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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a beta-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in delta pH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-beta-D-thiogalactoside.
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PMID:Inhibition of growth of Escherichia coli by lactose and other galactosides. 703 92

A study of the beta-galactosidase produced by the psychrotrophic bacterium Buttiauxella agrestis has been carried out. This micro-organism was isolated from raw milk and the enzyme isolated using standard methods. Molecular mass was estimated to be 515 kDa. The isoelectric point was close to 4.45. Optimum pH was 7.25. Maximal activity was observed at 50 degrees C and activation energy was estimated to be 39.1 kJ mol-1. Lactose enhanced thermal stability. Using p-nitrophenyl-beta-D-galactopyranoside as the substrate, the Km was 11 mumol l-1 and Vmax was 85 U mg-1 protein. beta-Mercaptoethanol and ethanol were inhibitors; glycerol acted as a complex effector. The enzyme required divalent cations for activity while it was inhibited by EDTA. When the enzyme was immobilized in diethyl aminoethylcellulose the optimum pH of activity was 8. Km was 47 mumol l-1 and Vmax was 96 U mg-1 protein.
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PMID:Isolation and properties of free and immobilized beta-galactosidase from the psychorotrophic enterobacterium Buttiauxella agrestis (strain NC4). 761 19

The fitnesses conferred by seven lactose operons, which had been transduced into a common genetic background from natural isolates of Escherichia coli, were determined during competition for growth rate-limiting quantities of galactosyl-glycerol, a naturally occurring galactoside. The fitnesses of these same operons have been previously determined on lactose and three artificial galactosides, lactulose, methyl-galactoside and galactosyl-arabinose. Analysis suggests that although marked genotype by environment interactions occur, changes in the fitness rankings are rare. The relative activities of the beta-galactosidases and the permeases were determined on galactosyl-glycerol, lactose, lactulose and methyl-galactoside. Both enzymes display considerable kinetic variation. The beta-galactosidase alleles provide no evidence for genotype by environment interactions at the level of enzyme activity. The permease alleles display genotype by environment interactions with a few causing changes in activity rankings. The contributions to fitness made by the permeases and the beta-galactosidases were partitioned using metabolic control analysis. Most of the genotype by environment interaction at the level of fitness is generated by changes in the distribution of control among steps in the pathway, particularly at the permease where large control coefficients ensure that its kinetic variation has marked fitness effects. Indeed, changes in activity rankings at the permease account for the few changes in fitness rankings. In contrast, the control coefficients of the beta-galactosidase are sufficiently small that its kinetic variation is in, or close to, the neutral limit. The selection coefficients are larger on the artificial galactosides because the control coefficients of the permease and beta-galactosidase are larger. The flux summation theorem requires that control coefficients associated with other steps in the pathway must be reduced, implying that the selection at these steps will be less intense on the artificial galactosides. This suggests that selection intensities need not be greater in novel environments.
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PMID:A molecular investigation of genotype by environment interactions. 770 23

The lacI gene has been used as a target gene in various mutation assays. We modified single strand conformation polymorphism (SSCP) analysis by introducing restriction digestion to detect mutations in the gene rapidly, and determined the sensitivity of the method. The entire coding sequence and partial promoter region of the lacI gene were amplified by the polymerase chain reaction with [alpha-32P]dCTP in a 1247 base pair fragment, digested into eight restriction fragments, and analyzed by SSCP. The sensitivity of the method was assessed using 160 phages with lacI mutations, which were selected by assay of expression of beta-galactosidase after their infection into E. coli. Of the 160 mutants, 146 (91.3%) showed shifted bands in the first condition of SSCP analysis (without glycerol, 20 degrees C). The remaining 14 mutants were analyzed in a second condition (with 5% glycerol, 20 degrees C), and eight of them showed shifted bands (cumulatively 96.3% of the 160 mutants). The remaining six mutants were analyzed in a third condition (with 5% glycerol, 10 degrees C), and all of them showed shifted bands (cumulatively 100%). Sequencing of the restriction fragments with mobility shifts in the 160 mutants revealed 108 kinds of mutations, 100 (92.6%) being detected in the first condition, seven (cumulatively 99.1%) in the second condition, and one (cumulatively 100%) in the third condition. This method greatly reduced the time to identify lacI mutations, and allowed the detection of multiple mutations in one lacI mutant. The results also show that in general PCR-SSCP analysis is very sensitive when test fragments are shorter than about 250 base pairs and electrophoresis is performed under at least two conditions.
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PMID:A rapid method for detection of mutations in the lacI gene using PCR-single strand conformation polymorphism analysis: demonstration of its high sensitivity. 775 92

Catabolite repression (CR) of xylose utilization by Bacillus subtilis involves a 14-bp cis-acting element (CRE) located in the translated region of the gene encoding xylose isomerase (xylA). Mutations of CRE making it more similar to a previously proposed consensus element lead to increased CR exerted by glucose, fructose, and glycerol. Fusion of CRE to an unrelated, constitutive promoter confers CR to beta-galactosidase expression directed by that promoter. This result demonstrates that CRE can function independently of sequence context and suggests that it is indeed a generally active cis element for CR. In contrast to the other carbon sources studied here, glucose leads to an additional repression of xylA expression, which is independent of CRE and is not found when CRE is fused to the unrelated promoter. This repression requires a functional xylR encoding Xyl repressor and is dependent on the concentrations of glucose and the inducer xylose in the culture broth. Potential mechanisms for this glucose-specific repression are discussed.
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PMID:Catabolite repression of the Bacillus subtilis xyl operon involves a cis element functional in the context of an unrelated sequence, and glucose exerts additional xylR-dependent repression. 813 69

