Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A simple and widely applicable method for cloning genes involved in glucan biosynthesis is described. An Escherichia coli genomic library was prepared in the low-copy plasmid, pLG339, and E. coli transformants from this library were screened by staining with iodine vapor. Colonies that stained darker than the control were isolated and characterized. The three classes of clones that were identified included: (i) plasmids encoding E. coli glycogen biosynthetic (glg) structural genes, (ii) clones that resulted in elevated glycogen levels, but did not encode glg structural genes or enhance the level of the first enzyme of the pathway, ADPglucose pyrophosphorylase (AGPP), and (iii) clones that enhanced the level of AGPP, but did not encode this enzyme. Two clones from the latter class also enhanced glgC'-'lacZ-encoded beta-galactosidase activity, and may encode factors that regulate the expression of glg structural genes. It should be possible to readily clone glycogen biosynthetic genes from other bacterial species via this method. The method could be made specific for a desired glg gene by using a recipient strain that is defective in the gene of interest.
 ...
PMID:A simple method for cloning genes involved in glucan biosynthesis: isolation of structural and regulatory genes for glycogen synthesis in Escherichia coli. 166 81

Gastric intubation was adopted as a means of comparing the effect of two feeding levels, continuous nutrient supply (C) and restricted nutrient supply (R), on the digestive development of pigs weaned at 14 d of age, during the first 5 d post-weaning. The absolute weights of the stomach and the pancreas were significantly greater (P less than 0.001) in C compared with R pigs. The effect was not significant for pancreas weight when expressed per kg body-weight but was significant (P less than 0.05) for stomach weight. The weights of the small intestine (SI), SI mucosa and total mucosal protein were significantly higher (P less than 0.001) in C pigs but protein content per g mucosa was similar in the C and R groups. There was no significant effect of treatment on the activity of lactase (beta-glucosidase; EC 3.2.1.23) or sucrase (sucrose-alpha-glucosidase; EC 3.2.1.48) irrespective of the basis of comparison used. The specific activity (mumol/min per g protein) of maltase (alpha-glucosidase; EC 3.2.1.20) and of glucoamylase (glucan-1,4-alpha-glucosidase; EC 3.2.1.3) were similar in C and R groups but activities of maltase (mumol/g mucosa) (P less than 0.05), and maltase and glucoamylase (mol/d) (P less than 0.01) were significantly higher in C pigs. Villous height and crypt depth were significantly greater in C pigs (P less than 0.001 and P less than 0.05 respectively). Enteroglucagon was significantly (P less than 0.05) higher in C compared with R pigs. Xylose absorption and the digestibility of energy were not affected by treatment. Digestibility of dry matter, organic matter, crude protein (nitrogen x 6.25) and carbohydrate were significantly higher (P less than 0.001, P less than 0.01, P less than 0.05 and P less than 0.001 respectively) in R pigs compared with C pigs but the differences were small, ranging from 1.3 to 2.5%. These results demonstrate that (1) nutrient intake in the weaned pig affects the anatomy, morphology and function of the gut, (2) there is considerable 'spare capacity' for digestion of cereal-based diets even in pigs weaned at 14 d of age, (3) measurements in vitro of digestive function are of limited value unless supported by information in vivo on absorption/digestibility.
 ...
PMID:Digestive development of the early-weaned pig. 2. Effect of level of food intake on digestive enzyme activity during the immediate post-weaning period. 204 2

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.
 ...
PMID:Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene. 285 22

The Saccharomyces diastaticus structural gene STA2, encoding an exracellular glucoamylase (1,4-alpha-D-glucan glycohydrolase, EC 3.2.1.3.), has been cloned by complementation of a stao strain. A genomic library was initially constructed from a STA2 yeast strain in the yeast Escherichia coli shuttle cosmid vector pYCl. The Sta+ complementing function was further delimited to an 8.3 kb BglII fragment whose restriction map was found to be similar to related genomic regions of STA1 and STA3. Fusions of several DNA fragments derived from the 8.3 kb BglII fragment with a truncated E. coli beta-galactosidase gene resulted in two overlapping fragments that could direct the production of large fusion proteins in E. coli. These fusion proteins were immunoprecipitable by anti-glucoamylase II antibodies, confirming that the Sta+ complementing fusion was due to the expression of a gene that coded for a yeast glucoamylase. Measurements of the STA1, STA2 and STA3 RNA transcripts by RNA-DNA hybridization using an internal fragment of the cloned STA2 gene as the probe indicated that a common transcript of 2.5 kb is produced by each of the STA genes. Integrative disruption of the STA2 gene through homologous recombination was achieved by transforming a STA2 yeast strain to Sta- using an in vitro constructed donor DNA fragment that has the URA3 gene inserted within the coding region of the cloned glucoamylase gene. This was confirmed by tetrad analysis of crosses between strains carrying a disrupted STA2 and a functional STA2. Southern blot analysis using BamHI digested genomic DNA from 15 tetrads demonstrated consistent co-segregation and Mendelian inheritance of the Sta- phenotype with STA2::URA3. These data further confirm that the cloned DNA that showed Sta+ complementing activity carries a functional STA2 gene that encodes the yeast extracellular glucoamylase II.
 ...
PMID:Molecular cloning and characterization of the STA2 glucoamylase gene of Saccharomyces diastaticus. 301 80

