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Alpha-factor structural gene mutations in Saccharomyces cerevisiae: effects on alpha-factor production and mating. 总被引:29,自引:19,他引:10
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J Kurjan 《Molecular and cellular biology》1985,5(4):787-796
The role of alpha-factor structural genes MF alpha 1 and MF alpha 2 in alpha-factor production and mating has been investigated by the construction of mf alpha 1 and mf alpha 2 mutations that totally eliminate gene function. An mf alpha 1 mutant in which the entire coding region is deleted shows a considerable decrease in alpha-factor production and a 75% decrease in mating. Mutations in mf alpha 2 have little or no effect on alpha-factor production or mating. The mf alpha 1 mf alpha 2 double mutants are completely defective in mating and alpha-factor production. These results indicate that at least one alpha-factor structural gene product is required for mating in MAT alpha cells, that MF alpha 1 is responsible for the majority of alpha-factor production, and that MF alpha 1 and MF alpha 2 are the only active alpha-factor genes. 相似文献
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S. Janecek 《Protein science : a publication of the Protein Society》1996,5(6):1136-1143
The question of parallel (alpha/beta)8-barrel fold evolution remains unclear, owing mainly to the lack of sequence homology throughout the amino acid sequences of (alpha/beta)8-barrel enzymes. The "classical" approaches used in the search for homologies among (alpha/beta)8-barrels (e.g., production of structurally based alignments) have yielded alignments perfect from the structural point of view, but the approaches have been unable to reveal the homologies. These are proposed to be "hidden" in (alpha/beta)8-barrel enzymes. The term "hidden homology" means that the alignment of sequence stretches proposed to be homologous need not be structurally fully satisfactory. This is due to the very long evolutionary history of all (alpha/beta)8-barrels. This work identifies so-called hidden homology around the strand beta 2 that is flanked by loops containing invariant glycines and prolines in 17 different (alpha/beta)8-barrel enzymes, i.e., roughly in half of all currently known (alpha/beta)8-barrel proteins. The search was based on the idea that a conserved sequence region of an (alpha/beta)8-barrel enzyme should be more or less conserved also in the equivalent part of the structure of the other enzymes with this folding motif, given their mutual evolutionary relatedness. For this purpose, the sequence region around the well-conserved second beta-strand of alpha-amylase flanked by the invariant glycine and proline (56_GFTAIWITP, Aspergillus oryzae alpha-amylase numbering), was used as the sequence-structural template. The proposal that the second beta-strand of (alpha/beta)8-barrel fold is important from the evolutionary point of view is strongly supported by the increasing trend of the observed beta 2-strand structural similarity for the pairs of (alpha/beta)8-barrel enzymes: alpha-amylase and the alpha-subunit of tryptophan synthase, alpha-amylase and mandelate racemase, and alpha-amylase and cyclodextrin glycosyltransferase. This trend is also in agreement with the existing evolutionary division of the entire family of (alpha/beta)8-barrel proteins. 相似文献
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A new signal peptide useful for secretion of heterologous proteins from yeast and its application for synthesis of hirudin. 总被引:2,自引:0,他引:2
The BGL2 gene from Saccharomyces cerevisiae encodes a beta-glucanase which is localized to the yeast cell wall. The ability of a 23-amino acid (aa) signal peptide derived from the BGL2 gene to direct a heterologous protein to the secretory pathway of yeast has been compared to that of the MF alpha 1-encoded signal peptide in a series of gene fusions. As a model protein, the leech anticoagulant, recombinant hirudin variant 2-Lys47 (HIR) has been studied. From a multicopy plasmid chimaeric proteins were produced which carry the BGL2 signal peptide (or the artificial BGL2 pre-Val7 variant) (i) in front of the MF alpha 1 pro sequence (or modified versions of MF alpha 1 pro), i.e., a prepro signal, or (ii) joined directly to the heterologous protein. Accumulation of active HIR in yeast culture supernatants was observed when the BGL2 (or the BGL2 pre-Val7) signal peptide were used in combination with either of three versions of the MF alpha 1 pro peptide: the authentic MF alpha 1 pro, a partially deleted MF alpha 1 pro-delta 22-61, or a pro bearing an aa change (MF alpha 1 pro-Gly22). In each case the BGL2 signal peptide (or its variant) has proven equally productive to the corresponding MF alpha 1 peptide. Four times more active HIR was detected in the culture supernatant when either signal peptide was fused directly to the recombinant protein, as compared to a prepro protein version. Correct signal peptide cleavage was obtained when HIR was produced as a BGL2 pre-Val7::fusion protein. 相似文献
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Wato S Kamei K Arakawa T Philo JS Wen J Hara S Yamaguchi H 《Journal of biochemistry》2000,128(1):139-144
