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)

The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins. We have used the signal sequence from the S. diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene. In frame STA2/lacZ gene fusions have been constructed and expressed in S. cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters. Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space. Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase.
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PMID:Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene. 251 42

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.
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PMID:GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae. 308 56

We have suggested previously from Northern blot analysis that transcription of the negative regulatory gene GAL80 was controlled positively by another regulatory gene GAL4, and negatively by GAL80 itself, in similar way to GAL1, GAL7 and GAL10 genes encoding galactose-metabolizing enzymes in Saccharomyces cerevisiae. To study further the controlled expression of GAL80, we have exploited the gene fusion technique. We constructed gene fusions consisting of 5' fragments of GAL80 and a 5' truncated lacZ of Escherichia coli, and introduced the GAL80'-'lacZ fusions into wild-type yeast or various GAL4 or GAL80 mutants using multiple-copy or single-copy plasmid vectors. We then studied beta-galactosidase activity in the resultant transformants under uninduced, induced or glucose-repressed conditions. Expression of the GAL80'-'lacZ fusions was clearly under the control of Gal4/Gal80. Next we constructed GAL7'-'lacZ fusions, whose upstream activating sequence (UAS) from GAL7 was replaced with a GAL80 fragment containing a UAS-like sequence located in the 5' flanking region of GAL80. Synthesis of beta-galactosidase directed by the hybrid genes was inducible by galactose exactly like the original GAL7'-'lacZ fusion with a UAS from GAL7. Finally we constructed a GAL7-GAL80 hybrid gene, in which the entire 5' flanking region was derived from GAL7. When the chromosomal GAL80 gene in wild-type yeast was replaced with the hybrid gene, the uninduced level, but not the induced level, of the GAL10-encoded enzyme (uridine diphosphoglucose-4-epimerase) was significantly increased.
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PMID:Autogenous regulation of the Saccharomyces cerevisiae regulatory gene GAL80. 330 97

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.
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PMID:Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage. 355 Apr 29

We isolated and identified mutant strains of Kluyveromyces lactis that are defective for the Leloir pathway enzymes galactokinase, transferase, and epimerase, and we termed these loci GAL1 , GAL7 , and GAL10 , respectively. Genetic data indicate that these three genes are tightly linked, having an apparent order of GAL7 - GAL10 - GAL1 . This same gene order has been observed in Saccharomyces cerevisiae. Strains harboring gal7 mutations have elevated levels of beta-galactosidase, coded by an unlinked gene, galactokinase, and epimerase activities under uninduced conditions. We investigated the genetic basis of this constitutive gene expression and found no recombinants between the constitutive and Gal- phenotypes among 76 tetrads, suggesting that either GAL7 or a tightly linked gene codes for a regulatory function. This is the second gene that has been shown to specifically coregulate expression of the genes coding for beta-galactosidase and the Leloir pathway enzymes.
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PMID:Genetic and biochemical characterization of the galactose gene cluster in Kluyveromyces lactis. 632 19

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.
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PMID:Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG. 639 52

An integrated GAL1-lacZ fusion provided a useful phenotypic marker for the gal80- regulatory mutation in Saccharomyces cerevisiae. On minimal glucose plates containing a beta-galactosidase indicator, a GAL80 strain containing the fusion gave white colonies, whereas a gal80- strain gave blue colonies. This color difference was used to isolate the GAL80 gene from a plasmid bank by complementation of the gal80- mutant. The putative GAL80 gene was located on a 2.6-kb HindIII-SalI fragment and has been subcloned into an integrating vector. Genetic analysis showed that the clone integrated at the GAL80 locus. A deletion that covered the entire GAL80 region was constructed in vitro and transplaced into the yeast genome to give an isogenic pair of GAL80 and gal80 deletion strains. Glucose repression of a GAL1-lacZ fusion was normal in the gal80 deletion strain, implying that the GAL80 gene product is not involved in glucose repression.
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PMID:Molecular cloning of the GAL80 gene from Saccharomyces cerevisiae and characterization of a gal80 deletion. 639 3

