Production of β-mannosidase and β-mannanase by an alkalophilic Bacillus sp.

Summary An alkalophilic bacterium producing high amounts of the cell-associated -mannosidase and extracellular -mannanase was isolated from soil. The isolate (AM-001) that grew well in alkaline pH media was identified as a strain of Bacillus sp. The optimal cultivation temperature for enzyme production was 31° C for -mannosidase and 37° C for -mannanase with the optimum production medium composed of 1% konjac powder, 0.2% yeast extract, 2% Polypepton, 0.1% K₂HPO₄, 0.02% MgSO₄ · 7H₂O and 0.5% Na₂CO₃. Optimum pH and temperature for -mannosidase were 7.0 and 55° C, and for -mannanase were 9.0 and 65° C.     

Nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167

The nucleotide sequence of the G6-amylase gene from alkalophilic Bacillus sp. H-167 was determined. The open reading frame of the gene consisted of 2865 base pairs, encoding 955 amino acids. The NH2-terminal amino acid sequence analysis of the G6-amylase indicated that the enzyme had a single peptide of 33 amino acid residues and the mature enzyme was composed of 922 amino acids, giving a molecular mass of 102,598. Identity of the NH2-terminal amino acid sequences among each component of the multiform G6-amylase suggested the proteolytic processing of the COOH-terminal side of the enzyme. The DNA sequence and the deduced amino acid sequence of the G6-amylase gene showed no homology with those of other bacterial alpha-amylases although the consensus amino acid sequences of the active center were well conserved.     

Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions

We have analyzed interactions between the mammalian TATA factor (TFIID) and derivatives of the yeast activator GAL4. The interaction of the TATA factor on the adenovirus E4 promoter with GAL4 binding sites adjacent to the TATA site was qualitatively altered in response to GAL4 binding. Alterations in the TFIID interactions were observed with two GAL4 derivatives that stimulated hybrid E4 promoter activity in vitro but not with a third derivative that bound to DNA but showed no activation. These results indicate that TFIID is a direct target for a GAL4 activation domain and suggest a simple general model for the activation mechanism.     

TFIIA induces conformational changes in TFIID via interactions with the basic repeat.

DNA-binding studies with Saccharomyces cerevisiae TFIID point mutants indicated that TFIIA interacts with the basic repeat region of TFIID and induces structural changes. The latter was shown by the ability of TFIIA to compensate for TFIID point mutants defective for DNA binding. Interaction with TFIIA also rendered TFIID binding temperature independent, thus mimicking the effect of removing the nonconserved N terminus of TFIID. In addition, N-terminal truncation of the TFIID point mutants defective for DNA binding mimicked the ability of TFIIA to restore DNA binding of those mutants. Taken together, these results suggest that TFIIA enhances TFIID binding to DNA by eliminating an otherwise inhibitory effect of the nonconserved N terminus of TFIID. Furthermore, analyses of TFIID contact points on DNA and binding studies with TATA-containing oligonucleotide probes showed that TFIIA decreases the effect of sequences flanking the adenovirus major late TATA element on TFIID binding to DNA, suggesting a possible role of TFIIA in allowing TFIID to recognize a wider variety of promoters.     

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.     

Molecular cloning and expression of the xylanase gene of alkalophilic Bacillus sp. strain C-125 in Escherichia coli.

The gene for xylanase A of alkalophilic Bacillus sp. strain C-125 was cloned in Escherichia coli with pBR322. The plasmid pCX311 contained 2.6- and 2.0-kilobase-pair HindIII fragments. The characteristics of the purified pCX311-encoded xylanase were the same as those of purified xylanase A from alkalophilic Bacillus sp. strain C-125.