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)

A CArG box motif [CC(A+T-rich)6GG] is one of the DNA elements required for muscle-specific gene transcription. Nuclear factors in mouse C2 myogenic cells strongly bind to the CArG box in the first intron of the gene (Sm alpha-A) encoding human smooth muscle alpha-actin. To clone cDNAs of the CArG box-binding factor (CBF), lambda gt11 cDNA expression libraries from C2 cells were screened for in situ DNA binding specific for this CArG box sequence. The 1.6-kb cDNA (CBF-A) encoding 285 amino acids (aa) was obtained, and a beta-galactosidase fusion protein, bacterially produced from the cDNA, bound to DNA fragments containing several CArG boxes. When the expression level of CBF-A in C2 cells increased by transfection of CBF-A expression plasmids, Sm alpha-A transcription was repressed. The deduced aa sequence of CBF-A is similar to some single-stranded (ss) nucleic acid-binding proteins. The fusion protein could bind to ssDNA, whereas CBF in C2 cell nuclear extracts could not. From these results, CBF-A is a novel CArG box-, ssDNA- and RNA-binding protein, as well as a repressive transcriptional factor.
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PMID:A protein binding to CArG box motifs and to single-stranded DNA functions as a transcriptional repressor. 139 4

To assess thyroid hormone receptor (TR)-mediated activation of transcription in yeast in the presence or absence of thyroid hormone (T3), we developed a co-expression system using a TR-beta 1 expression vector and a reporter plasmid containing a 16 base pair palindromic thyroid hormone response element (TRE) upstream from a proximal CYC1 promoter that was fused to the beta-galactosidase lac Z gene of Escherichia coli. Although TR-beta 1 functions as a repressor in most mammalian systems, using our system we observed a unique thyroid hormone-independent transcriptional response indicating that wild TR-beta 1 acted as a constitutive activator in yeast; the addition of 1 microM T3 induced a moderate but significant (p less than 0.01) 25-40% further increase in transcriptional activity. Using a series of rat TR-beta 1 mutant constructs, we found that deletion of domain D and portions of E completely eliminated transcriptional activity, whereas truncations of domain F and E permitted a partial (20-40%) response compared to wild TR-beta 1 in the presence or absence of T3. These observations indicate that TR-beta 1 functions as an activator in yeast and that domains D,E and F play important interactive roles in its hormone-independent gene activation with the D domain likely being the most essential. Furthermore, our results suggest that the different transcriptional property of TR-beta 1 in yeast compared to mammalian cells i.e. activator vs repressor function, is likely determined by transcriptional factor differences which are dependent upon cellular origin.
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PMID:Rat liver c-erb A beta 1 thyroid hormone receptor is a constitutive activator in yeast (Saccharomyces cerevisiae): essential role of domains D,E and F in hormone-independent transcription. 165 14

ompC expression in Escherichia coli K-12 is known to be regulated by the ompB locus, comprising the ompR and envZ genes, and the OmpR protein is believed to act as a positive transcriptional factor. We examined the transcriptional capability of the ompC gene in vitro and found that RNA polymerase could transcribe ompC without a requirement for other transcriptional factors. Furthermore, transcripts from three tandem promoters in ompC were identified in vitro. We employed oligonucleotide-directed site-specific mutagenesis to dissect the promoter region of the gene and assayed the promoters separately for transcriptional ability using fusions to the lacZ gene. The levels of beta-galactosidase indicate that ompC expression in vivo is dependent on the function of at least one of the upstream promoters. The function of OmpR appears to be the enhancement of a basal level of ompC expression. From the results of our experiments, the site of action of OmpR was deduced to be in the vicinity of the upstream promoters of ompC.
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PMID:Regulation of the ompC gene of Escherichia coli. Involvement of three tandem promoters. 301 84

We have independently identified and DNA sequenced the INO2 locus by its close proximity to the KIN1 locus in Saccharomyces cerevisiae. Mutant strains in which the INO2 chromosomal locus has been deleted show pleiotropic phenotypes under growth conditions of inositol/choline availability. Many ino2 delta cells show delocalized cell growth resulting in large cells having aberrant shapes. These mutant cells may display nuclear segregation or positioning defects as well as defects in bud formation. Furthermore, homozygous ino2 delta-1 diploids fail to sporulate. Previous studies have shown that INO2 mutants are defective in phospholipid synthesis due to an inability to derepress the INO1 gene, the structural gene for inositol-1-phosphate synthase. To identify and determine the function of Ino2p in yeast cells, we raised antibodies to a beta-galactosidase/Ino2 fusion protein. The INO2 open reading frame codes for a 304 amino acid protein with a calculated molecular weight of 39.7 kDa. Immunoblot analysis reveals two Ino2-specific proteins of approximately 44 and 46 kDa. The 44 kDa species is localized to the nucleus. Ino2p is believed to function as a positive transcriptional activator in phospholipid synthesis. Our results suggest that it affects additional pathways important to polarized cell growth and division perhaps by functioning as a more general transcriptional factor.
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PMID:INO2, a regulatory gene in yeast phospholipid biosynthesis, affects nuclear segregation and bud pattern formation. 801 48

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

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient Drosophila cell line cotransfected with Msx-1-luciferase and an Sp1 expression vector pPacSp1. The transgenic mice embryos containing -165/106-bp Msx-1 promoter-LacZ DNA in their genomes abundantly expressed beta-galactosidase in maxillae and mandibles and in the cellular primordia involved in the formation of the meninges and the bones of the skull. Thus, the truncated murine Msx-1 promoter can target expression of a heterologous gene in the craniofacial tissues of transgenic embryos known for high level of expression of the endogenous Msx-1 gene and found to be severely defective in the Msx-1 knock-out mice.
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PMID:A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice. 927 25

