Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Integrative transformation of yeast with gapped DNA fragments results in single or multiple integration into the yeast genome via homologous recombination. A sequence of yeast DNA was found which favours multiple integration even when the strategy of gene replacement is used. This strategy by which the transformed DNA fragment replaces its chromosomal homologue rather than simply integrating into the genome usually occurs as a single exchange event. The described region is unique and lies near a telomere about 5 kb proximal to the SUC4 locus on chromosome XIII. DNA from this region was used as a vehicle for the integration of different SUC genes coding for invertase. Most of the sucrose fermenting transformants isolated carried between two and seven copies of the SUC genes. These transformants overproduced invertase even though there was no selective pressure for high invertase activity in these experiments. I conclude that this region is highly recombinogenic and favours multiple integration of DNA fragments. This region could be used for stable multiple integration of heterologous genes into the yeast genome for over-production of the respective gene product.
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PMID:A region in the yeast genome which favours multiple integration of DNA via homologous recombination. 283 99

The scr genes located on plasmid pUR400 and responsible for sucrose (Scr) metabolism of Escherichia coli K12 and other enteric bacteria have been cloned on a 9.3 kb DNA fragment. The different genes were mapped by transposon insertion mutagenesis, by restriction endonuclease and deletion mapping, and the corresponding gene products were identified. Besides the known structural genes scrA, coding for an EnzymeII(Scr) (45 kD) of the phosphoenolypyruvate-dependent phosphotransferase system (PTS), and scrB, coding for a sucrose 6-phosphate hydrolase (invertase) (55 kD), two new structural genes were discovered. Gene scrK apparently codes for an intracellular and ATP-dependent fructokinase (39 kD), while scrY seems to code for a sucrose porin (58 kD) in the outer cell membrane. No genes for an Enzyme III(Scr) of the PTS or for (a) glycosyltransferase(s) were detected. The four genes form an scr operon (gene order, scrK scrY scrA scrB, transcription from K to B), regulated by a repressor (gene scrR, 37 kD) and inducible by sucrose, fructose and fructose-containing oligosaccharides.
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PMID:Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400. 283 84

The interaction between malnutrition and exposure to a mucosal damaging agent, difluoromethylornithine (DFMO), was examined by monitoring the small-intestinal changes in weanling rats. Malnutrition as induced by the expanded-litter method resulted in severe reduction in body weights in the expanded litters as compared to normal litters. Subsequent treatment of malnourished and well-nourished pups with DFMO for 7 days resulted in decreases in small-intestinal weights and enzyme contents. A 2 factors (well-nourished and malnourished) by 2 factors (DFMO-treated and nontreated) analysis of variance showed no interaction between malnutrition and DFMO treatment in terms of food intake, total mucosal protein, and contents of enterokinase, leucine aminopeptidase and sucrase. Very slight and insignificant interactions (p less than or equal to 0.2) were found for body weights, intestinal weights and total DNA content. Only one parameter studied, the maltase content, showed significant interaction between malnutrition and DFMO treatment (p less than 0.05). Three weeks after the withdrawal of DFMO, essentially all the changes caused by DFMO recovered. But those changes caused by malnutrition did not, such that the malnourished group, whether treated with DFMO or not, still remained significantly less than the control group in their small-intestinal parameters. Analysis of variance showed no interaction between malnutrition and DFMO treatment in the recovery phase. The results suggest that malnutrition is a more important factor in determining the intestinal damage and that malnutrition in the immediate postnatal period does not increase the sensitivity of the small intestine to the damaging effect of DFMO.
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PMID:Interaction of malnutrition and difluoromethylornithine-induced intestinal mucosal damage: degree of severity and subsequent recovery. 285 68

Clathrin-coated membranes are intimately associated with a variety of protein transport processes in eukaryotic cells, yet no direct test of clathrin function has been possible. The data presented demonstrate that Saccharomyces cerevisiae does not require clathrin for either cell growth or protein secretion. Antiserum to the yeast clathrin heavy chain has been used to isolate a molecular clone of the heavy chain gene (CHC1) from a library of yeast DNA in lambda gt11. Clathrin-deficient mutant yeast have been obtained by replacing the single chromosomal CHC1 gene with a disrupted version of the cloned DNA. Cells harboring a nonfunctional chc1 allele produce no immunoreactive heavy chain polypeptide, and vesicles prepared from mutant cells are devoid of clathrin heavy and light chains. Although clathrin-deficient cells grow two to three times more slowly than normal, secretion of invertase occurs at a nearly normal rate. Therefore protein transport through the secretory pathway is not obligately coupled to the formation of clathrin-coated vesicles.
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PMID:A test of clathrin function in protein secretion and cell growth. 286 11

Fractions of isolated epithelial cells were harvested from a segment of porcine jejunum by ten successive incubations with a chelating buffer. The cell fractions showed a progressive decrease in the activity of the brush-border enzymes, alkaline phosphatase and sucrase, with increasing incubation number but a progressive increase in the ability to incorporate labelled thymidine into DNA. Fractions enriched in cells from the crypt region (fractions 9 and 10) contained higher concentrations per mg protein of somatostatin-like immunoreactivity (1.8-fold), glucagon-like immunoreactivity (5.3-fold) and serotonin (3.0-fold) than fractions enriched in cells from the villus tip (fractions 1 and 2). Analysis of extracts of the fractions by gel filtration/radioimmunoassay showed that somatostatin-28 represented the predominant molecular form of somatostatin-like immunoreactivity in all cell fractions but the relative proportion of somatostatin-14 (and related metabolites) to somatostatin-28 was significantly higher (P less than 0.05) in fractions enriched in villus cells (fraction 1 and 2) than in fractions enriched in crypt cells (fractions 5-10). This result suggests that metabolism of somatostatin-28 to somatostatin-14 takes place during migration of the D cell from the crypt base to the villus tip. Heterogeneity in the somatostatin-14 region of the chromatograms indicates that the peptide may be further metabolized by the action of aminopeptidases.
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PMID:Conversion of somatostatin-28 to somatostatin-14 during maturation of epithelial cells in the porcine jejunum. 286 59

