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At least 1,387 records · Page 77Linked to original sources
PubMed1997

Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro.

A microtiter assay for drug evaluation has been developed with a strain of Toxoplasma gondii that expresses bacterial beta-galactosidase. By using chlorophenol red-beta-D-galactopyranoside (CPRG) as the substrate for beta-galactosidase, the efficacy of a drug against the parasite can be determined with a colorimetric readout. Drugs known to have activity against T. gondii (specifically, pyrimethamine, sulfadiazine, atovaquone, and clindamycin) were tested, and efficacies were determined by CPRG cleavage. The 50% inhibitory concentrations determined by the CPRG-based colorimetric assay were similar to those determined by the traditional radiolabelled uracil incorporation assay. Since CPRG is nontoxic to the parasite, viable drug-treated parasites can be obtained at the conclusion of the assay for further evaluation if desired. This assay provides a high-throughput and nonradioactive alternative for the identification of anti-T. gondii compounds.

Animals↗
PubMed2001

Use of a recombinant strain of Mycobacterium avium expressing beta-galactosidase to evaluate the activities of antimycobacterial agents inside macrophages.

A reliable and low-cost method that enables rapid screening of the activity exerted by new antimicrobial agents on intracellularly growing Mycobacterium avium has been developed. To this aim, a recombinant (lacZ) strain of M. avium expressing the Escherichia coli beta-galactosidase gene was used to evaluate, in murine macrophages, the susceptibility of M. avium to common antimycobacterial agents. beta-Galactosidase levels, measured in the presence of each of the antibiotics tested, were closely correlated with the number of CFU recovered from the M. avium lacZ strain-infected macrophages.

Animals↗
PubMed1997

Inhibition of beta-galactosidase biosynthesis in Escherichia coli by tetracycline residues in milk.

Low levels of tetracyclines found as residues in milk inhibited the biosynthesis of beta-galactosidase in Escherichia coli. To produce the same effect, other antibacterials had to occur in concentrations that were more than 10-fold higher. This relative selectivity was exploited for the development of a screening test for tetracyclines in milk based on a chemiluminometric assay of beta-galactosidase. The method was validated with spiked samples of raw milk and applied to field samples contaminated with tetracyclines.

Animals↗
PubMed2000

Evaluation of p-naphtholbenzein-beta-D-galactoside as a substrate for bacterial beta-galactosidase.

We describe the synthesis of a new substrate for the detection of beta-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) and cyclohexenoesculetinbeta-D-galactoside (CHE-Gal). Of 206 Enterobacteriaceae strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed p-naphtholbenzein-beta-D-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of beta-galactosidase.

Chromogenic Compounds↗
PubMed1971

Salmonella screening procedure with tests for beta-galactosidase and flagellar antigens.

Differentiation of Salmonella from other gram-negative bacilli requires several biochemical and serological tests. A simplified 24-hr screening procedure has been devised which allows discarding of large numbers of isolates (picked from selective plating media) before they are subjected to this extensive testing. Cultures of gram-negative organisms isolated to triple sugar-iron slants during routine examination of products for Salmonella were tested for the presence of beta-galactosidase and Salmonella flagellar antigens. beta-Galactosidase-positive cultures which did not agglutinate in polyvalent flagellar antiserum were considered to be nonsalmonellae. Of 1,103 Salmonella cultures tested, none of the 61 different serotypes was missed by this procedure, whereas 673 (82.3%) of 818 nonsalmonellae were excluded from further testing. This screening procedure eliminates most nonsalmonellae and augments the proportion of cultures undergoing further biochemical and serological testing which will be confirmed as Salmonella.

Agglutination Tests↗
PubMed1998

Expression of the extracellular domain of the human immunodeficiency virus type 1 envelope protein and its fusion with beta-galactosidase in Saccharomyces cerevisiae.

