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)

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.
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PMID:Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides. 776 34

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

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of beta-galactosidase with symmetric variants of alpha- and beta-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the alpha-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the alpha-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. A180, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for alpha-centered trp operator variants with exchanges in positions 3, 4 and 5.
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PMID:The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition. 786 89

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

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.
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PMID:Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter. 820 May 8

Middle transcription of bacteriophage Mu requires Escherichia coli RNA polymerase holoenzyme and a Mu-encoded protein, Mor. Consistent with these requirements, the middle promoter, Pm, has a recognizable -10 region but lacks a -35 region. Mutagenesis of this promoter (from -70 to +10) was performed using mutagenic oligonucleotide-directed PCR. The resulting fragments were cloned into a promoter-lacZfusion vector and analyzed for promoter activity by assaying beta-galactosidase production. Single point mutations with a Down phenotype were clustered in three regions: the -10 region, the Mor footprint region and the spacer between them. Gel retardation experiments with purified Mor protein and promoter mutants demonstrated that sequences important for Mor binding are located within the Mor footprint region and lead us to propose the existence of a dyad symmetry element involved in Mor binding. In agreement with this prediction, glutaraldehyde crosslinking of Mor in solution generated a species with the size of a dimer. These experiments also identified an unusual group of mutations located in the spacer region adjacent to the Mor footprint. These mutations alter promoter activity without affecting Mor binding. A circular permutation assay revealed that Mor does not introduce a significant bend upon binding to its target sequence.
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PMID:Transcription activation by the bacteriophage Mu Mor protein: analysis of promoter mutations in Pm identifies a new region required for promoter function. 860 57

A well-defined set of isogenic yeast strains has been constructed whereby each strain contains a different HXK2::lacZ gene fusion integrated at the URA3 locus. These HXK2::lacZ fusions differ in the amount of the HXK2 gene (encoding hexokinase 2 isoenzyme) that is fused to the lacZ reporter gene. Comparison of the beta-galactosidase activities of each strain during growth on glucose or ethanol revealed that some part of the coding region between +39 and +404 bp is involved in repressing gene expression in a carbon source dependent manner. A series of deletions of this HXK2 coding region were constructed and fused upstream of a minimal CYC1::lacZ promoter. beta-Galactosidase activities on glucose or ethanol growth yeast calls revealed that two different regulatory elements are present in this DNA region. Gel mobility shift analysis and in vitro DNase I footprinting have shown that proteins bind specifically to two downstream repressor sequences (DRS1 located from +140 to +163 and DRS2 located between +231 and +251) that influence the rate of HXK2 transcription when ethanol is used as carbon source by Saccharomyces cerevisiae. We identified and partially purified a 18 kDa protein that binds specifically to synthetic double-stranded oligonucleotides containing the (A/C)(A/G)GAAAT box sequence. Our data suggest that p18 synthesis is under the control of genes involved in glucose repression (MIG1 = CAT4) and glucose derepression (SNF1 = CAT1).
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PMID:Identification and characterisation of two transcriptional repressor elements within the coding sequence of the Saccharomyces cerevisiae HXK2 gene. 865 61

The adult neuromuscular junction displays an accumulation of both the acetylcholine receptor (AChR) protein in the subneural domain of the post-synaptic membrane and the mRNAs coding for all its subunits at the level of the subjunctional "fundamental nuclei." In the course of end plate development, the epsilon-subunit, at variance with other subunits, becomes exclusively expressed at the level of the fundamental nuclei, yet at a rather late stage (around birth). To analyze the promoter region of the epsilon-subunit gene which directs its specific expression at the synapse, we used a quantitative transient in vivo expression assay in intact muscle tissue using constructs of the epsilon-subunit promoter placed upstream of the beta-galactosidase reporter gene. One crucial element for synapse-specific expression was detected between the -11 and -6 positions. Disruption of this element, either by a scanning mutation or single base mutations, greatly diminishes, or even completely inhibits, preferential expression of the transgene at the end plate. Gel shift experiments reveal the presence of a complex in nuclear muscle extracts that bind the core sequence of this element. The identification of such a site opens the possibility to identify regulatory factors responsible for compartmentalized expression at the neuromuscular junction.
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PMID:Identification of an element crucial for the sub-synaptic expression of the acetylcholine receptor epsilon-subunit gene. 866 16

A regulatory region of the temperate Lactococcus lactis bacteriophage r1t chromosome has been cloned and characterized. It encompasses the two divergently oriented genes rro, encoding the phage repressor, and tec. Both genes, of which the transcription start sites have been mapped, are preceded by consensus -35 and -10 promoter sequences. The region contains three 21 bp direct repeats with internal dyad symmetry which probably act as operators. Two of these repeats partially overlap the two promoter sequences. The distant third repeat is located within the tec coding sequence. Gel mobility shift assays demonstrated that Rro specifically binds to this sequence. To study possible transcriptional regulation of the region, a lacZ translational fusion with an open reading frame following tec was constructed. Under conditions that favour the lysogenic life cycle of r1t, beta-galactosidase activity was very low. Expression of the lacZ fusion could be induced 70-fold by the addition of mitomycin C at a concentration which promotes the switch of r1t from the lysogenic to the lytic life cycle. In non-induced cells, promoter activity was repressed by Rro, as a frameshift mutation in rro resulted in constitutive expression of the lacZ gene fusion.
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PMID:Inducible gene expression mediated by a repressor-operator system isolated from Lactococcus lactis bacteriophage r1t. 873 Aug 74

Adherence to eukaryotic cells is essential in the pathogenesis of Neisseria meningitidis. Pilus-mediated adhesion has been shown to play an essential role in this process. Pilin, the pilus major subunit, and two pilus associated proteins, PilC1 and PilC2, are key components in meningococcal adhesiveness. Phase and/or antigenic variation of these molecules are the only identified means by which N. meningitidis modulates pilus-mediated adhesion. PilA/PilB is a pleiotropic regulatory system first characterized in Neisseria gonorrhoeae where it controls pilin gene transcription. Similar alleles are found in N. meningitidis. To address the role of this regulatory pathway in N. meningitidis, we engineered a meningococcal pilA mutant strain and analysed the consequences of this mutation on pilus-mediated adhesion using epithelial Hec-1-B cells. This mutation resulted in a threefold reduction in adhesiveness. As no change in the amount of pilin nor in pilin gene mRNA was detected, we compared the expression of the pilC genes in both pilA and parental strains. Two transcriptional fusions pilC1-lacZ and pilC2-lacZ were constructed. A threefold reduction in beta-galactosidase activity was observed in the pilA mutant strain harbouring the pilC1-lacZ fusion. No effect of the pilA mutation on beta-galactosidase activity was observed in the strain carrying the pilC2-lacZ fusion. Gel retardation experiments confirmed that the PilA protein binds to the promoter region of pilC1 but not of pilC2. Taken together, these data demonstrate that PilA modulates meningococcal adhesiveness via the transcription of pilC1. Thus, in addition to phase variation, a more co-ordinate and responsive system may allow a fine adaptation of adhesiveness of meningococci to various environmental signals.
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PMID:The pilA regulatory gene modulates the pilus-mediated adhesion of Neisseria meningitidis by controlling the transcription of pilC1. 883 Feb 64


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