The ability to introduce DNA into mammalian cells has provided a powerful means to examine the regulation of gene expression and the function of gene products. However, the most commonly used techniques for DNA transfection are not always suitable for primary cells. Primary human keratinocytes are particularly stringent in their growth requirements and are also very refractory to transfection, rendering transient gene expression studies difficult. We have investigated the ability of several polycationic lipids to promote DNA uptake into human epidermal keratinocytes, as monitored with the bacterial beta-galactosidase reporter gene. We report that the cationic lipopolyamine dipalmitoyl phosphatidylethanolamine spermine as well as another procedure using Polybrene can achieve a 20% to 30% transfection efficiency, superior to any other agent tested on these cells. Gene transfer was accomplished by a 3-h exposure of monolayer cells to DNA complexes formed with either reagent by simple mixing in a serum-free medium, followed by a brief osmotic shock with glycerol. Neither DNA carrier showed any toxicity at the effective concentrations nor interfered with cell attachment, growth or differentiation. The use of a fully biodegradable lipopolyamine as DNA carrier should make it possible to extend this transfection method to gene transfer for in vivo therapeutic applications.
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PMID:High-efficiency transfection of primary human keratinocytes with positively charged lipopolyamine:DNA complexes. 817 62

The gene YAT1 from Saccharomyces cerevisiae encodes a protein of 688 amino acids which displays significant sequence similarity to vertebrate L-carnitine acyltransferases and yeast inner mitochondrial L-carnitine acetyltransferase. Steady state levels of the respective mRNA are low during growth on glucose or galactose, derepressed on glycerol, and significantly induced when ethanol or acetate are the only carbon sources. The YAT1 promotor region confers the identical carbon source dependence also on the expression of beta-galactosidase when cloned 5' to the Escherichia coli lacZ-coding region. An antiserum directed against a beta-galactosidase/Yat1p fusion protein recognizes a protein of 78-kDa molecular mass in the mitochondrial fraction from yeast. In vitro translated Yat1p, which lacks a cleavable presequence, binds to mitochondria in a protease-sensitive location in a standard in vitro import assay. Deletion of the single copy gene in a haploid yeast strain yields no obvious phenotype with any carbon source. Carnitine acetyltransferase activities of intact mitochondria from a YAT1 deletion mutant are slightly lower than wild type, but are approximately alike in lysed mitochondria from mutant and control cells. Thus, the novel gene is nonessential and likely to code for a minor mitochondrial outer carnitine acetyltransferase.
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PMID:The ethanol-inducible YAT1 gene from yeast encodes a presumptive mitochondrial outer carnitine acetyltransferase. 826 85

A thioic O-acid ester-containing sulfolipid (thionsulfolipid) was isolated from cells of picoplankton cyanobacterium, Synechococcus sp. The lipid accounted for about 0.2% of the lyophilized cells. The lipid was subjected to mild alkaline hydrolysis, and the structures of the hydrolysis products were identified by infrared spectra, mass and nuclear magnetic resonance spectrometries, as fatty acids and hexadecane-, hexadecene- and tetradecanethioic S-acids. Thioic S-acid was further confirmed by the synthesis of hexadecanethioic S-acid from palmitoylchloride and hydrogen sulfide. The positional distribution of the thioic acid ester in the lipid was determined by beta-galactosidase, sulphur-oxygen exchange reaction using silver nitrate, and lipase hydrolysis of the diacylglycerol derived from the lipid. The structure of the thionsulfolipid was identified as 6-sulfo-alpha-D-quinovopyranosyl(1-->1')-2'-O-acyl-3'-O-thioacy l -2-glycerol. When cells of HL 60, as a human lymphoma, were cultured with thionsulfolipid, 61% of the cell growth was inhibited at the concentration of 200 micrograms/ml. The lipid was toxic against minnows (Tanichtys albonubes). The LD50 was 20 ppm. Thioic O-acid ester-containing lipid (thionsulfolipid) has not been found in any other photosynthetic organisms.
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PMID:Thioic O-acid ester in sulfolipid isolated from freshwater picoplankton cyanobacterium, Synechococcus sp. 833 48

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.
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PMID:Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli. 841 12

In order to study transcriptional regulation of hepatic genes during development, a method for transfer of fusion genes to primary cultures of fetal hepatocytes was required. The aim of this study was to assess currently available transfection methods and optimize the best method for use with cultured fetal hepatocytes. The Rous sarcoma virus 5' long terminal repeat controlling transcription of the beta-galactosidase reporter gene (pRSV lac Z II) was used to assess electroporation, lipofection, DEAE-dextran and calcium phosphate transfection in cultured primary fetal hepatocytes. The success of transfection was determined by histochemical detection and quantitation of beta-galactosidase activity. Results showed that calcium phosphate transfection was optimal for fetal hepatocytes with respect to beta-galactosidase activity and cell survival. For maximum transfection of cells, 10 micrograms/ml DNA, HEPES buffered saline transfection buffer at pH 7.05 and a 24 hr expression period for the reporter gene were employed. Glycerol shock did not increase transfection efficiency significantly. The method was simplified by adding calcium chloride solution to DNA diluted in transfection buffer and the resulting co-precipitate added directly to the medium covering the cells. Transfection 24 hr after initial culture and a precipitate incubation time of 20 hr were optimal. The suitability of this method was confirmed with a liver-specific promoter controlling beta-galactosidase and chloramphenicol acetyltransferase expression. In conclusion this study shows that a modified calcium phosphate transfection method is most effective for transferring DNA to primary cultured fetal hepatocytes. It is concluded that this method is appropriate for use with fetal hepatocytes and will facilitate studies of gene regulation during liver development.
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PMID:Calcium phosphate transfection and cell-specific expression of heterologous genes in primary fetal rat hepatocytes. 867 28


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