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)

Hybrid transducing phages were constructed in vitro that carry the galK gene fused to each of three ribosomal promoters: the promotor for an rRNA operon (rrnE); the promoter for the spec r protein operon and the promotor for the alpha r protein operon. We also constructed hybrid transducing phages that carry the IacZ gene fused to the promoter for the rrnE operon or to the promoter for the spc r protein operon. The amounts of galactokinase (or beta-galactosidase) were analyzed in lysogens carrying these various transducing phages grown in several different growth media. The synthesis rate of galactokinase (or beta-galactosidase) from the fused rrn-gal (or rrn-lac) operon relative to the total protein synthesis rate increased with increasing growth rate, as expected from the transcriptional activity of rRNA operons. In contrast, the relative synthesis rate of galactokinase (or beta-galactosidase) from the operon fused to alpha or spc r protein promoter remained approximately constant with increasing growth rate. These results were interpreted to mean that the characteristic increase in the relative synthesis rate of r protein with increasing growth rate is determined not by transcription regulatory mechanisms, but by posttranscriptional mechanisms, which presumably involve the feedback inhibition of r protein mRNA translation by free r proteins.
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PMID:Growth-rate-dependent regulation of ribosome synthesis in E. coli: expression of the lacZ and galK genes fused to ribosomal promoters. 679 40

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.
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PMID:Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid. 681 93

Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were not observed. However, a typical biphasic growth was observed on a mixture of glucose and galactose under aerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploid distiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higher ethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeast grown under aerobic conditions. A constant proportion (10%) of the population retained the plasmid throughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobic conditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount of lactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partial galactokinase activity.
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PMID:Fermentation of lactose by yeast cells secreting recombinant fungal lactase. 828 14

In the budding yeast Kluyveromyces lactis glucose repression of genes involved in lactose and galactose metabolism is primarily mediated by LAC9 (or K1GAL4) the homologue of the well-known Saccharomyces cerevisiae transcriptional activator GAL4. Phenotypic difference in glucose repression existing between natural strains are due to differences in the LAC9 gene (Breunig, 1989, Mol.Gen.Genet. 261, 422-427). Comparison between the LAC9 alleles of repressible and non-repressible strains revealed that the phenotype is a result of differences in LAC9 gene expression. A two-basepair alteration in the LAC9 promoter region produces a promoter-down effect resulting in slightly reduced LAC9 protein levels under all growth conditions tested. In glucose/galactose medium any change in LAC9 expression drastically affects expression of LAC9 controlled genes e.g. those encoding beta-galactosidase or galactokinase revealing a strong dependence of the kinetics of induction on the LAC9 concentration. We propose that in tightly repressible strains the activator concentration drops below a critical threshold that is required for induction to occur. A model is presented to explain how small differences in activator levels are amplified to produce big changes in expression levels of metabolic genes.
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PMID:Glucose repression of lactose/galactose metabolism in Kluyveromyces lactis is determined by the concentration of the transcriptional activator LAC9 (K1GAL4) [corrected]. 844 21

In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed. Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the -35 region of the metR promoter. Starting with a metE-lacZ.metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence. Each mutation, along with the wild-type metE-lacZ.metR-galK gene fusion, was cloned into phage lambda gt2. Regulation of the metE and metR genes was examined by measuring beta-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions. Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.
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PMID:MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region. 848 40

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.
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PMID:Expression of the extracellular domain of the human immunodeficiency virus type 1 envelope protein and its fusion with beta-galactosidase in Saccharomyces cerevisiae. 966 73

Thermotoga maritima represents one of the few hyperthermophilic bacteria currently known. The chromosomal alpha-galactosidase gene of T. maritima strain MSB8 has been cloned and its nucleotide sequence was determined. The gene, designated galA, has coding capacity for a 552 residue polypeptide with a calculated molecular mass of 63,653 Da. GalA was found to be flanked by other genes probably involved in galactoside breakdown and utilization. The previously sequenced beta-galactosidase gene, lacZ, is localized immediately upstream of galA while two open reading frames that putatively encode enzymes of galactose catabolism, i.e. galactose-1-phosphate uridylytransferase (galT) and galactokinase (galK), were found downstream of galA. The identified genes are extremely close together or even overlap and have the same orientation, so they could all be part of one galactoside utilization operon of T. maritima MSB8. GalA displayed low-level amino acid sequence similarity with alpha-galactoside of glycosyl hydrolase family 36. However, GalA is smaller than the other members of this enzyme family. The galA gene was expressed in Escherichia coli and the recombinant alpha-galactosidase was purified and characterized. The molecular mass of the recombinant enzyme was estimated at about 62 kDa by denaturting gel electrophoresis. Maximal hydrolysis of the chromogenic substrate p-nitrophenyl-alpha-D-galactopyranoside was measured at pH 5.0-5.5 and 90-95 degrees C (5 min assay). Divalent cations were not required for activity. The enzyme released galactose from raffinose, melibiose and the synthetic substrates p-nitrophenyl-and omicron-nitrophenyl-alpha-D-galactopyranoside. The T. maritima alpha-galactosidase thus was highly specific for the galactose moiety and the alpha-anomeric configuration of the glycosidic linkage. Its extreme thermal stability (t 1/2 = 6.5 h at 85 degrees C) makes this enzyme an interesting candidate for biotechnological applications.
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PMID:Properties of an alpha-galactosidase, and structure of its gene galA, within an alpha-and beta-galactoside utilization gene cluster of the hyperthermophilic bacterium Thermotoga maritima. 974 Nov 5

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.
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PMID:Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius. 1055 56

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.
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PMID:Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis. 1062 Jul 56

A plasmid clone that suppresses galactose toxicity in a gal7 yeast strain has been isolated from a multicopy genomic DNA library. Molecular analysis revealed that the region responsible for the suppression of galactose toxicity corresponds to the ORF YPR030w, which was named MRG19. A CEN-based plasmid carrying the above ORF was unable to suppress the toxicity. Galactokinase activity was substantially reduced in cell extracts obtained from transformants bearing multiple copies of MRG19. Multiple copies of MRG19 were also able to suppress galactokinase expression driven by the CYC1 promoter but not the TEF1 promoter. Multiple copies of MRG19 could not suppress GAL1-driven galactokinase expression in a gal80 strain. However, MRG19-mediated suppression of CYC1-driven galactokinase expression was independent of GAL80 function. These results imply that multiple copies of MRG19 suppress galactokinase expression probably at the level of transcription. In agreement with this idea, multiple copies of MRG19 also suppress beta-galactosidase expression driven by the GAL1 promoter in a GAL80-dependent manner. Disruption of MRG19 leads to an increase in the cell density at stationary phase in synthetic complete medium. MRG19 encodes a previously uncharacterised 124-kDa protein that shows no sequence homology to any known proteins.
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PMID:Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80-dependent manner in Saccharomyces cerevisiae. 1066 72


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