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 Cdc25p and Sdc25p proteins were the first members of the family of guanine nucleotide exchange factors to be identified. These proteins promote the formation of active Ras-GTP complex from inactive Ras-GDP complex by exchange of GDP for GTP. Therefore Cdc25p which is the main positive regulator of Ras, regulates through Ras the activity of adenylate cyclase in Saccharomyces cerevisiae. The amino-terminal part of Cdc25p has a sequence similar to the cyclin destruction box (CDB) of mitotic cyclins. This sequence has been reported to be required for ubiquitin-dependent proteolysis. In this study we show that Cdc25p is an unstable polypeptide with a half-life of 15-20 min. Its instability depends upon the presence of the CDB which can also confer instability to other proteins. Degradation of Cdc25p and CDB containing beta-galactosidase was found to be independent of various cell cycle arrest points. The fast degradation of Cdc25p opens the possibility that Ras and the cAMP cascade in yeast are directly modulated by the cellular content of the guanine nucleotide exchange factor rather than variation in activity or localization control.
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PMID:The cellular content of Cdc25p, the Ras exchange factor in Saccharomyces cerevisiae, is regulated by destabilization through a cyclin destruction box. 765 56

Current assays for functional activation of Gs-coupled receptors usually involve quantitation of adenylyl cyclase or measurement of cAMP concentration by radioimmunoassay. The activation of Gq-coupled receptors is commonly assayed by measurement of the production of inositol triphosphate or diacylglycerol from phosphatidylinositol 4,5-bisphosphate or of changes in intracellular calcium. These assays generally require large numbers of cells (10(5)-10(6)) and/or the use of radioactive materials. We have developed a rapid nonradioactive colorimetric assay that utilizes a beta-galactosidase (lacZ) gene fused to five copies of the cyclic AMP response element (CRE) to detect the activation of CRE-binding protein that results from an increase in intracellular cAMP or calcium. This assay can be performed using as few as 30,000 cells in a 96-well format with the end products measured simultaneously in a microplate reader. Consequently, a single individual can readily assay 1000 samples a day. Using this assay, the fold increase in beta-galactosidase activity was similar in magnitude to increases in cAMP or adenylyl cyclase activity and was approximately linear from 0.01 to 0.27 fmol/cell of intracellular cAMP. Furthermore, pharmacological characterization of one of the melanocortin receptors, mMC5-R, using this assay resulted in a similar order of potency for several melanocortin peptides to that obtained with a commonly used adenylyl cyclase enzyme assay. This assay is also useful for the characterization of Gq-coupled receptors as is demonstrated here using cells transfected with the mouse bombesin receptor. The large-scale capacity of this assay makes it an excellent method for screening molecules of interest acting on Gs- and Gq-coupled receptors.
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PMID:A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. 779 37

A corticotropin-releasing hormone (CRH) and cAMP-responsive region (-236/-133) in the rat POMC gene promoter previously reported to confer CRH/cAMP responsiveness to heterologous reporter constructs has been characterized. DNAse footprint analysis revealed that multiple elements in this region were bound by nuclear proteins from the POMC expressing AtT20 cells. When these individual DNA elements were separately tested in heterologous reporter constructs for CRH induction, only one element, designated PCRH-RE (POMC CRH responsive element, -171/-160) was found to give strong CRH stimulation (5- to 7-fold). This element appears novel as to the possible binding factors, although it has homology to the mouse metallothionein metal regulatory element. Gel shift analyses of the PCRH-RE with AtT20 cell nuclear extracts showed marked stimulation of retarded nucleoproteins following CRH stimulation, suggesting that the possible binding factor(s) may mediate transcriptional regulation at this site. The activity of PCRH-RE binding protein was inhibited by divalent cations, with Cu2+ and Cd2+ being most effective; Zn2+ had no effect, indicating that this binding factor(s) is functionally distinct from the metallothionein metal regulatory element binding protein. A 2.6 kilobase cDNA clone encoding a protein (PCRH-REB-1) binding to this element was isolated by Southwestern screening of an AtT20 expression library with radiolabeled PCRH-RE oligonucleotides. This clone was used to isolate several other cDNA clones to determine the sequence corresponding to the entire coding region of the protein (PCRH-REB), which proved to be identical to a recently described DNA binding protein of the replication factor C complex, mRFC140/Mouse Southwestern. Primer extension and Northern blot analysis revealed that the size of the full length mRNA is about 4.9 kilobases. PCRH-REB mRNA expression is not restricted to corticotrophs but is present in a broad tissue distribution as evaluated by reverse transcription polymerase chain reaction analysis. A bacterially expressed beta-galactosidase-PCRH-REB-1 fusion protein was shown to bind PCRH-RE efficiently. Furthermore, binding of the PCRH-REB-1 fusion protein to the POMC CRH-responsive element was inhibited by divalent cations with similar sensitivities to those observed using AtT20 nuclear extracts. The predicted PCHR-REB protein sequence presents several interesting motifs: one p-Loop motif (ATP binding site), nine protein kinase A phosphorylation sites (implying a possible role in responding to the CRH-induced cAMP signal), and regions of homology to proteins involved in DNA replication and repair. PCRH-REB is, therefore, a potential transacting factor binding to a major CRH-responsive element in the POMC promoter.
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PMID:Characterization of a corticotropin-releasing hormone-responsive element in the rat proopiomelanocortin gene promoter and molecular cloning of its binding protein. 785 55

