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)

The galactose metabolism positive regulatory gene from Kluyveromyces lactis, LAC9, has been isolated through its ability to activate expression of galactose metabolism enzyme genes in Saccharomyces cerevisiae. The LAC9 gene also activates expression of the S. cerevisiae alpha-galactosidase (MEL1) and K. lactis beta-galactosidase (LAC4) genes in S. cerevisiae. Although LAC9-activated gene expression in K. lactis is not glucose repressed, activation of MEL1 gene expression by LAC9 in S. cerevisiae is. The LAC9 gene is expressed at an extremely low level as a approximately 2.9-kb mRNA, and encodes a protein of 865 amino acids. Although the LAC9 gene is functionally analogous to the S. cerevisiae GAL4 gene, the bulk of its protein sequence shows little homology to that of GAL4. Two of the three regions of homology that do exist, however, are restricted to areas of GAL4 protein already implicated in nuclear localization, DNA binding, and transcriptional activation.
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PMID:Analysis of the Kluyveromyces lactis positive regulatory gene LAC9 reveals functional homology to, but sequence divergence from, the Saccharomyces cerevisiae GAL4 gene. 302 34

A Kluyveromyces lactis mutant defective in lac9 cannot induce beta-galactosidase or galactokinase activity and is unable to grow on lactose or galactose. When this strain was transformed with the GAL4 positive regulatory gene of Saccharomyces cerevisiae it was able to grow on lactose or galactose as the sole carbon source. Transformants bearing GAL4 exhibited a 4.5-h generation time on galactose or lactose, versus 24 h for the nontransformed lac9 strain. A K. lactis lac9 strain bearing two integrated copies of GAL4 showed 3.5-fold induction of beta-galactosidase activity and 1.8-fold induction of galactokinase activity compared with 15.6-fold and 4.4-fold induction, respectively, for the LAC9 wild-type strain. In transformants bearing 10 integrated copies of GAL4, the induced level of beta-galactosidase was nearly as high as in the LAC9 wild-type strain. In addition to restoring lactose and galactose gene expression, GAL4 in K. lactis lac9 mutant cells conferred a new phenotype, severe glucose repression of lactose and galactose-inducible enzymes. Glucose repressed beta-galactosidase activity 35- to 74-fold and galactokinase activity 14- to 31-fold in GAL4 transformants, compared with the 2-fold glucose repression exhibited in the LAC9 wild-type strain. The S. cerevisiae MEL1 gene was repressed fourfold by glucose in LAC9 cells. In contrast, the MEL1 gene in a GAL4 lac9 strain was repressed 20-fold by glucose. These results indicate that the GAL4 and LAC9 proteins activate transcription in a similar manner. However, either the LAC9 or GAL4 gene or a product of these genes responds differently to glucose in K. lactis.
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PMID:GAL4 of Saccharomyces cerevisiae activates the lactose-galactose regulon of Kluyveromyces lactis and creates a new phenotype: glucose repression of the regulon. 310 45

The Gal4, Gal80, and Gal3 proteins of Saccharomyces cerevisiae constitute a galactose-responsive regulatory switch for GAL gene promoters. The low cellular levels of these proteins have hampered mechanistic studies and limit the utility of the GAL gene promoters for high-yield production of endogenous and exogenous proteins. We have constructed two new vectors, pMEGA2 and pMEGA2-DeltaURA3, that increase the level of the Gal4p-Gal80p-Gal3p switch proteins under conditions that preserve the Gal3p-Gal80p-Gal4p stoichiometries required for normal switch function. Cells carrying pMEGA2 show 15- to 20-fold more Gal4p and 30- to 40-fold more Gal3p and Gal80p than cells lacking pMEGA2. These high levels of Gal4p, Gal80p, and Gal3p do not perturb the integrity of galactose-inducible regulation. Cells that carry pMEGA2 exhibit normal galactose-induction kinetics for the chromosomal MEL1 gene expression and normal, albeit slower, log-phase growth. Insertion of the MEL1 gene into pMEGA2 provides a 24- to 30-fold increase in the Mel1 protein. Cells carrying a 2-microm-based URA3-selectable plasmid containing a GAL1pro:lacZ reporter gene and a second plasmid, pMEGA2-DeltaURA3, produce 12-fold more beta-galactosidase than cells carrying only the GAL1pro:lacZ reporter plasmid. The performance of the MEGA plasmids in providing amplified production of the Gal3, Gal80, and Gal4 proteins should prove useful in investigations of the mechanistic aspects of these transcription switch proteins and in work aimed at achieving high-level, galactose-regulatable production of proteins in yeast.
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PMID:Vectors allowing amplified expression of the Saccharomyces cerevisiae Gal3p-Gal80p-Gal4p transcription switch: applications to galactose-regulated high-level production of proteins. 1068 51

UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators. The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene. Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator. Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration. Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations. It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids.
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PMID:Zero background yeast reporter plasmids. 1077 44

We describe a novel assay format for the Gal4-based yeast two-hybrid-system, in which the readout from three different reporter genes is measured sequentially in a single microplate. Activation of the URA3, MEL1, and lacZ reporters in response to a protein-protein interaction is monitored by measuring sequentially: (i) growth in medium lacking uracil, (ii) alpha-galactosidase activity, and (iii) beta-galactosidase. The data thus generated permit elimination of many false positive signals and provide a preliminary measurement of reporter activation-strength that may be confirmed by further analysis. The assay procedure is inexpensive and requires few liquid-handling steps. It is appropriate for automated high-throughput interaction mating assays, validation of putative interactor strains and hybrid-protein self-activator tests.
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PMID:"One plate/three-reporter" assay format for the detection and validation of yeast two-hybrid interactions. 1556 Jan 40