Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.
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PMID:Isolation and characterization of protoplasts from Saccharomyces rouxii. 43 20

Raffinose hydrolysis was studied in Saccharomyces rouxii. The responsible enzyme was identified as a beta-fructofuranosidase (EC 3.2.1.26), which has a pH optimum of 5.5 and a K(m) of 83 mM for raffinose. This enzyme was cryptic in cells from a 3-day culture. A 2-min treatment with 0.1 volume of ethyl acetate in sodium acetate buffer (pH 6) gave complete expression of the enzyme, which was still retained by the cell. Ghosts were prepared by modifying membrane structure with small basic proteins in distilled water, and after washing they showed the full complement of enzymatic activity. The enzyme remained cryptic in osmotically protected spheroplasts; however, after lysis (by dilution) release, as well as expression, was effected. Mechanical disruption of fresh cells revealed and released all of the enzyme. Cells in early stationary phase had all of their beta-fructofuranosidase in a cryptic state. Aging yielded fractional expression of activity; initially this was proportional to cell death, but later the degree of expression exceeded the death rate. Media from aged cultures or cell-free extracts of aged cells were not effective in revealing the cryptic enzyme of younger cells. S. rouxii beta-fructofuranosidase has a different autolytic-release pattern from its counterpart in S. cerevisiae. Also, high concentrations of glucose do not repress the S. rouxii enzyme. The beta-fructofuranosidase in young cells of S. rouxii must be enclosed by the protoplasmic membrane or a special vesicular structure. This system was compared with other Saccharomyces species in connection with the translocation of enzymes across the protoplasmic membrane.
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PMID:Expression of cryptic beta-fructofuranosidase in Saccharomyces rouxii. 445 87

A recombinant cosmid carrying the sucrase gene (sacA) was obtained from a colony bank of E. coli harboring recombinant cosmids representative of the B. subtilis genome. It was shown that the sacA gene is located in a 2kb EcoRI fragment and that the cloned sequence is homologous to the corresponding chromosomal DNA fragment. A fragment of 2kb containing the gene was subcloned in both orientations in the bifunctional vector pHV33 and expression was further looked for in B. subtilis and E. coli. Complementation of a sacA mutation was observed in Rec+ and REc- strains of B. subtilis. Expression of sucrase was also demonstrated in E. coli, which is normally devoid of this activity, by SDS-polyacrylamide gel electrophoresis, specific immunoprecipitation and assay of the enzyme in crude extracts. The specific activity of the enzyme depended on the orientation of the inserted fragment. The saccharolytic activity was found to be cryptic in E. coli since the presence of the recombinant plasmids did not allow the transport of [U14C] sucrose and the growth of the cells. It was shown also that the recombinant cosmid contained part of the neighboring locus (sacP) which corresponds to a component of the PEP-dependent phosphotransferase system of sucrose transport of B. subtilis.
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PMID:Cloning and expression in Escherichia coli of the sucrase gene from Bacillus subtilis. 681 36

The synthesis of beta-fructofuranosidase in synchronously dividing cells of S. rouxii was continuous (as opposed to periodic) throughout the budding cycle and followed the increase in cell mass. Similar patterns for cell mass and enzyme increases were observed even in phosphate-deprived cells which did not divide. The beta-fructofuranosidase activity remained physically cryptic throughout the cell cycle as evidenced by analyses on equilibrium density gradient fractions. The beta-fructofuranosidase activity released from mechanically disrupted cells resisted sedimentation when subjected to 131 000 g for 1 h, thus ruling out membrane association. Ethyl acetate was routinely employed to break the crypticity barrier. Enzyme in cell-free extract or in cells was equally sensitive to inactivation at pH values below 5 in the presence of ethyl acetate, which suggested that this is an inherent property of the enzyme in question and not a reflection of proteolytic inactivation. The status of beta-fructofuranosidase in selected species of Saccharomyces was compared with that for S. rouxii and a close similarity with S. bisporus var. mellis was noted. The degree of crypticity encountered in genetically defined strains of S. cerevisiae (e.g. X2180 a/alpha) was relatively high (42%) compared with that for commercially derived bakers' and brewers' strains (about 6%). Extant data on the cryptic beta-fructofuranosidase of S. rouxii are evaluated and the utility of this system for studying enzyme translocation is discussed.
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PMID:The cryptic beta-fructofuranosidase of Saccharomyces rouxii. 711 Jan 26

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6, 868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 x 10(6) CFU/microg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10(4) to 10(7) CFU/microg of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.
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PMID:Characterization of pRGO1, a plasmid from Propionibacterium acidipropionici, and its use for development of a host-vector system in propionibacteria. 1105 11