White kidney bean (Phaseolus vulgaris) contains two kinds of alpha-amylase inhibitors, one heat-stable (alpha AI-s) and one heat-labile (alpha AI-u). alpha AI-s has recently been revealed to be a tetrameric complex, alpha(2)beta(2), with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of alpha AI-u, which is composed of three kinds of subunits, alpha, beta, and gamma. The gamma-subunit, in contrast to the alpha- and beta-subunits that are indistinguishable from the alpha- and beta-subunits of alpha AI-s, was found to correspond to a subunit of an alpha-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the alpha-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of alpha AI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that alpha AI-u is a trimeric complex, alpha beta gamma. The inhibition of alpha AI-u by increasing amounts of porcine pancreatic alpha-amylase (PPA) indicates that an inactive 1:1 complex is formed between alpha AI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of alpha AI-u with one PPA molecule. Thus it seems probable that alpha AI-u is an evolutionary intermediate of the P. vulgaris alpha-amylase inhibitor. 相似文献
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Cloning and expression of the alpha-amylase gene from Bacillus stearothermophilus in several staphylococcal species 总被引:2,自引:0,他引:2
The plasmid-coded alpha-amylase gene of Bacillus stearothermophilus (amy) was cloned in Staphylococcus carnosus using plasmid pCA43 as a vector. The amy gene was located on a 5.4-kb HindIII DNA fragment of the hybrid plasmid pamy7. When transformed into other staphylococcal species, plasmid pamy7 exhibited marked differences in the production of alpha-amylase (alpha Amy). Most active for heterospecific alpha Amy production was Staphylococcus aureus. In its culture supernatant nearly half as much alpha Amy activity was found as for the donor strain B. stearothermophilus. All staphylococcal species were able to secrete alpha Amy, since more than 80% of the enzyme activity was found in the culture supernatant. The extracellular alpha Amy of S. aureus [pamy7] was purified to homogeneity. The enzyme exhibited an Mr of approx. 58 000, an optimum activity at pH 5.3-6.3 and at 65 degrees C. Although the enzyme was stable at 65 degrees C for at least 3 h, its thermostability was not unusual. The enzymatic properties of the alpha Amy from S. aureus were similar to those previously reported for various B. stearothermophilus strains. 相似文献
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The present study investigates the effects of a weak (+/-200 microT(pk)), pulsed, extremely low frequency magnetic field (ELF MF) upon the human electroencephalogram (EEG). We have previously determined that exposure to pulsed ELF MFs can affect the EEG, notably the alpha frequency (8-13 Hz) over the occipital-parietal region of the scalp. In the present study, subjects (n = 32) were exposed to two different pulsed MF sequences (1 and 2, used previously) that differed in presentation rate, in order to examine the effects upon the alpha frequency of the human EEG. Results suggest that compared to sham exposure, alpha activity was lowered over the occipital-parietal regions of the brain during exposure to Sequence 1, while alpha activity over the same regions was higher after Sequence 2 exposure. These effects occurred after approximately 5 min of pulsed MF exposure. The results also suggest that a previous exposure to the pulsed MF sequence determined subjects' responses in the present experiment. This study supports our previous observation of EEG changes after 5 min pulsed ELF MF exposure. The results of this study are also consistent with existing EEG experiments of ELF MF and mobile phone effects upon the brain. 相似文献
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An expression system for high-level expression of the native Hordeum vulgare alpha-amylase/subtilisin inhibitor (BASI) has been developed in Pichia pastoris, using the methanol inducible alcohol oxidase 1 (AOX1) promoter. To optimize expression, two codon-optimized coding regions have been designed and expressed alongside the wild-type coding region. To ensure secretion of the native mature protein, a truncated version of the alpha mating factor secretion signal from Saccharomyces cerevisiae was used. In order to be able to compare expression levels from different clones, single insertion transformants generated by gene replacement of the AOX1 gene was selected by PCR screening. Following methanol induction, expression levels reached 125 mgL(-1) from the wild-type coding region while expression from the two codon-optimized variants reached 65 and 125 mgL(-1), respectively. The protein was purified and characterized by Edman degradation, liquid chromatography mass spectrometry and insoluble blue starch assay, and was shown to possess the same characteristics as wild-type protein purified from barley grains. 相似文献
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The expression and secretion of Bacillus amyloliquefaciens alpha-amylase was studied in yeast Saccharomyces cerevisiae. The Bacillus promoter was removed by BAL 31 digestion and three forms of the alpha-amylase gene were constructed: the Bacillus signal sequence was either complete (YEp alpha a1), partial (YEp alpha a2) or missing (YEp alpha a3). Secretion of alpha-amylase into the culture medium was obtained with the complete signal sequence only. The secreted alpha-amylase was glycosylated and its signal peptide was apparently processed. The glycosylated alpha-amylase remained active. The enzyme produced by the other constructions was not glycosylated and thus probably remained in the cytoplasm. 相似文献
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The gene amyE(TV1) codes for a nonglucogenic alpha-amylase from Thermoactinomyces vulgaris 94-2A in Bacillus subtilis.
