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)

To analyze regulation of the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR), cell lines were generated from LTR-tax x LTR-beta-galactosidase (beta-Gal) doubly transgenic mouse fibroblastic tumors. The HTLV-I LTR directs expression of both the tax and lacZ genes, and Tax up-modulates both promoters in primary cells. However, once cells were transformed by tax, beta-Gal but not tax expression was suppressed. Supertransformation of these cells with v-src suppressed both beta-Gal and tax expression. This suppression was reversed by treatment with the tyrosine kinase inhibitor herbimycin A or protein kinase A inhibitor H8. Electrophoretic mobility shift assays demonstrated augmented binding in the R but not U3 region. This binding was competitively inhibited by a high-affinity CREB oligodeoxynucleotide and super-shifted with a specific CREB antibody. Treatment of cells with the cyclic AMP analog dibutyryl cyclic AMP also transiently increased the R region binding dramatically. In vitro DNase I footprint analysis identified a protein-binding sequence in the R region which corresponded with suppression. However, this target sequence lacked a conventional CREB-binding site. A 70.5-kDa DNA-binding protein was partially purified by affinity chromatography, along with a 49-kDa protein which reacted with CREB-specific sera. These data demonstrate that HTLV-I LTR suppression is associated with CREB factor binding in the R region, probably by direct interaction with a 70.5-kDa protein, and provide a novel mechanism for maintenance of viral latency.
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PMID:Transcriptional suppression of the human T-cell leukemia virus type I long terminal repeat occurs by an unconventional interaction of a CREB factor with the R region. 803 15

The transcription factor Tax of the oncogenic human T-cell leukemia virus type 1 is likely to be responsible for viral replication in the host organism and for the induction of proliferation in infected cells. To investigate Tax-mediated transcription in vivo, we expressed Tax as well as CREB in Saccharomyces cerevisiae. The activity of these proteins was monitored by expression of a beta-galactosidase reporter gene, which was fused to two viral 21-bp repeats located upstream of the yeast cytochrome c1 oxidase minimal promoter. Coexpression of Tax and CREB in S. cerevisiae led to a 20-fold increase in beta-galactosidase activity in comparison with that in strains expressing either Tax or CREB alone. By screening a human cDNA library, we were able to demonstrate that the Tax transactivation assay using S. cerevisiae can be successfully applied to identify other cellular proteins forming ternary complexes with Tax and 21-bp repeats in vivo. Upon transformation in S. cerevisiae, 1 of 13,500 clones tested positive. Sequencing of the cDNA insert of the rescued plasmid revealed that this DNA encoded the ATF-1 protein. beta-Galactosidase induction was comparable to that of the Tax/CREB coexpression system. This indicates that Tax-mediated transcription is critically dependent on the presence of cellular CREB or ATF-1 in vivo. Stimulation of transcription initiation required an unmasked NH2 terminus of Tax. Fusion of Tax to the yeast Gal4 protein abolished the transactivation potential of Tax. Reconstitution of the transcriptional properties of viral Tax together with the cellular proteins of the ATF-1/CREB family in S. cerevisiae allows the functional characterization of these proteins in vivo.
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PMID:The oncoprotein Tax of the human T-cell leukemia virus type 1 activates transcription via interaction with cellular ATF-1/CREB factors in Saccharomyces cerevisiae. 889 66

Herpes simplex virus 1 (HSV1) ICP34.5 deletion mutants are avirulent upon inoculation of both the central and peripheral nervous systems of mice, but they replicate to near wild-type titres in a number of non-neuronally derived cell lines in culture. Thus these mutants might be suitable for development as safe vectors for gene transfer to the nervous system. However, the mechanism of this avirulent phenotype in neuronal cells is at present poorly understood, although it has been suggested that nonpermissive cells infected with these mutants may undergo apoptosis, the function of ICP34.5 being to prevent this response and to allow continued virus replication. If this were the case ICP34.5 null mutants might be unsuitable for gene transfer as infected cells would quickly die, limiting the expression of a transgene. Here we have inserted a beta-galactosidase marker gene into a nonessential gene of an HSV1 strain 17+ mutant in which ICP34.5 has been deleted and also into a second mutant in which the virion transactivator protein VMW65 is also inactive. While all the mutants grew to high titre in tissue culture, mice inoculated by the foot-pad or intracranial route at high titre remained healthy until the end of the experiment. Moreover, beta-galactosidase was expressed either in the brain or in the dorsal root ganglia, depending on the site of inoculation. This suggests that in vivo the absence of ICP34.5 does not prevent the expression of a transgene in neuronal tissue and indicates that non-neurovirulent mutants lacking this gene may be suitable for further development as safe vectors for gene therapy in vivo.
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PMID:Gene delivery to the central and peripheral nervous systems of mice using HSV1 ICP34.5 deletion mutant vectors. 890 2