Aeromonas caviae, often reported to be associated with diarrhoeal patients, elaborates several virulence factors as well as catabolic enzymes such as xylanase and beta-galactosidase. Studies on the kinetics of growth of A. caviae and synthesis of beta-galactosidase suggested that the activity was cell associated and reached a peak during the late logarithmic phase of growth. The optimum pH for beta-galactosidase activity was 7.0 and required Ca2+ and glutathione for enhancement of its activity; IPTG also slightly improved the activity. Aerobic cultivation of A. caviae in LB containing glucose, fructose, maltose and sucrose completely inhibited the activity possibly due to acetic acid production. Addition of 100 mM cAMP to the media containing glucose (0.25%, w/v) restored the relative activity by 8.8%; however, the final pH of the media remained acidic. Aerobic growth of A. caviae with other carbon sources did not affect beta-galactosidase activity, probably as there was no acid production and thereby the final pH of the media unaltered. Arabinose, xylose and galactose induced the A. caviae beta-galactosidase activity by several folds and lactose moderately enhanced its activity.
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PMID:Factors influencing beta-galactosidase activity of Aeromonas caviae. 793 8

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.
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PMID:A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli. 793 25

The Escherichia coli serC-aroA operon encodes biosynthetic enzymes for unrelated amino acid biosynthetic pathways leading to the synthesis of serine and the aromatic amino acids. A serC-aroA-lac translational fusion was constructed in the vector pMC1403. Synthesis of beta-galactosidase from the serC-aroA-lac fusion was found to be enhanced in the presence of lactose as the sole carbon source. This enhancement was not observed in strains containing a cya or crp mutant. However, the exogenous addition of cAMP greatly increased the beta-galactosidase synthesis in the cya mutant strain. The serC-aroA mRNA content, analyzed by a dot blot assay, also appeared to increase in the serC+ aroA+ cells after the exogenous addition of cAMP. These findings unambiguously indicate that the expression of the serC-aroA operon is positively controlled by cAMP.
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PMID:Cyclic AMP-dependent expression of the Escherichia coli serC-aroA operon. 801 34

Escherichia coli induces the expression of more than 50 proteins in response to starvation for a carbon source. Strains MC7 (csi7::phoA) and MC19 (csi19::phoA) contain fusions of a signal peptide-deficient phoA reporter sequence to a csi (carbon starvation-inducible) gene. PhoA expression increased when these strains were deprived of a carbon source or entered stationary phase but did not when the cells were deprived of a nitrogen source or subjected to osmotic, oxidative or thermal stress. Mapping and sequence analysis of the cloned phoA fusions in strains MC7 and MC19 indicated that they had occurred in different locations within the same previously unidentified gene. The wild-type allele of this gene was cloned and the encoded protein was found to be a new lipoprotein. Therefore we propose to call this locus slp (starvation lipoprotein). The 22 kDa Slp protein is associated with the outer membrane fraction. The slp gene was located at 78.6 centisomes on the E. coli genetic map. The -10 and -35 regions upstream of the mRNA start site were characteristic of a sigma 70 promoter. The major transcript from this promoter was sufficiently large to contain slp sequences but not the downstream open reading frame. Induction of beta-galactosidase activity from a slp::lacZ translational fusion during carbon starvation or stationary phase was independent of cAMP, RpoS (KatF) and DnaK, all of which are known to affect the expression of certain starvation-inducible or stationary phase-inducible proteins.
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PMID:Characterization of the carbon starvation-inducible and stationary phase-inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli. 802 77