A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the beta-galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.
 ...
PMID:A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation. 311 16

Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.
 ...
PMID:Yeast exo-beta-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae. 797 93

Two adjacent, highly homologous endoglucanase genes, celD and celE from Fibrobacter succinogenes S85, which were separated by an AT-rich 223-nucleotide intergenic region were characterized. The celD gene codes for endoglucanase D (EGD), a protein of 668 residues with a molecular mass of 71.7 kDa, while the celE gene encodes endoglucanase E, a protein of 467 amino acids with a molecular mass of 50.7 kDa. Both gene products belong to family 9 of glycosyl hydrolases. EGD displays an array of serine-rich periodic sequences (SRPS) near its C terminus which separate the catalytic domain from a basic terminal domain (BTD) rich in positively charged amino acids. Endoglucanase E has a BTD which is homologous to that of EGD, but it lacks the SRPS and 151 residues present at the N terminus of EGD. The SRPS structures may function as flexible linkers which facilitate interactions between the BTDs and acidic membrane proteins from F. succinogenes S85. The recombinant EGD showed pH and temperature optima of 5.5 and 35 degrees C, respectively. The enzyme cleaved barley-beta-glucan, carboxymethyl cellulose, and acid-swollen cellulose with specific activities of 19.1, 11.5 and 1.7 micromol x min-1 x mg of protein-1, respectively. There was a rapid drop in viscosity during hydrolyses of carboxymethyl cellulose, which is characteristic of an endoglucanase. Glucose was the main hydrolysis product of acid-swollen cellulose. Monospecific polyclonal antibodies against EGD detected the expression of a 68-kDa cellulose-inducible protein corresponding in size to the recombinant EGD in the culture fluid of F. succinogenes S85 and several larger proteins. The celE gene appeared to have little activity when expressed from the beta-galactosidase promoter in pBluescript in Escherichia coli; however, reverse transcriptase PCR analysis with internal primers for the gene revealed that a cellulose-inducible message was made in F. succinogenes, thereby documenting expression of the gene.
 ...
PMID:A novel family 9 endoglucanase gene (celD), whose product cleaves substrates mainly to glucose, and its adjacent upstream homolog (celE) from Fibrobacter succinogenes S85. 897 18

