (Mottagui-Tabar et al., 1994; Bj?rnsson et al., 1996). We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal. The peptide effect from the penultimate amino acid on translation termination in B. subtilis is similar to that seen in E. coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S. typhimurium (with 95.3% similarity to E. coli) is weaker. The effect of changing the -1 codon (P-site) is weaker in S. typhimurium as compared to those in E. coli and B. subtilis. RF-2s from E. coli and S. typhimurium have a threonine or alanine at position 246, respectively. This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E. coli and S. typhimurium are compared (Uno et al., 1996). However, B. subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E. coli. Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E. coli and B. subtilis is similar in peptide sensitivity while being different from that in S. typhimurium. Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.     

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.     

The Bacillus subtilis nucleotidyltransferase is a tRNA CCA-adding enzyme

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3' poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the beta and gamma subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca, following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3' polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.     

Pretreatment with intralesional hyaluronidase prior to excision of dermatofibrosarcoma protuberans

A DNA fragment from Pseudomonas fluorescens DSM50106 containing the genes for the uptake and utilization of mannitol, arabitol and glucitol was cloned in Escherichia coli and sequenced. Seven open reading frames (mtlEFGKDYZ) were identified on the 10031 bp fragment. The deduced amino acid sequences of the first four open reading frames (mtlEFGK) revealed significant similarity to the components of the maltose transport system in E. coli and Salmonella typhimurium. The gene mtlD encoding a polyol dehydrogenase was located downstream of mtlK. The deduced proteins of the last two genes on the fragment showed a high similarity to a fructokinase from Vibrio alginolyticus (MtlZ) and a xylulose kinase from Streptomyces rubiginosus (MtlY), respectively. Both genes were expressed in E. coli. MtlZ phosphorylated fructose, glucose and glucitol whereas MtlY was highly specific for xylulose. Upstream of mtlE, a putative promoter/operator region was identified by promoter probe studies which was active in P. fluorescens but not in E. coli.