Nucleotide sequence and cloning in Bacillus subtilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT
54
Citation
44
Reference
10
Related Paper
Citation Trend
Abstract:
The regulatory gene (degT) from Bacillus stearothermophilus NCA1503 which enhanced production of extracellular alkaline protease (Apr) was cloned in Bacillus subtilis with pTB53 as a vector. When B. subtilis MT-2 (Npr- [deficiency of neutral protease] Apr+) was transformed with the recombinant plasmid, pDT145, the plasmid carrier produced about three times more alkaline protease than did the wild-type strain. In contrast, when B. subtilis DB104 (Npr- Apr-) was used as a host, the transformant with pDT145 could not exhibit any protease activity. After construction of the deletion plasmids, DNA sequencing was done. A large open reading frame was found, and nucleotide sequence analysis showed that the degT gene was composed of 1,116 bases (372 amino acid residues, molecular weight of 41,244). A Shine-Dalgarno sequence was found nine bases upstream from the open reading frame. A B. subtilis strain carrying degT showed the following pleiotropic phenomena: (i) enhancement of production of extracellular enzymes such as alkaline protease and levansucrase, (ii) repression of autolysin activity, (iii) decrease of transformation efficiency for B. subtilis (competent cell procedure), (iv) altered control of sporulation, (v) loss of flagella, and (vi) abnormal cell division. When B. stearothermophilus SIC1 was transformed with the recombinant plasmid carrying degT, the transformants exhibited abnormal cell division. These phenomena are similar to those of the phenotypes of degSU(Hy) (hyperproduction), degQ(Hy), and degR mutants of B. subtilis. However, the amino acid sequence of the degT product (DegT) is different from those of the reported gene products. Furthermore, DegT includes a hydrophobic core region in the N-terminal portion (amino acid numbers 50 to 160), a consensus sequence for a DNA binding region (amino acid numbers 160 to 179), and a region homologous to transcription activator proteins (amino acid numbers 351 to 366). We discuss the possibility that the membrane protein DegT functions as a sensor protein and transfers the signal of environmental stimuli to the regulatory region of target genes to activate or repress transcription of the genes.Keywords:
Autolysin
Bacillaceae
Cite
Citations (1)
Summary Nucleotide sequencing of the celZ gene encoding the extracellular endoglucanase Z of Erwinia chrysanthemi indicated the presence of an open reading frame encoding 428 amino acids. The mature protein appeared to be extended by a signal peptide of 43 amino acids; this sequence is unusually long and positively charged (+5). It was shown to function as a signal peptide by fusing it to a truncated phoA gene encoding Escherichia coli alkaline phosphatase. Comparison of the encoded sequence with those of the endoglucanases of Bacillus subtilis and alkalophilic Bacillus revealed the existence of a region of extensive homology occurring in all three proteins at about the same distance from the NH 2 ‐terminal end. These regions may be involved in substrate binding and/or catalytic sites.
Homology
Bacillaceae
Cite
Citations (55)
The nucleotide sequence of the genes encoding the canine herpesvirus (CHV) gB, gC and gD homologues was determined. These genes are predicted to encode polypeptides of 879, 459 and 345 amino acids, respectively. Comparison of the predicted amino acid sequences of CHV gB, gC and gD with the homologous sequences from other herpesviruses indicates that CHV is an alphaherpesvirus, a conclusion that is consistent with the previous classification of this virus according to biological properties. Alignment of the homologous gB, gC and gD amino acid sequences indicates that most of the cysteine residues are conserved, suggesting that these glycoproteins possess similar tertiary structures. The nucleotide sequence of the open reading frame downstream from the CHV gC gene was also determined. The predicted amino acid sequence of this putative polypeptide appears to be homologous to a family of proteins encoded downstream from the gC gene in most, although not all, alphaherpesviruses.
Sequence (biology)
Homology
Cite
Citations (32)
A temperature-sensitive mutant of Bacillus subtilis which has impaired ability to autolyze was isolated. The growth rate of mutant cells at high temperature can be increased by the addition of egg-white lysozyme or a B. subtilis autolysin. Experiments with this mutant show that lysis is essential for cell growth.
Autolysin
Temperature-sensitive mutant
Cite
Citations (46)
The nucleotide and deduced amino acid sequences of three genes of turkey rhinotracheitis virus (TRTV) together with the nucleotide sequences of the relevant intergenic regions were determined. The deduced amino acid sequence of one of the genes shows significant identity (42%) to that of the 22K protein of human respiratory syncytial virus (RSV). The TRTV 22K gene, like that of RSV, has a second open reading frame, although the amino acid sequence deduced from this reading frame does not show any similarity to the equivalent predicted RSV protein. The other two genes and their deduced amino acid sequences do not show any sequence similarity to the genes of other pneumoviruses. However, the hydrophobicity profiles of the predicted proteins do show similarities to those of the small hydrophobic (SH) and attachment protein (G) genes of RSV. The TRTV G gene is 1193 nucleotides in length and encodes a protein of 391 amino acids (M r 42984), which is rather larger than the RSV G protein (predicted M r 36000). The TRTV SH gene is 589 nucleotides in length, encoding a protein of 174 amino acids (M r 18797), which is considerably larger than the size of the RSV SH protein (M r 7500). The sequences of the intergenic regions derived from clones of polycistronic mRNAs and polymerase chain reaction products obtained with primers from different genes reveal the order on the virus genome to be 3′ F-22K-SH-G 5′. This differs from the gene order of paramyxoviruses and morbilliviruses, which lack a 22K gene (and in some cases a SH gene), and the pneumoviruses RSV and pneumonia virus of mice, which have the F and 22K genes located after the G gene.