The immediate-early transactivator protein BICPO is a key regulatory element of bovine herpesvirus 1 (BHV-1) replication based on transient expression assays. To examine BICPO function in the context of the viral genome, we created recombinant BHV-1 expressing beta-galactosidase instead of BICPO. To complement the defect, a neomycin resistant MDBK cell line (M164) expressing BICPO was established, permitting selection of a blue-staining BHV-1 recombinant (A2G2). Southern blot and PCR analysis confirmed that the BICPO gene was interrupted by the beta-galactosidase gene and that wt progeny was absent. Compared with wt BHV-1, A2G2 reached lower titers in M164 cells but replicated with similar kinetics. Once isolated, A2G2 also grew in MDBK cells although the titer was reduced a further 10-fold and the virus remained strongly cell-associated. Thus, BICPO is not absolutely required for replication in cell culture. Gene expression of A2G2 was investigated by Western blots and immunofluorescence. Surprisingly, not only was BICPO absent, but glycoprotein C (gC) was also missing. Other viral genes were expressed normally. Semiquantitative PCR showed that A2G2 produced similar amounts of viral DNA as wt but a much smaller number of infectious particles. Cotransfection of A2G2 DNA and a plasmid containing the BICPO gene yielded revertant virus with fully restored wt properties. We conclude that BICPO is required for gC expression, and that the missing gC partly accounts for the reduced A2G2 infectivity.
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PMID:Recombinant bovine herpesvirus-1 (BHV-1) lacking transactivator protein BICPO entails lack of glycoprotein C and severely reduced infectivity. 899 89

Proliferating, activated, hepatic stellate cells have a high level of collagen type I expression. Therefore, stellate cell proliferation is a critical step in hepatic fibrosis. Here we show that proliferation of activated primary rat stellate cells was blocked by elevation of cAMP with 8 Br-cAMP or isomethylbutyl xanthine, a phosphodiesterase inhibitor, and by stimulation of Ca2+ fluxes with the Ca2+ ionophore A-23187. Because phosphorylation of CREB on Ser133 is an important mediator of cAMP-protein kinase (PKA) and Ca2+-calmodulin kinase II (CAMK-II) activation, we tested whether CREB-PSer133 was essential for stellate cell quiescence. Nuclear extracts from quiescent, but not from activated, stellate cells contained CREB-PSer133. Moreover, the phosphorylation of CREB on Ser133 was stimulated in activated cells by inducing the activity of PKA or CAMK-II. In addition, coexpression of CREB and either a constitutively active PKA or a constitutively active CAMK-II inhibited the proliferation of activated stellate cells. In contrast, expression of CREB alone, PKA or CAMK-II alone, CREB-Ala 133 (which lacks the Ser133 phosphoacceptor) with PKA or CAMK-II, or CREB with inactive PKA or CAMK-II mutants did not affect stellate cell proliferation, suggesting that CREB-PSer133 is necessary for blocking the stellate cell cycle. Conversely, expression of a trans-dominant negative CREB-Ala 133 mutant (which competes with CREB/CREB-PSer133 for cognate DNA binding sites and presumably for protein interactions) induced a greater than fivefold entry into S-phase of quiescent stellate cells, compared with control cells expressing either beta-galactosidase or wt CREB, indicating that CREB-PSer133 may be indispensable for the quiescent stellate cell phenotype. This study suggests that PKA and CAMK-II play an essential role on stellate cell activation through the induction of CREB phosphorylation on Ser133, and provides potential approaches for the treatment of hepatic fibrogenesis in patients with chronic liver diseases.
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PMID:Proliferation of hepatic stellate cells is inhibited by phosphorylation of CREB on serine 133. 907 42

The herpes simplex virus type 1 (HSV-1) mutant in 1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in 1820. In almost all cell types tested, in 1820 initiated infection less efficiently than in 1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in 1820 was less impaired than in 1814. A rescuant of in 1820 at the VP16 locus, in 1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In 1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in 1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1 tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in 1820 to produce in 1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in 1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative of in 1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in 1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in 1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors.
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PMID:Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression. 912 65

