Multiple sequence alignment of protein families showing low sequence homology: a methodological approach using database pattern-matching discriminators for G-protein-linked receptors.

A multiple alignment has been constructed, containing 37 sequences from related families of membrane-bound receptors believed to share the same structural framework as rhodopsin. Sequence homology within families was high (occasionally greater than 90%), but homology between them was generally low (20% or less). Database pattern-scanning methods were therefore used to construct a set of discriminators to aid both the task of alignment and the identification of distantly related sequences showing similar rhodopsin-like transmembrane helices. The results indicate that these discriminators are uniquely able to identify each of the transmembrane helices without major cross-reaction with similar regions in unrelated integral membrane proteins. This ability engenders more accurate alignments of the sequences and facilitates structural analysis and model building of the receptors.     

Methanol metabolism in thermotolerant methylotrophic Bacillus strains involving a novel catabolic NAD-dependent methanol dehydrogenase as a key enzyme

The enzymology of methanol utilization in thermotolerant methylotrophic Bacillus strains was investigated. In all strains an immunologically related NAD-dependent methanol dehydrogenase was involved in the initial oxidation of methanol. In cells of Bacillus sp. C1 grown under methanol-limiting conditions this enzyme constituted a high percentage of total soluble protein. The methanol dehydrogenase from this organism was purified to homogeneity and characterized. In cell-free extracts the enzyme displayed biphasic kinetics towards methanol, with apparent K _m values of 3.8 and 166 mM. Carbon assimilation was by way of the fructose-1,6-bisphosphate aldolase cleavage and transketolase/transaldolase rearrangement variant of the RuMP cycle of formaldehyde fixation. The key enzymes of the RuMP cycle, hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), were present at very high levels of activity. Failure of whole cells to oxidize formate, and the absence of formaldehyde-and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formaldehyde via HPS. A comparison of the levels of methanol dehydrogenase and HPS in cells of Bacillus sp. C1 grown on methanol and glucose suggested that the synthesis of these enzymes is not under coordinate control.Abbreviations RuMP ribulose monophosphate - HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - MDH methanol dehydrogenase - ADH acohol dehydrogenase - PQQ pyrroloquinoline, quinone - DTT dithiothreitol - NBT nitrobluetetrazolium - PMS phenazine methosulphate - DCPIP dichlorophenol indophenol     

Purification and properties of myo-inositol-1-phosphatase from bovine brain.

myo-Inositol-1-phosphatase from bovine brain was purified over 2000-fold. The native enzyme has a Mr of 59,000, and on SDS/polyacrylamide-gel electrophoresis the subunit Mr was 31,000. Thus the native enzyme is a dimer of two apparently identical subunits. The enzyme, purified to a specific activity of more than 300 units/mg of protein (1 unit of enzyme activity corresponds to the release of 1 mumol of Pi/h at 37 degrees C), catalysed the hydrolysis of a variety of phosphorylated compounds, the best one, in terms of V/Km, being D-myo-inositol 1-phosphate. Kinetic constants of compounds tested, including both isomers of glycerophosphate and two deoxy forms of beta-glycerophosphate, were measured. They show the importance of the two hydroxyl groups which are adjacent to the phosphate in myo-inositol 1-phosphate. With a wide variety of substrates Li+ was found to be an uncompetitive inhibitor whose Ki varied with substrate structure.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

OWL--a non-redundant composite protein sequence database.

A comprehensive, non-redundant composite protein sequence database is described. The database, OWL, is an amalgam of data from six publicly-available primary sources, and is generated using strict redundancy criteria. The database is updated monthly and its size has increased almost eight-fold in the last six years: the current version contains > 76,000 entries. For added flexibility, OWL is distributed with a tailor-made query language, together with a number of programs for database exploration, information retrieval and sequence analysis, which together form an integrated database and software resource for protein sequences.     

Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene.

Heterologous expression of the Clostridium cellulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTL500E).     

Intraspecific diversity in the fungal speciesChaunopycnis alba: Implications for microbial screening programs

Intraspecific variation among 84 isolates of the anamorphic fungusChaunopycnis alba from 26 different geographical locations was analyzed by investigating optimal growth temperatures, differences in the production of secondary metabolites and presence or absence of the cyclosporin synthetase gene. The genetic diversity was assessed using random amplified polymorphic DNA (RAPD). Analysis of these data showed high genetic, metabolic and physiological diversity within this species. Isolates from the Antarctic represented the most homogeneous group withinC. alba and together with isolates from the Arctic these polar strains differed from alpine, temperate and tropical strains by low optimal growth temperatures and by low production of secondary metabolites. Isolates from tropical climes were characterized by high optimal growth temperatures and by the production of comparatively diverse metabolite spectra. Most of the isolates that were similar in the combination of their physiological and metabolic characters were also genetically related. Isolates from different geographical origins did not show many similarities, with the exception of the cyclosporin A-producing isolates, and large diversity could be observed even within a single habitat. This leads us to the suggestion that for pharmaceutical screening programs samples should be collected from a diversity of different geographical and climatic locations. For the selection of strains for screening the RAPD assay seems to be the most powerful tool. It reflected the highest intraspecific diversity and the results corresponded well with the other characteristics.     

Interaction of formycin A-5'-triphosphate with pyruvate carboxylase

Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a competitive inhibitor of chicken liver pyruvate carboxylase with respect to ATP. The chicken liver enzyme is unable to utilise FTP as a substrate at a measureable rate, but FTP is a poor substrate for the sheep liver enzyme. When FTP binds to the enzyme, its fluorescence is enhanced and in this way the formation of enzyme-FTP complexes can be monitored. Using this property of FTP, the effect of Mg2+ and acetyl-CoA on the binding of nucleoside triphosphates to the chicken liver enzyme was examined. Mg2+ was found to enhance the binding of FTP whilst acetyl-CoA reduced the fluorescence intensity of a mixture of Mg2+, enzyme and FTP. Most probably, this was caused by a conformational change in the enzyme which changed the environment of the fluorophore.