The yeast Saccharomyces cerevisiae contains a flavohemoglobin, encoded by the gene YHB1, whose function is unclear. Previous reports presented evidence that its maximal expression requires disruption of mitochondrial respiration and that it plays a role in the response to oxidative stress. We have studied the expression of YHB1 in respiratory deficient cells and in cells exposed to various compounds causing oxidative stress. Several different strains and approaches (spectroscopic detection of the oxygenated form of Yhb1p, beta-galactosidase activity of a YHB1-lacZ fusion, and Northern blot analysis) were used to demonstrate that YHB1 expression and Yhb1p production are not increased by respiration deficiency. YHB1 expression was unchanged in cells challenged with antimycin A or menadione, while it decreased in cells exposed to H2O2, diamide, dithiothreitol, and Cu2+. Transcription of YHB1 is not under the control of the transcriptional factor Yap1p. These results do not support a participation of YHB1 in the genetic response to oxidative stress. We also analyzed the growth phenotypes associated with altered Yhb1p production using YHB1-deleted strains and strains that greatly overproduced Yhb1p. Yhb1p appears to protect cells against the damage caused by Cu2+ and dithiothreitol, while sensitizing them to H2O2. Yhb1p overproduction in a glucose-6-phosphate dehydrogenase-deficient mutant decreased its growth rate. These data indicate that there is a complex relationship(s) between Yhb1p function(s) and cell defense reactions against various stresses.
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PMID:Flavohemoglobin expression and function in Saccharomyces cerevisiae. No relationship with respiration and complex response to oxidative stress. 954 81

The retinoblastoma protein (Rb), a key regulator of cell cycle progression, can bind the transcription factor E2F converting it from a positive transcriptional factor capable of driving cells into S phase into a negative complex which arrests cells in G1. We have created a potent transcriptional repressor of E2F-dependent transcription by fusing the C-terminal fragment of Rb (p56) to the DNA and DP1-binding domains of E2F. Because the expression of E2F/56 fusion protein from a constitutive promoter was incompatible with virus growth, adenovirus constructs were prepared where transgenes were expressed from a fragment of the smooth muscle alpha-actin (SMA) promoter. Immunoblot and beta-galactosidase staining demonstrated smooth muscle-specific expression of this transcriptional element in vitro. The SMA-p56 and SMA-E2F/p56 adenoviral constructs also induced G0/G1 cell cycle arrest specifically in smooth muscle cells. Following administration to rat tissues, the SMA-beta-galactosidase construct exhibited expression in balloon-injured carotid arteries, but not in liver, bladder or skeletal muscle. Local delivery of the SMA-E2F/p56 adenoviral construct to balloon-injured carotid arteries inhibited intimal hyperplasia. Our results demonstrate that local delivery of the SMA-E2F/p56 adenoviral construct can limit intimal hyperplasia in balloon-injured vessels, while avoiding toxicity that could occur from the dissemination and expression of the viral transgene.
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PMID:Tissue-specific expression of an anti-proliferative hybrid transgene from the human smooth muscle alpha-actin promoter suppresses smooth muscle cell proliferation and neointima formation. 1182 38

Fungal denitrification is a dissimilating metabolic mechanism for nitrate and was first described in Fusarium oxysporum. Here we investigated regulatory systems of expression of CYP55, which encodes cytochrome P450 (P450nor) and is essential for the fungal denitrification. Promoter-reporter analysis of F. oxysporum CYP55 using Escherichia coli beta-galactosidase showed that the region between nucleotides -526 and -515 was critical for induction by nitrate. It contained a nucleotide sequence similar to the binding consensus sequence of the pathway-specific transcriptional factor NirA, which induces expression of the nitrate-assimilatory genes of Aspergillus nidulans in the presence of nitrate. This indicates that expression of the nitrate dissimilatory gene (CYP55) is concomitantly regulated with the nitrate-assimilatory genes. The deletion studies also indicated that the nucleotide sequence between -118 and -107, which was similar to the binding consensus of the yeast Rox1p, which represses the anoxic genes under aerobic conditions, was responsible for repression of CYP55 under aerobic conditions. These results indicate that the fungus adapts to the denitrifying conditions by a combination of NirA- and Rox1-like transcription factors.
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PMID:Transcriptional control of nitric oxide reductase gene (CYP55) in the fungal denitrifier Fusarium oxysporum. 1209 13

Interferon regulatory factor 3 (IRF3) is a transcriptional factor that plays a crucial role in activation of innate immunity and inflammation in response to viral infection. We investigated the biological function of IRF3 overexpressed in somatic cells such as fibroblasts and astrocytes. Similar to overexpression of oncogenic H-ras in the normal human fibroblast, overexpression of IRF3 in human fibroblast BJ cells was shown to decrease cell growth and increase senescence-associated beta-galactosidase activity by activating a p53 tumor suppressor. BCNU, a DNA damage agent, further accelerated p53 function and cell death in the IRF3-overexpressed BJ cells compared to control BJ cells, without increased expression of IRF3 target genes. IRF3 failed to activate p53 function and cell growth inhibition in BJ cells downregulating p53 by RNAi-mediated p53 knockdown. Furthermore, enforced expression of IRF3 did not show any effect of cell growth inhibition in astrocytes or embryonic fibroblasts derived from the p53(-/-) mouse. When compared to control BJ cells, BJ cells which downregulated IRF3 by RNAi-mediated IRF3 knockdown showed extended in vitro life span. Taken together, the present study indicates that IRF3 should be a novel inducer of cell growth inhibition and cellular senescence through activation of p53 tumor suppressor.
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PMID:Interferon regulatory factor 3 activates p53-dependent cell growth inhibition. 1651 54


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