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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Shuttle vectors expressing resistance to hygromycin B in both E. coli and in mammalian cells were constructed. A combination of the simian virus 40 early promoter upstream of the native bacterial promoter of the neo gene from transposon Tn5 was found to express hygromycin B resistance better in both types of host cells than a combination of the Tn5 promoter followed by the promoter of the Herpes simplex virus thymidine kinase gene. Hygromycin phosphotransferase fusion proteins with extensions at the carboxyterminus were also tested and found to be marginally less effective as selection markers in eukaryotic cells but virtually inactive in E. coli.
Mol Biol Rep 1992 Nov
PMID:Shuttle vectors conferring hygromycin B resistance to E. coli and to mammalian cells. Differential expression of carboxyterminal fusion proteins. 133 77

Immortalized cell lines derived from sensory neurons are relatively non-permissive for lytic infection with herpes simplex virus (HSV) and fail to transcribe the viral immediate-early genes following infection. Treatment of these cells with agents which raise the intra-cellular level of cyclic AMP results in increased activity of the IE1 gene which contains a cyclic AMP response element within its promoter and produces a consequent increase in permissivity for HSV infection. The significance of these effects for the regulation of HSV infection of neuronal cells are discussed in the light of the finding that cyclic AMP treatment can reactivate latent HSV infections.
Brain Res Mol Brain Res 1992 Jan
PMID:Elevation of cyclic AMP levels in cell lines derived from latently infectable sensory neurons increases their permissivity for herpes virus infection by activating the viral immediate-early 1 gene promoter. 137 62

The sexually dimorphic expression of the urinary protein genes of mice (Mup genes) in the liver is mediated by the different male and female temporal patterns of circulating GH. Normal females were induced to male levels when GH was administered by injection to mimic the male GH pattern, showing that expression at the male level does not require a male sex steroid status in addition to intermittent GH. Two Mup-alpha 2u-globulin hybrid transgenes with different Mup gene promoters showed sexually dimorphic expression, and their expression in females increased to male levels upon testosterone treatment. GH-deficient (lit/lit) mice did not express these transgenes, and GH-deficient females did not respond to testosterone treatment, showing that GH was required for induction. Both normal and GH-deficient females were induced to male levels when GH was administered by injection. This is the first report of a transgene responsive to GH. A transgene consisting of a Mup promoter fused to a Herpes simplex virus thymidine kinase reporter sequence also showed sexual dimorphism, although to a lesser degree. It was expressed at the same level in normal females and GH-deficient mice of both sexes and was induced when GH-deficient mice were treated with GH. We propose that this transgene has a basal constitutive expression, possibly due to the absence of any rodent DNA downstream of the promoter. Since expression of the transgene was significantly induced by GH, the GH response is due at least in part to sequences in the promoter region.
Mol Endocrinol 1992 Feb
PMID:Sexual dimorphism and growth hormone regulation of a hybrid gene in transgenic mice. 137 18

The ubiquitous and constitutive octamer transcription factor OTF-1 (Oct 1) is the target of positive regulation by the potent herpes simplex virus trans-activator VP16, which forms a complex with the homeodomain of OTF-1. Here we present evidence that the glucocorticoid receptor can negatively regulate OTF-1 function by a mechanism that is independent of DNA binding. In vivo-expressed glucocorticoid receptor inhibited in a hormone-dependent manner activation of a minimal promoter construct carrying a functional octamer site. Moreover, expression of the receptor in vivo resulted in hormone-dependent repression of OTF-1-dependent DNA-binding activity in nuclear extract. In vitro, the DNA-binding activity of partially purified OTF-1 was repressed following incubation with purified glucocorticoid receptor. Cross-linking and immunoprecipitation experiments indicated that the functional interference may be due to a strong association between these two proteins in solution. Finally, preliminary evidence indicates that the homeo subdomain of OTF-1 that directs formation of a complex with VP16 may also be critical for interaction with the glucocorticoid receptor. Thus, OTF-1 is a target for both positive and negative regulation by protein-protein interaction. Moreover, the functional interference between OTF-1 and the glucocorticoid receptor represents a novel regulatory mechanism in the cross-coupling of signal transduction pathways of nuclear receptors and constitutive transcription factors.
Mol Cell Biol 1992 Nov
PMID:Functional interference between the ubiquitous and constitutive octamer transcription factor 1 (OTF-1) and the glucocorticoid receptor by direct protein-protein interaction involving the homeo subdomain of OTF-1. 140 72

A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker--the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications.
Somat Cell Mol Genet 1992 Jul
PMID:Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene. 144 55