In order to evaluate the efficiency of the tetracycline-regulated gene expression system in Drosophila, we have generated transgenic lines expressing a tetracycline-controlled transactivator protein (tTA), with specific expression patterns during embryonic and larval development. These lines were used to direct expression of a tTA-responsive promoter fused to the coding region of either the beta-galactosidase or the homeotic protein Antennapedia (ANTP), under various conditions of tetracycline treatment. We found that expression of beta-galactosidase can be efficiently inhibited in embryos and larvae with tetracycline provided in the food, and that a simple removal of the larvae from tetracycline exposure results in the induction of the enzyme in a time- and concentration-dependent manner. Similar treatments can be used to prevent the lethality associated with the ectopic expression of ANTP in embryos and, subsequently, to control the timing of expression of the homeoprotein ANTP specifically in the antennal imaginal disc. Our results show that the expression of a gene placed under the control of a tetracycline-responsive promoter can be tightly controlled, both spatially by the regulatory sequences driving the expression of tTA and temporally by tetracycline. This provides the basis of a versatile binary system for controlling gene expression in Drosophila, with an additional level of regulation as compared to the general method using the yeast transcription factor GAL4.
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PMID:Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. 958 19

The "reverse" tetracycline repressor (rtR) binds a specific DNA element, the tetracycline operator (tetO), only in the presence of tetracycline, or derivatives such as doxycycline (dox). Fusion of rtR to the transcriptional activation domain of herpes virus protein VP16 produces a eukaryotic transactivator protein (rtTA). rtTA has previously been shown to allow dox-dependent transcription of transgenes linked to tetO sequences in mammals. To adapt this system to Drosophila, the Actin5C promoter was used to drive constitutive expression of rtTA in transgenic flies. Three reporter constructs, each encoding E. coli beta-galactosidase (beta-gal), were also introduced into transgenic flies. In one reporter seven tetO sequences were fused to the Adh core promoter. The other two reporter constructs contain seven tetO sequences fused to the hsp70 core promoter. Feeding of transgenic Drosophila containing the rtTA construct and any one of the three reporter constructs with dox caused up to 100-fold induction of beta-gal. Dox induced beta-gal expression in all tissues, in larvae and in young and senescent adults. Induction of beta-gal in adults had no detectable effect on life span. These results suggest the potential usefulness of this system for testing specific genes for effects on Drosophila development and aging.
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PMID:Doxycycline-induced transgene expression during Drosophila development and aging. 967 Oct 25

Although usually considered to be a constitutively expressed protein, in the primate ovary the expression of CREB (cAMP response element-binding protein) is extinguished after ovulation, and its loss is temporally associated with the cessation of proliferation of luteal cells and the ultimate commitment of the corpus luteum to undergo regression. To determine the cellular consequences of the loss of CREB expression, we expressed a nonphosphorylatable mutant of CREB (CREB M1) in primary cultures of rat granulosa cells using a replication-defective adenovirus vector. Expression of CREB M1 did not block granulosa cell differentiation as assessed by acquisition of the ability to produce estrogen and progesterone in response to FSH or forskolin. However, granulosa cells expressing CREB M1, but not adenovirus-directed beta-galactosidase or enhanced green fluorescent protein, exhibited a 35% reduction in viability that was further reduced to 65% after stimulation with 10 microM forskolin. These results demonstrate that the trophic effects of cAMP (proliferation and survival) on ovarian granulosa cells are functionally separate from the effects of cAMP on differentiation and provide novel evidence that CREB may function as a cell survival factor in the ovary. The separation of signaling pathways that govern differentiation and survival in the ovary thereby provides a mechanism by which progesterone production, which is absolutely essential for the maintenance of pregnancy, can continue despite the cessation of proliferation of luteal cells and their commitment to cell death (luteolysis).
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PMID:Adenovirus-directed expression of a nonphosphorylatable mutant of CREB (cAMP response element-binding protein) adversely affects the survival, but not the differentiation, of rat granulosa cells. 1044 9

PMP22, one of the major components of myelin, is overexpressed in Charcot-Marie-Tooth type 1A (CMT1A) patients. In an attempt to determine the mechanisms by which the expression of this gene is regulated (with a view to lowering its expression in CMT1A patients), we subcloned genomic fragments covering 6kb of the promoter region in an expression vector containing the beta-galactosidase gene as reporter, and used these in transfection assays. We show that the 300bp upstream of the transcription start contain the elements required for Schwann cell specific expression of the reporter gene. This minimal promoter activity appears to be under the control of a silencer element sensitive to cAMP, located between -0.3kb and -3. 5kb from the start of transcription. Computer analysis of 2kb of the promoter predicted the presence of transcription factor binding sites, including CREB (which may be involved in the response of PMP22 expression to cAMP stimulation) and steroid receptors. Using constructs with or without the CREB sites, we were able to demonstrate that these sites are involved in silencing the PMP22 promoter activity. Lastly, we identified a region containing blocks of polymorphic CA repeats, located close to the CREB binding site, which may further influence the transcriptional activity of PMP22.
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PMID:Molecular dissection of the Schwann cell specific promoter of the PMP22 gene. 1080 67


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