We have established a transgenic model to facilitate the study of stress-induced gene regulation in the hypothalamus. This model, which uses a human proenkephalin-beta-galactosidase fusion gene, readily permits anatomic and cellular colocalization of stress-regulated immediate early gene products (e.g. Fos) and other transcription factors [e.g. cAMP response element-binding protein (CREB)] with the product of a potential target gene. Moreover, Fos provides a marker of cellular activation that is independent of the transgene. Hypertonic saline stress induced Fos in almost all cells in the PVN that exhibited basal expression of the proenkephalin transgene; however, all cells in which the transgene was activated by stress also expressed Fos. CREB was found in essentially all neurons. Gel shift analysis with and without antisera to Fos and CREB showed that AP-1 binding activity, containing Fos protein, was induced by hyperosmotic stress. However, Fos was not detected binding to the proenkephalin second messenger-inducible enhancer even in hypothalamic cell extracts from stressed animals. In contrast, CREB formed specific complexes with both the proenkephalin enhancer and a cAMP- and calcium-regulated element (CaRE) within the c-fos gene. Moreover, we found that hypertonic saline induced CREB phosphorylation in cells that express the transgene within the paraventricular nucleus and supraoptic nucleus. These results suggest a model in which proenkephalin gene expression in the paraventricular nucleus is regulated by CREB in response to hypertonic stress.
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PMID:Molecular mechanisms of stress-induced proenkephalin gene regulation: CREB interacts with the proenkephalin gene in the mouse hypothalamus and is phosphorylated in response to hyperosmolar stress. 817 Apr 80

The cAMP-inducible prespore gene, PL3, encodes a protein which is a novel component of the spore coat. Unlike the well-characterized spore coat proteins (SP60, SP70, and SP96) which are found in the outer layer of the coat, the PL3 gene product is localized to a subregion of the coat beneath the outer proteinaceous layer. Moreover, a substantial portion of the PL3 protein is tightly associated with the spore coat and not released under the conditions that led to the identification of the other coat proteins. The promoter for this novel spore coat gene is described. Unlike the other coat-protein gene promoters, it lacks the extensive CA-type elements. It contains two short CA boxes and five prominent G-rich regions. Sequential deletions from the 5' end of the promoter which remove both CA boxes as well as two of the G-rich regions reduce the level of expression but do not alter the spatial regulation of expression. Despite the sequence differences, the PL3 promoter still confers correct spatial, temporal, and cell type-specific regulation on a reporter gene. Escherichia coli beta-galactosidase enzyme activity expressed under the control of this PL3 promoter first appears in randomly isolated cells at the loose mound stage. Because of the sensitivity of the assay, beta-galactosidase activity is detectable prior to the appearance of the PL3 protein on Western blots and by immunofluorescence. Later the number of cells staining for beta-galactosidase activity and the intensity of staining increases. During tipped mound, slug, and culminant stages, cells expressing beta-galactosidase under the control of the PL3 promoter are localized to prespore regions and are spatially coincident with cells expressing the PL3 protein.
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PMID:The promoter of a gene encoding a novel Dictyostelium spore coat protein. 817 86

We and others have previously shown that cAMP-dependent protein kinase (PKA) activity is essential for aggregation, induction of prespore gene expression and multicellular development in Dictyostelium. In this manuscript, we further examine this regulatory role. We have overexpressed the Dictyostelium PKA catalytic subunit (PKAcat) in specific cell types during the multicellular stages, using prestalk and prespore cell-type-specific promoters to make PKA activity constitutive in these cells (independent of cAMP concentration). To examine the effects on cell-type differentiation, we cotransformed the PKAcat-expressing vectors with reporter constructs expressing lacZ from four cell-type-specific promoters: ecmA (specific for prestalk A cells); ecmB (specific for prestalk B and anterior-like cells in the slug); ecmB delta 89 (specific for stalk cells); and SP60 (prespore-cell-specific). By staining for beta-galactosidase expression histologically at various stages of development in individual strains, we were able to dissect the morphological changes in these strains, examine the spatial localization of the individual cell types, and understand the possible roles of PKA during multicellular development. Expression of PKAcat from either the ecmA or ecmB prestalk promoters resulted in abnormal development that arrested shortly after the mound stage, producing a mound with a round apical protrusion at the time of tip formation. Prestalk A and prestalk B cells were localized in the central region and the apical mound in the terminal differentiated aggregate, while prespore cells showed an aberrant spatial localization. Consistent with a developmental arrest, these mounds did not form either mature spores or stalk cells and very few cells expressed a stalk-cell-specific marker. Expression of PKAcat from the prespore promoter resulted in abnormal morphogenesis and accelerated spore cell differentiation. When cells were plated on agar, a fruiting body was formed with a very large basal region, containing predominantly spores, and a small, abnormal sorocarp. Mature spore cells were first detected by 14 hours, with maximal levels reached by 18-20 hours, in contrast to 24-26 hours in wild-type strains. When cells were plated on filters, they produced an elongated tip from a large basal region, which continued to elongate as a tubular structure and produce a 'slug-like' structure at the end. The slug was composed predominantly of prestalk cells with a few prespore cells restricted to the junction between the 'slug' and tube. As the slug migrated, these prespore cells were found in the tube, while new prespore cells appeared at the slug/tube junction, suggesting a continual differentiation of new prespore cells at the slug's posterior.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:cAMP-dependent protein kinase differentially regulates prestalk and prespore differentiation during Dictyostelium development. 827 51


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