Intergenic region
Cite
Citations (103)
Summary: It has been found that human renal adenocarcinoma ACHN cells synthesize and secrete immunoreactive endothelin (ir-ET) in the culture medium. Partial characterization of this material with reverse-phase high-performance liquid chromatography (RP-HPLC) suggested that ACHN cells synthesized only human endothelin-2 (ET-2). Isolation and characterization of this ir-ET-2 has revealed that this peptide has almost the same amino acid sequence and molecular weight as that of human ET-2 deduced from the nucleotide sequence of cloned human ET-2 gene. To delineate the precise structure of human ET-2 precursor, ET-2 cDNAs were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA that has the longest open reading frame encodes prepro-ET-2 protein, consisting of 178 amino acid residues. The ET-like sequence found in the prepro-ET-1 and -ET-3 was also conserved in this prepro-ET-2. The Northern blot analysis of mRNA revealed that the transcript of the human ET-2 gene was 1.4 kb.
Cite
Citations (8)
A number of mutants of Bacillus subtilis producing high levels of extracellular protease have been isolated. Analysis of culture supernatants of these mutants has shown that the total amount of proteolytic activity is elevated from 16- to 37-fold over the wild strain. The elevated activity was due to a simultaneous increase in both the neutral and alkaline protease. All of the mutants genetically analyzed were found linked to the argC4 marker by PBS-1 transduction analysis.
Neutral protease
Bacillaceae
Strain (injury)
Transduction (biophysics)
Cite
Citations (78)
One pair of primer was designed and synthesized according to the feline interferon alpha gene(IFN-α) nucleic acid sequence.Wild cat IFN-α was amplified by PCR,then cloned and sequenced.The nucleotide sequence of the amplified IFN-α gene and the amino acid sequence deduced from the gene were then analyzed by the DNAstar software and on-line tools,and the similarity and evolution of IFN-α gene were analyzed among wild cat,human and other animals.The results showed the full length sequence of wild cat IFN-α gene was 631 bp,which includes one open-reading frame,encoding 194 amino acid residues,one potential N-glycosalation site and seven O-glcnac sites in deduced amino acid sequence.The results of comparative sequence analysis and phyloge-netic tree analysis indicated there was difference of genus in IFN-α gene,the nearer their relationship,the higher the homology.This study paved the way for future study of biological function and application of feline IFN-α.
Homology
Primer (cosmetics)
Cloning (programming)
Cite
Citations (0)
Objective:To investigate the reasons for high-yield of Bacillus subtilis mutant ZC-7 obtained by implantation with 30 keV N+ ions beam to B.subtilis AS1.398. Methods:The DNA fragments of the neutral protease genes in the original strain and the mutant were cloned and sequenced,respectively. Then,they were compared to analyze the gene difference between ZC-7 and AS1.398 neutral protease. Subsequently,the two protein secondary structures were simulated on CPHmodels Server net. Results:The alignment results showed that five amino acids of the mature peptide changed and three of them were in the key region. The protein secondary structures prediction results indicated that some conformation changed in the fixed range because of the mutation sites in the central region occurred. Conclusion:In previous research,the neutral protease productions of the two strains were almost the same. Therefore,these mutant amino acids located in the key domain of substrate binding led to the conformation change of ZC-7 neutral protease,and it was responsible for more appropriate for substrate binding,in another word,higher activity.
Strain (injury)
Neutral protease
Cite
Citations (0)
The extracellular acid protease of Candida tropicalis was purified from the supernatant fraction of culture medium containing bovine serum albumin as nitrogen source and the NH 2 ‐terminal amino acid (aa) sequence of the protein was determined. The gene for the acid protease (ACP) was isolated using a pool of synthetic oligonucleotides as a probe and a segment of the deduced aa sequence was found to be in agreement with the NH 2 ‐terminal aa sequence of the protein. The deduced aa sequence of ACP is similar to the aa sequence of proteases of the pepsin family. The nucleotide sequence of the 5′ portion of this gene revealed a coding sequence for a 60 residue propeptide containing two Lys—Arg amino acid pairs that have been identified as sites for peptidase processing of several exported peptides and proteins. The final Lys—Arg site occurs at the junction with the mature extracellular form or the acid protease.
Cite
Citations (90)