Previous studies using in vitro procedures have not clearly established whether the estrogen receptor (ER) acts as a monomer or dimer in the cell. We have used the yeast two-hybrid system as an in vivo approach to investigate the dimerization of the estrogen receptor in the absence and presence of estrogen and anti-estrogens. This system is independent of ER binding to the estrogen response element. Two vectors, expressing GAL4 DNA binding domain-human ER and GAL4 transactivation domain-human ER, were constructed. Control experiments showed that each fusion protein had a high affinity binding site for estradiol-17 beta and could transactivate an ERE-LacZ reporter gene in yeast similar to the wild type ER. The two fusion proteins, GAL4 DB-hER and GAL 4 TA-hER, were expressed in the yeast strain, PCY2, which carries a GAL1 promoter-lacZ reporter. ER dimerization was measured via reconstitution of GAL4 through interaction of the fusion proteins, which transactivates LacZ through the GAL1 promoter. When both ER fusion proteins were expressed, beta-galactosidase activity was estradiol-17 beta-inducible. Furthermore, we showed that both tamoxifen and ICI 182,780 also induced beta-galactosidase activity, albeit lower than that induced by estradiol-17 beta. These results strongly argue that ER dimerization is ligand-dependent and the dimer can be induced by estradiol-17 beta, tamoxifen, or ICI 182,780. We also treated the yeast containing the two fusion proteins with estradiol-17 beta and tamoxifen or ICI 182,780 simultaneously to determine the effects on ER dimerization. beta-Galactosidase activity was lower when the yeast was treated with a higher ratio of tamoxifen or ICI 182,780 to estrogen than estradiol-17 beta alone. Taken together, we conclude that ER dimerization is ligand (estradiol-17 beta, tamoxifen, or ICI 182, 780)-dependent, and we suggest that estradiol-17 beta-induced dimers are destabilized when estradiol-17 beta is used with tamoxifen or ICI 182,780 simultaneously.
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PMID:Yeast two-hybrid system demonstrates that estrogen receptor dimerization is ligand-dependent in vivo. 755 88

The advantages of gratuitous induction for GAL-regulated cloned gene (lacZ) product synthesis were evaluated for the yeast Saccharomyces cerevisiae. The growth, yield, and productivity of a gratuitous (gal1) strain were compared with those of an otherwise isogenic, nongratuitous (GAL1) strain. Batch studies clearly demonstrated the improvements possible in product synthesis when the inducer is not metabolized by the yeast cells; both beta-galactosidase specific and volumetric activities were superior for the gal1 strain. At equivalent metabolizable sugar concentrations, the productivity of the gratuitous strain exceeded that of the nongratuitous strain by 180%. The effects of initial inducer concentration and induction time were also examined. For the gratuitous strain, galactose:glucose ratios as low as 0.1 still gave maximum beta-galactosidase volumetric activity. A 5-fold higher ratio was necessary for full induction with the nongratuitous strain, and productivity was substantially lower relative to the gal1 strain. A comparison of various times for galactose addition indicated that productivity is highest when the gratuitous culture is induced for the entire batch fermentation.
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PMID:Enhanced productivity through gratuitous induction in recombinant yeast fermentations. 776 24

The effects of residual catabolite repression and the importance of induction timing were determined for a temperature-sensitive (ts) GAL-regulated stable yeast expression system. The Saccharomyces cerevisiae strain employed carries a reg1 mutation inhibiting catabolite repression, and a ts mutation enabling induction of the regulated GAL promoters by a temperature shift to 35 degrees C. Despite the reg1 mutation and induction method, glucose depressed lacZ expression from a GAL1 promoter during batch culture. beta-Galactosidase specific activity was consistently lower at higher initial glucose concentrations in both SDC (semi-defined) and YPDa (complex) media; decreases of 18-36% were observed as glucose concentration was increased between 1, 3, 5, and 10 g l-1. However, the reductions in beta-galactosidase specific activity due to residual catabolite repression were more than balanced by substantial improvements in biomass yield at higher glucose levels. Therefore, productivity rose with increasing glucose concentration; in YPDa medium, increasing initial glucose from 1 to 10 g l-1 resulted in a 2.6-fold increase in beta-galactosidase volumetric activity. Due to the negative effects of shifting temperature to 35 degrees C, the trade-offs between optimum growth and a lengthy induction period were also evaluated. Delaying the time of induction reduced final specific activities but improved cell yield, and waiting 14 h into batch culture to induce lacZ expression provided modest 9-15% improvements in overall productivity.
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PMID:Catabolite repression and induction time effects for a temperature-sensitive GAL-regulated yeast expression system. 776 17


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