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B Hofemeister S Knig V Hoang J Engel G Mayer G Hansen J Hofemeister 《Applied microbiology》1994,60(9):3381-3389
We isolated the gene amyE(TV1) from Thermoactinomyces vulgaris 94-2A encoding a nonglucogenic alpha-amylase (AmyTV1). A chromosomal DNA fragment of 2,247 bp contained an open reading frame of 483 codons, which was expressed in Escherichia coli and Bacillus subtilis. The deduced amino acid sequence of the AmyTV1 protein was confirmed by sequencing of several peptides derived from the enzyme isolated from a T. vulgaris 94-2A culture. The amino acid sequence was aligned with several known alpha-amylase sequences. We found 83% homology with the 48-kDa alpha-amylase part of the Bacillus polymyxa beta-alpha-amylase polyprotein and 50% homology with Taka amylase A of Aspergillus oryzae but only 45% homology with another T. vulgaris amylase (neopullulanase, TVA II) recently cloned from strain R-47. The putative promoter region was characterized with primer extension and deletion experiments and by expression studies with B. subtilis. Multiple promoter sites (P3, P2, and P1) were found; P1 alone drives about 1/10 of the AmyTV1 expression directed by the native tandem configuration P3P2P1. The expression levels in B. subtilis could be enhanced by fusion of the amyE(TV1) coding region to the promoter of the Bacillus amyloliquefaciens alpha-amylase gene. 相似文献
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A novel yeast secretion vector utilizing secretion signal of killer toxin encoded on the yeast linear DNA plasmid pGKL1 总被引:4,自引:0,他引:4
M Tokunaga N Wada F Hishinuma 《Biochemical and biophysical research communications》1987,144(2):613-619
The NH2-terminal signal region comprising of approximately 70% length of the prepro-sequence of the pGKL killer precursor protein was found to direct an efficient secretion of the mouse alpha-amylase into the culture medium of Saccharomyces cerevisiae. The alpha-amylase molecule secreted into the culture medium was identified by both immuno-blotting and assay of the enzyme activity. The amount of alpha-amylase secreted via the killer toxin signal was comparable to that directed by the leader sequence of mating factor alpha. The secretion of alpha-amylase using the killer toxin signal was blocked at 37C but not at 25C in sec18-1 host, indicating that alpha-amylase is exported through the normal secretion pathway of S. cerevisiae. 相似文献
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Cloning, sequencing, and expression of the gene encoding extracellular alpha-amylase from Pyrococcus furiosus and biochemical characterization of the recombinant enzyme. 总被引:2,自引:1,他引:2
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The gene encoding the hyperthermophilic extracellular alpha-amylase from Pyrococcus furiosus was cloned by activity screening in Escherichia coli. The gene encoded a single 460-residue polypeptide chain. The polypeptide contained a 26-residue signal peptide, indicating that this Pyrococcus alpha-amylase was an extracellular enzyme. Unlike the P. furiosus intracellular alpha-amylase, this extracellular enzyme showed 45 to 56% similarity and 20 to 35% identity to other amylolytic enzymes of the alpha-amylase family and contained the four consensus regions characteristic of that enzyme family. The recombinant protein was a homodimer with a molecular weight of 100,000, as estimated by gel filtration. Both the dimer and monomer retained starch-degrading activity after extensive denaturation and migration on sodium dodecyl sulfate-polyacrylamide gels. The P. furiosus alpha-amylase was a liquefying enzyme with a specific activity of 3,900 U mg-1 at 98 degrees C. It was optimally active at 100 degrees C and pH 5.5 to 6.0 and did not require Ca2+ for activity or thermostability. With a half-life of 13 h at 98 degrees C, the P. furiosus enzyme was significantly more thermostable than the commercially available Bacillus licheniformis alpha-amylase (Taka-therm). 相似文献
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Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin 总被引:18,自引:4,他引:14
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《The Journal of cell biology》1995,129(3):867-879
Rabbit synovial fibroblasts (RSF) express basal levels of the metalloproteinases (MMP) collagenase, stromelysin-1 and 92-kD gelatinase when plated on intact fibronectin (FN), but elevated levels when plated on either the central RGD-containing cell-binding region of FN (120FN) or antibody against the alpha 5 beta 1 integrin, suggesting that domains outside 120FN may suppress the induction of MMP (Werb, Z., P. M. Tremble, O. Behrendtsen, E. Crowley, and C.H. Damsky. 1989. J. Cell Biol. 109:877-889). We therefore attempted to reconstitute the basal signaling of intact FN by plating RSF on 120FN together with domains of FN outside this region. Large COOH-terminal fragments containing both the heparin-binding and HICS domains suppressed MMP when combined with 120FN. To map the active sequences, peptides from this region and larger fragments that did, or did not, include the CS-1 portion of IIICS were tested. Only CS-1 peptide, or larger fragments containing CS-1, suppressed MMP expression induced by 120FN. In contrast, peptide V from the heparin-binding region, shown previously to stimulate focal contact formation, further enhanced MMP expression by RSF when present on the substrate with 120FN. RSF expressed alpha 4 beta 1 integrin, the receptor for CS-1, and the anti-alpha 4 mAb blocked the ability of CS-1 to suppress MMP induction by 120FN. These results show that signals modulating MMP expression and focal contact assembly are regulated independently, and that cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins plays a dominant role in regulating expression of these extracellular matrix-remodeling genes in response to FN. This work demonstrates directly the modular way in which information in the extracellular matrix is detected and processed by cell surface receptors. 相似文献