Two envelope glycoprotein gene fragments were cloned from the proviral genome of the HXB2 isolate of human immunodeficiency virus (HIV). For the production of the two domains of the envelope gene product these cloned gene fragments were inserted into an Escherichia coli-yeast inducible shuttle vector fused to the galactokinase (GAL1) promoter. Cell extracts from strains of Saccharomyces cerevisiae harboring these two vectors (pYENV1 and pYENV2) were found to contain a specific protein with a size of 50 kDa when induced by galactose, while the protein could not be detected in extracts from control cells containing only the E. coli-yeast vector in the presence of galactose. Furthermore, another expression plasmid coding for fusion proteins from the majority of the external envelope glycoprotein (gp120) moiety and a large part of the beta-galactosidase was constructed. Antibodies from HIV type 1-positive sera could react with recombinant fusion polypeptides. Transformants could produce this fusion protein to a level of about 1.6% of the total protein content, as deduced from beta-galactosidase activity.

Cloning, Molecular↗
PubMed1972

Induction and characterization of -galactosidase in an extreme thermophile.

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.

Acetone↗
PubMed1972

High-level production of -galactosidase by Escherichia coli merodiploids.

Two merodiploids of Escherichia coli that contain genes for the lac operon on both chromosome and episome were tested for production of lac enzymes after growth on various carbon sources. The specific activity of beta-galactosidase (and of thiogalactoside transacetylase) was about twice that from haploid cells when grown on glycerol. With succinate as carbon source, the specific activity increased by an additional factor of 3. Up to 25% of the soluble cell protein is beta-galactosidase in these strains, one of which is inducible and the other constitutive. The enzyme is purified easily in high yield by ammonium sulfate fractionation and electrophoresis.

Acetyltransferases↗
PubMed1974

Beta-galactosidase from termination and deletion mutant strains.

beta-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the beta-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg(-)) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg(-) and Deg(+) strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.

Amino Acid Sequence↗
PubMed1976

Transcriptional and post-transcriptional control of beta-galactosidase synthesis.

lac messenger ribonucleic acid synthesis is dissociated from its translation in certain lac operon mutants. More beta-galactosidase messenger ribonucleic acid (as determined by hybridization) is made in fully induced lacOc mutants and in trp-lac fusions than is eventually translated into enzyme. Consequently, beta-galactosidase synthesis may be post-transcriptionally controlled under these conditions.

Cyclic AMP↗
PubMed1977

Degradation of Escherichia coli beta-galactosidase fragments in protease-deficient mutants of Salmonella typhimurium.

The degradation rates of several mutationally generated fragments of Escherichia coli beta-galactosidase were determined in wild-type strains of Salmonella typhimurium and in mutant Salmonella strains lacking several proteases and peptidases. Three termination fragments (produced by lacZ545, lacZ521, and lacZX90) and one internal reinitiation (restart) fragment [lacZpi(1)] are degraded in wild-type Salmonella strains at the same rates observed in wild-type Escherichia coli strains. Mutations that lead to loss of peptidases N, A, B, P, and Q or to loss of protease I or II do not affect the decay rates of any of these fragments. In addition, all of these peptidases and proteases are present in E coli mutants carrying deg mutations (deg mutations are known to stabilize beta-galactosidase fragments). Apparently, none of the proteases and peptidases that are currently accessible to direct genetic analysis plays a role in the early steps of the degradation of protein fragments.

Escherichia coli↗
PubMed1999

Regulation of beta-galactosidase expression in Bacillus megaterium DSM319 by a XylS/AraC-type transcriptional activator.

The beta-galactosidase-encoding bgaM gene of Bacillus megaterium DSM319 and the divergently orientated bgaR operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of bgaM; a potential regulator binding site overlaps the bgaM promoter. A mechanism for regulation of beta-galactosidase expression in B. megaterium is proposed.

Bacillus megaterium↗
PubMed2006

Identification of a novel alpha-galactosidase from the hyperthermophilic archaeon Sulfolobus solfataricus.

Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable alpha-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for alpha-linked galactosides, which are optimally hydrolyzed at pH 5 and 90 degrees C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable alpha-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.

Enzyme Activation↗
PubMed1965

Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli.