We used a shuttle vector based on the Epstein-Barr virus origin of plasmid replication (oriP) to determine the types of mutations induced by depurination in human cells. Plasmid DNA was incubated at pH 2 at 40 degrees C for various times to induce up to 20 apurinic (AP) sites per 9.7-kb plasmid and electroporated into lymphoblastoid cells derived from either a normal individual or an ataxia telangiectasia patient. After replication of the vector in the human cells, plasmid DNA was isolated and analyzed for mutations induced in the plasmid-encoded herpes simplex virus type 1-thymidine kinase gene. Both the frequencies and types of mutations induced by depurination were essentially identical for normal and ataxia telangiectasia cells. The mutant frequency at 20 AP sites/plasmid was 10-fold to 13-fold greater than that observed for untreated DNA. Deletion and frameshift events accounted for 46-55% of the mutants induced by depurination. The induced deletions were relatively small (median size, 100-150 bp) and characterized by short (1-5 bp) regions of sequence homology at the endpoints. These mutations and the frameshifts, a majority of which occurred in runs of identical nucleotides, are consistent with a model involving AP-site-induced template dislocation during DNA synthesis. A broad spectrum of base-substitution mutations, which accounted for 19-36% of the induced mutants, was observed. The apparent preference for insertion opposite AP sites in human cells was G (43-55%) greater than A approximately C (18-21%) greater than T (9-14%). Our results in human cells contrast markedly with those published previously for the mutational specificity of AP sites in Escherichia coli, in which a large majority of the mutants resulted from insertion of an A opposite the abasic site.
Mol Carcinog 1992
PMID:Mutagenesis by apurinic sites in normal and ataxia telangiectasia human lymphoblastoid cells. 150 43

The heat shock transcription factor (HSF) of the yeast Saccharomyces cerevisiae is posttranslationally modified. At low growth temperatures, it activates transcription of heat shock genes only poorly; after shift to high temperatures, it activates transcription readily. In an effort to elucidate the mechanism of this regulation, we constructed a series of HSF-VP16 fusions that join the HSF DNA-binding domain to the strong transcriptional activation domain from the VP16 gene of herpes simplex virus. Replacement of the endogenous C-terminal transcriptional activation domain with that of VP16 generates an HSF derivative that exhibits behavior reminiscent of HSF itself: low transcriptional activation activity at normal growth temperature and high activity after heat shock. HSF can thus restrain the activity of the heterologous VP16 transcriptional activation domain. To determine what is required for repression of activity at low temperature, we deleted portions of HSF from this HSF-VP16 fusion to map the regulatory domain. We also isolated point mutations that convert the HSF-VP16 fusion into a constitutive transcriptional activator. We conclude that the central, evolutionarily conserved domain of HSF, encompassing the DNA-binding and multimerization domains, contains a major determinant of temperature-dependent regulation.
Mol Cell Biol 1992 Mar
PMID:Temperature-dependent regulation of a heterologous transcriptional activation domain fused to yeast heat shock transcription factor. 154 86

The major DNA-binding protein, or infected-cell protein 8 (ICP8), encoded by herpes simplex virus can localize to the cell nucleus independently of other viral proteins. To define the nuclear localization signals within ICP8, we performed several forms of mutagenesis on the cloned ICP8 gene. Deletion analysis of the ICP8 gene showed that several portions of ICP8 are involved in its nuclear localization. To determine whether these regions were independent localization signals, we introduced various portions of the ICP8 gene into a series of cassette plasmids which allowed expression of fusion proteins containing pyruvate kinase, normally a cytoplasmic protein, fused to various portions of ICP8. These results showed that the carboxyl-terminal 28 residues are the only portion of ICP8 capable of targeting protein kinase into the nucleus. However, inclusion of certain additional regions of ICP8 into the fusion protein led to an inhibition of nuclear localization. Therefore, the carboxyl-terminal 28 residues of ICP8 can act independently as a nuclear localization signal, but certain conformational constraints or folding or assembly requirements in the remainder of the protein can affect the nuclear localization of the protein. Our results demonstrate that sequences distant from a nuclear localization signal can affect its ability to function. A set of fusion vectors has been isolated which should be of general use for making 5' or 3' fusions in any reading frame to rapidly map localization signals.
Mol Cell Biol 1992 Mar
PMID:Distal protein sequences can affect the function of a nuclear localization signal. 154 14

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.
Mol Endocrinol 1992 Apr
PMID:Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1. 158 26

The hygromycin phosphotransferase gene was fused in-frame with the herpes simplex virus type 1 thymidine kinase gene. The resulting fusion gene (termed HyTK) confers hygromycin B resistance for dominant positive selection and ganciclovir sensitivity for negative selection and provides a means by which these selectable phenotypes may be expressed and regulated as a single genetic entity.
Mol Cell Biol 1991 Jun
PMID:Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene. 164 50


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