Analysis of flagellin genes was carried out on strains of Salmonella Typhimurium, Salmonella Hadar, Salmonella Abortusequi, Salmonella Enteritidis and Salmonella Gallinarum serovars, using a PCR system designed in this study. The purpose of these studies was to explore the flagellin genes of biphasic and monophasic Salmonellae for future targeted genetic interventions. The PCR primers were designed for two different structural genes of flagellin (fliC, fljB), for the repressor of fliC (fljA), for the operator region of fliC, and for the invertase system responsible for phase variation in Salmonella (hin, hixL, hixR). PCR analysis revealed that all of the examined genes (fliC, fliC-operator, fljB, fljA, hin, hixL, hixR) were present in all S. Typhimurium (n = 10) and S. Hadar (n = 10) strains tested. The results obtained on S. Typhimurium and S. Hadar strains confirmed their biphasic character at DNA level. However, the S. Enteritidis (n = 46) and S. Gallinarum (n = 5) strains lacked the invertase system (hin, hixL, hixR) as well as the fljA and fljB genes, while fliC and its operator were detectable. Consequently, the S. Enteritidis strains could only express fliC gene resulting in phase H1 flagellin. The examined S. Gallinarum strains were also demonstrated to have a cryptic flagellin gene (fliC). On the other hand, PCR results on S. Abortusequi (n = 2) indicated that both flagellin genes (fliC, fljB) and the whole phase variation system were present in both strains tested but only the H2 phase gene (fljB) was expressed. The phenotype of these strains could be clarified by motility test and/or by classical flagellar serology. The findings are also substantiated by the results of serovar-specific PCR for S. Typhimurium and S. Enteritidis. In conclusion, the PCR system developed in this study proved to be suitable for characterisation of Salmonella flagellin genes and confirmed serological results regarding all S. Typhimurium, S. Hadar and S. Enteritidis strains. This system could also identify cryptic flagellar genes of S. Abortusequi and S. Gallinarum.
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PMID:Development of a PCR system for the characterisation of Salmonella flagellin genes. 1595 75

The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells.
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PMID:Effects of Polyenes, Detergents, and Other Potential Membrane Perturbants on an Osmotolerant Yeast, Saccharomyces rouxii. 1634 36

We report the complete nucleotide sequence and characterization of a cryptic plasmid, pHLHK26, recovered from a strain of Laribacter hongkongensis isolated from a patient with community acquired gastroenteritis. pHLHK26 consists of 8700 bp, with G + C content 51.3%. The copy number (mean +/- SD) is 0.57 +/- 0.07 and it is stable after four passages (about 240 generations) in the absence of selection. There is a predicted origin of replication that consists of a DnaA box and five 22-bp direct repeats. pHLHK26 has four ORFs with two genes encoded in the sense direction and the other two in antisense direction. These four ORFs encode a putative plasmid partitioning protein of the ParA family, a putative protein that contains putative ADP-ribose 1"-phosphatase activity belonging to the Appr-1-p processing enzyme family, a putative recombinase (TniR) of the resolvase/invertase family, and a putative replication protein, respectively. We speculate that pHLHK26 is a theta, possibly Class A, replicative plasmid, as it contains an origin of replication with AT-rich region, a number of iterons and a DnaA box and a gene that encodes a replicative protein most homologous to those of other theta replicative plasmids and it shares eight of the nine positions of the consensus sequence TTAT(C/A)CA(C/A)A (TTTTCCACA in pHLHK26) in the DnaA boxes observed in other classical examples of Class A plasmids of this group.
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PMID:Characterization of a novel cryptic plasmid, pHLHK26, in Laribacter hongkongensis. 1761 58

Suppressor tRNAs induce expression of additional (off-frame) genes coded by stopless genetic codes without lengthening genomes, decreasing DNA replication costs. RNA 3'-to-5' polymerization by tRNAHis guanylyltransferase suggests further cryptic code: hypothetical 'invertases' polymerizing in the 3'-to-5' direction, advancing in the 5'-to-3' direction would produce non-complementary RNA templated by regular genes, with different coding properties. Assuming 'invertase' activity, BLAST analyses detect GenBank-stored RNA ESTs and proteins (some potentially coding for the hypothesized invertase) for human mitochondrial genes. These peptides' predicted secondary structures resemble their GenBank homologues'. 3'-to-5' EST lengths increase with their self-hybridization potential: Single-stranded RNA degradation perhaps limits 3'-to-5' elongation. Independent methods confirm predicted 3'-to-5' overlapping genes: (a) Presumed 3'-to-5' overlapping genes avoid codons belonging to circular codes; (b) Spontaneous replicational deamination (mutation) gradients occur at 3rd codon positions, unless these are involved in overlap coding, because mutations are counter selected in overlapping genes. Tests a and b converge on predicted 3'-to-5' gene expression levels. Highly expressed ones include also fewer stops, and mitochondrial genomes (in Primates and Drosophila) adapt to avoid dependence of 3'-to-5' coding upon antitermination tRNA activity. Secondary structure, circular code, gradient and coevolution analyses yield each clear positive results independently confirming each other. These positive results (including physical evidence for 3'-to-5' ESTs) indicate that 3'-to-5' coding and invertase activity is an a priori improbable working hypothesis that cannot be dismissed. Note that RNAs produced by invertases potentially produce triple-stranded DNA:RNA helices by antiparallel Hoogsteen pairings at physiological pH, as previously observed for mitochondrial genomes.
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PMID:Overlapping genes coded in the 3'-to-5'-direction in mitochondrial genes and 3'-to-5' polymerization of non-complementary RNA by an 'invertase'. 2299 21