Ben-Hamida, Fakher (Washington University School of Medicine, St. Louis, Mo.), and David Schlessinger. Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli. J. Bacteriol. 90:1611-1616. 1965.-Synthesis of beta-galactosidase stops within several minutes when preinduced, permeaseless cultures are diluted into medium containing 40 mug/ml of 5-fluorouracil (5-FU) but no inducer. However, if inducer (isopropylthiogalactoside) is left in the medium, enzyme formation in the presence of 5-FU continues for at least 11 min. Thus, inducer may increase the differential metabolic stability of the corresponding messenger ribonucleic acid (RNA; defined as the capacity to produce measurable enzyme) in inducible strains. However, such an interpretation requires that 5-FU rapidly arrest the further synthesis of messenger RNA competent to form active enzyme. C(14)-5-FU, like uracil, does appear to enter cells without measurable lag, saturating the pool of uracil nucleotides, and thereby the messenger RNA being formed, within several minutes. That 5-FU acts very quickly is also supported by the similar continuation of enzyme synthesis in the presence of inducer and antibiotics (actinomycin D and proflavine) which shut off all RNA synthesis, as well as by the response to 5-FU of enzyme synthesis in various constitutive mutants.

Acridines↗
PubMed1966

Properties and regulation of the beta-D-galactosidase in Shigella dysenteriae and in Escherichia coli-Shigella dysenteriae hybrids.

Sarkar, S. (Massachusetts Institute of Technology, Cambridge). Properties and regulation of the beta-d-galactosidase in Shigella dysenteriae and in Escherichia coli-Shigella dysenteriae hybrids. J. Bacteriol. 91:1477-1488. 1966.-Shigella dysenteriae strain 60 has a beta-d-galactosidase related to that of Escherichia coli but more heat-sensitive and with a turnover number about 10 times lower. Hybridization by transduction produces strains with enzymes of intermediate properties by recombination within the z gene. Both E. coli and S. dysenteriae have a regulatory i(+) gene. Recombination between i(-) mutants of the two organisms leads to restoration of the i(+) genotype. In S. dysenteriae 60, most of the i(-) mutants are subject to genetic suppression by suppressor mutations at unlinked loci. The effect of these suppressors on the products of the suppressed i(-) genes is discussed.

Centrifugation↗
PubMed1969

Characterization of a beta-galactosidase formed between a complementary protein and a peptide synthesized de novo.

In a cell-free system, phi80dlac can be transcribed, and the resulting ribonucleic acid can be translated to yield a product which interacts with an enzymatically inactive z protein to produce active enzyme. The inactive z protein is produced by Escherichia coli strain 21, which contains a deletion in the first part of the gene for beta-galactosidase and appears to exist as a dimer. The enzyme formed in the cell-free system appears to be composed of one strain 21 z protein dimer and one newly synthesized polypeptide chain with a molecular weight of about 3 x 10(4). The estimated size of this complementing segment is in good agreement with Ullmann, Jacob, and Monod's estimate of the size of the alpha region of beta-galactosidase. Using alpha fragments produced by autoclaving or guanidine treatments, we found that the active portion of alpha seems to be smaller than the full alpha region. We also found, using alpha produced by the autoclaving technique, that active dimer undergoes conversion to tetramer as the amount of alpha is increased. Evidently, the binding of alpha favors this conversion, but it is unlikely that the conversion of dimer to tetramer per se results in increased enzyme activity.

Bacterial Proteins↗
PubMed1993

Evaluation of a genetically engineered cell line and a histochemical beta-galactosidase assay to detect herpes simplex virus in clinical specimens.

A novel histochemical method was compared with a cytopathic effect (CPE) assay for the identification of herpes simplex virus (HSV) in clinical specimens. The method utilizes a stably transformed cell line (BHKICP6LacZ-5) that expresses beta-galactosidase only after infection with HSV. A total of 96 specimens submitted to our diagnostic virology laboratory were analyzed. Thirty-one specimens contained HSV as evidenced by positive CPE, and all were positive for beta-galactosidase staining. CPE were not evident for 2 or more days in 15 of the 31 positive specimens, whereas the histochemical stain was positive in all 31 positives by 16 to 24 h. This preliminary study shows that the BHKICP6LacZ-5 cell line can be used in a rapid, sensitive, and specific assay for the detection of HSV in clinical specimens.

Animals↗