A beta-D-glucosidase has been purified to apparent homogeneity from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seedlings during the mobilization of the xyloglucan stored in the cotyledonary cell walls. The purified protein (Mr 76, 000; a glycoprotein; pl > 9.5; apparent pH optimum 4.5; temperature optimum 30 degrees C) catalysed the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside, cello-oligosaccharides, beta-linked glucose disaccharides, and certain xyloglucan oligosaccharides. Glucose disaccharides with different linkages were hydrolysed at different rates [(1-->3) > (1-->4) > (1-->2) > (1-->6)] with significant transglycosylation occurring in the early stages of the reaction. Cello-oligosaccharide hydrolysis was also accompanied by extensive transglycosylation to give transitory accumulations of higher oligosaccharides. At least some of the glycosyl linkages formed during transglycosylation were (1-->6)-beta. Xyloglucan oligosaccharides xylose-substituted at the non-reducing terminal glucose residue (XXXG, XXLG, XLXG and XLLG, where G is an unsubstituted glucose residue, X is a xylose-substituted glucose residue, and L is a galactosylxylose-substituted glucose residue) were not hydrolysed. Some xyloglucan oligosaccharides with an unsubstituted non-reducing terminal glucose residue (GXXG, GXLG and GXG) were hydrolysed, but others (GLXG and GLLG) were not. This indicated steric hindrance by L but not X substitution at the glucose residue next to the one at the non-reducing end of the oligosaccharide. Hydrolysis of xyloglucan oligosaccharides was not accompanied by transglycosylation. Natural xyloglucan subunit oligosaccharides (XXXG, XXLG, XLXG, XLLG) were totally degraded to their monosaccharide components when treated with nasturtium beta-D-galactosidase. (Edwards et al (1988) J. Biol. Chem. 263, 4333-4337), followed by alternations of nasturtium xyloglucan-specific alpha-xylosidase (Fanutti et al (1991) Planta 184, 137-147) and this enzyme. Several extensively overlapping cDNA clones were obtained by RT-PCR and by screening cDNA libraries. A composite, full-length DNA had an open reading frame of 1962 bp, encoding a polypeptide of 654 amino acids, including all N-terminal and internal sequences obtained from the purified beta-glucosidase protein, and a motif resembling plant signal sequences thought to direct proteins to the cell wall. Database searches revealed homology with beta-glucosidases from several sources (plant, bacteria, yeast), notably with glycosylhydrolases of 'Family 3', according to the classification of Henrissat (Henrissat (1991) Biochem. J. 280, 309-316). There was strong sequence homology with a beta-glucan exo-hydrolase from barley (Hrmova et al. (1996) J. Biol. Chem. 271, 5277-5286). The nasturtium beta-glucosidase is ascribed a role in xyloglucan mobilization, and its interaction with the alpha-xylosidase and the beta-galactosidase is modelled.
 ...
PMID:A xyloglucan oligosaccharide-active, transglycosylating beta-D-glucosidase from the cotyledons of nasturtium (Tropaeolum majus L) seedlings--purification, properties and characterization of a cDNA clone. 974 92

Brucella abortus cyclic glucan synthase (Cgs) is a 316-kDa (2,831-amino-acid) integral inner membrane protein that is responsible for the synthesis of cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. B. abortus Cgs uses UDP-glucose as a sugar donor and has the three enzymatic activities necessary for synthesis of the cyclic polysaccharide (i.e., initiation, elongation, and cyclization). Cyclic glucan is required in B. abortus for effective host interaction and complete expression of virulence. To gain further insight into the structure and mechanism of action of B. abortus Cgs, we studied the membrane topology of the protein using a combination of in silico predictions, a genetic approach involving the construction of fusions between the cgs gene and the genes encoding alkaline phosphatase (phoA) and beta-galactosidase (lacZ), and site-directed chemical labeling of lysine residues. We found that B. abortus Cgs is a polytopic membrane protein with the amino and carboxyl termini located in the cytoplasm and with six transmembrane segments, transmembrane segments I (residues 419 to 441), II (residues 452 to 474), III (residues 819 to 841), IV (residues 847 to 869), V (residues 939 to 961), and VI (residues 968 to 990). The six transmembrane segments determine four large cytoplasmic domains and three very small periplasmic regions.
 ...
PMID:Membrane topology analysis of cyclic glucan synthase, a virulence determinant of Brucella abortus. 1548 31

We have identified a number of ecto-glycanases (glycosylhydrolases) associated with the capsule and/or the cell wall of Cryptococcus neoformans. The enzyme activities detected included alpha-mannosidase, alpha-, and beta -glucosidase, alpha-, and beta-galactosidase, beta-xylosidase, beta-glucuronidase, and endo-beta-1,3-glucanase. Small portions of the enzymes were also secreted into the growth medium. Cell-wall associated endo-beta-1,3-glucanases exhibited highest activity in the acidic range between pH 2.5 and 5.0. The products of laminarin hydrolysis by the enzymes located on the cell surface were glucose and beta-1,3-linked glucooligosaccharides. The same products were released from isolated cell walls incubated in the buffer. Endo-beta-1,3-glucanase activity extracted from the cell surfaces by mild sonication consisted of six isoforms separable by isoelectric focusing. In spite of the presence of the whole array of glycanase activities on the cell surfaces, capsular polysaccharides released from C. neoformans cells into the growth medium were practically metabolically stable. From the defined polysaccharides tested, only laminarin (beta-1,3-glucan) and to some extent also mixed-linkage beta-1,3/beta-1,4-glucan and/or 4-O-methyl-D-glucurono-D-xylan were able to support the yeast growth. The activities of majority of identified ecto-glycanases were low when the yeast was grown on glucose but were considerably elevated when the cells were grown on glycerol indicating that their synthesis is regulated by catabolite repression.
 ...
PMID:Ecto-glycanases and metabolic stability of the capsule in Cryptococcus neoformans. 1713 12


1 2 Next >>