A genetic library consisting of over 5000 clones with an average insert size of 6.9 kilobasepairs (kbp) of Streptococcus mutans GS-5 has been constructed in a bivalent plasmid vector pMK3, which is capable of replicating in Escherichia coli and Bacillus subtilis. The recombinant plasmid pSUCRI, containing a 6.0 kbp fragment of S. mutans GS-5 DNA, was the focus of this study. Using Southern hybridization, in vitro and in vivo gene expression techniques, and biochemical analysis, this clone was shown to encode the 55 kiloDalton (kDal) GS-5 gtfA gene product, as well as a 38 and a 66 kDal polypeptide. In addition to the gtfA gene, pSUCRI encodes a dextranase activity with specificity for alpha(1----6)-linked glucans, and with no detectable activity on mutan. The dextranase enzyme had an apparent molecular weight of 66 kDal as demonstrated by SDS-PAGE analysis of the proteins produced by a dextranase-negative deletion derivative. The pH optimum of the enzyme was approximately 6.0, and there was no detectable activity below pH 5.0. By subcloning various combinations of DNA fragments from pSUCRI, it was demonstrated that the dextranase gene (designated dexB) can be separated from the gtfA gene and still be efficiently expressed in both E. coli and B. subtilis. The dexB gene contained its own promoter and ribosome-binding site. The genetic linkage of the gtfA and dexB genes in the S. mutans GS-5 chromosome was confirmed by Southern hybridization and by the independent isolation of four distinct clones containing the gtfA gene and common flanking sequences. In addition to a glucosyltransferase and dextranase, an invertase-like activity is also encoded on pSUCRI, indicating that there is a cluster of genes on the S. mutans GS-5 chromosome which is devoted to the dissimilation of sucrose and concomitant synthesis or modification of glucans into a water-insoluble form, perhaps constituting an operon for glucan modification which can be coordinately regulated in response to environmental alterations.
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PMID:Tight genetic linkage of a glucosyltransferase and dextranase of Streptococcus mutans GS-5. 294 34

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation.
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PMID:Isolation and characterization of unusual gin mutants. 297 1

A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis.
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PMID:Molecular cloning and characterization of scrB, the structural gene for the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system sucrose-6-phosphate hydrolase. 300 99

The Streptococcus mutans GS-5 gene, scrB, coding for sucrose 6-phosphate hydrolase activity has been cloned into Escherichia coli utilizing the bacteriophage replacement vector lambda L47.1. DNA sequences containing the gene were initially subcloned into the moderate-copy-number plasmid vector pLG339 to yield active subclones. However, due to the instability of the resultant chimeric plasmids, the gene was subsequently subcloned into the low-copy-number vector pOU61 to yield the stable hybrid plasmid pMH613. Both plasmids contain a 6.6-kilobase EcoRI fragment from strain GS-5 and express both hydrolase and sucrase activities. The relative position of the gene in the insert has been determined after Tn5 mutagenesis and deletion analysis. The cloned enzyme was purified to near homogeneity after gel filtration and anion-exchange chromatography, chromatofocusing, and preparative polyacrylamide gel electrophoresis. The purified enzyme displayed a molecular mass of 58 kilodaltons, which is significantly higher than the 48-kilodalton enzyme previously purified from S. mutans GS-5. These results suggest that processing of the hydrolase occurs in S. mutans.
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PMID:Isolation and characterization of the sucrose 6-phosphate hydrolase gene from Streptococcus mutans. 301 64

The Streptococcus mutans LM7 gene gtfA was cloned in Escherichia coli along with flanking regions of the chromosome as a fragment representing 10.3 kilobases (kb) of streptococcal DNA. Restriction endonuclease mapping revealed that the cloned DNA consisted of four EcoRI fragments with gtfA sucrase activity localized to one fragment, EcoRI-B (2.4 kb). Subsequent analysis with E. coli minicells indicated that three polypeptides were encoded on the 10.3-kb insert (55 [GtfA], 45, and 35 kilodaltons). Neither the 45- nor 35-kilodalton polypeptide exhibited any detectable sucrase activity. The approximate positions and directions of transcription of the two larger proteins were determined from minicell protein profiles displaying truncated versions of these polypeptides. The restriction endonuclease data for the cloned gtfA gene were used to develop a strategy for insertional inactivation of this locus in vivo. An internal HincII fragment of the gtfA gene was removed and replaced with a DNA fragment containing a tetracycline resistance determinant. This new recombinant plasmid was linearized and then transformed into S. mutans GS5 and S. mutans V403 where it was incapable of replication. It was predicted that Tcr colonies would result from double-crossover recombinational events involving homologous regions flanking the gtfA gene. This was verified by Southern DNA hybridization analyses. The inactivation of the gtfA gene in both S. mutans GS5 and S. mutans V403 resulted in a decrease of water-soluble exopolysaccharide but no detectable changes in the amounts of water-insoluble polymers.
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PMID:Molecular organization and expression of the gtfA gene of Streptococcus mutans LM7. 301 93


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