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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcriptional enhancers for genes transcribed by RNA polymerase II may be localized upstream or downstream of the stimulated promoter in their normal chromosomal context. They stimulate transcription in an orientation-independent manner when assayed on circular plasmids. We describe a transient transformation system to evaluate the orientation preference of transcriptional enhancers in Drosophila. To accomplish this, the gypsy insulator element was used to block bidirectional action of an enhancer on circular plasmids. In this system, as in the chromosome, blocking of enhancer activity requires wild-type levels of the su(Hw) protein. We evaluated the orientation preference for the relatively large (4.4 kb) Adh larval enhancer from Drosophila melanogaster, used in conjunction with a luciferase reporter gene under the control of a minimal Adh promoter. An orientation preference was revealed by insertion of a single copy of the insulator between the enhancer and the promoter. This orientation effect was greatly amplified when the promoter was weakened by removing binding sites for critical transcription factors, consistent with a mechanism of insulator action in which the insulator intercepts signals from the enhancer by competing with the promoter. The orientation preference, as much as 100-fold, is a property of the enhancer itself because it is displayed by gene constructions introduced into the chromosome regardless of the presence of the insulator in a distal location. These findings are most easily reconciled with a facilitated tracking mechanism for enhancer function in a native chromosomal environment.
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PMID:Polarity of transcriptional enhancement revealed by an insulator element. 1111 71

Dendritic cell (DC)-mediated cancer immunotherapy is a very promising alternative approach to cancer treatment. In a previous study, we successfully transfected bone marrow-derived dendritic progenitors (BMDDPs) with a T7 vector--a nonviral, cytoplasmic-based autogene expression system--encoding a model tumor antigen, firefly luciferase, and subsequently stimulated the transfected cells to differentiate into DCs. When injected into experimental mice, those DCs generated a strong immune response against tumor cells bearing luciferase, which not only prevented occurrence of metastasis but also eradicated existing tumors. In the present study, we constructed a T7 vector encoding mouse tyrosinase, a well--known melanoma associated tumor antigen, and used it to transfect BMDDPs. Reverse transcriptase polymerase chain reaction and Western analysis confirmed the expression of tyrosinase by DCs differentiated from transfected BMDDPs. Two immunizations of these DCs at a dose of 2 x 10(6) of each successfully prevented tumor growth. More importantly, one injection of 2 x 10(6) of these DCs into mice followed by five doses of recombinant human interleukin-2 administration effectively eradicated existing tumors as indicated by pulmonary metastasis assay.
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PMID:Murine tyrosinase expressed by a T7 vector in bone marrow-derived dendritic progenitors effectively prevents and eradicates melanoma tumors in mice. 1112 87

We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.
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PMID:Kaposi's sarcoma-associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site. 1116 Jun 85

The Ly49 multigene family consists of at least 14 closely related genes located in the natural killer (NK) gene complex on mouse Chromosome 6. Reverse transcriptase (RT)-PCR on single NK cells has shown that Ly49c is expressed on approximately 50% of NK cells, whereas the closely related Ly49j gene is expressed on 5-8% of NK cells. In this study, we examined three regions to determine whether they contain cis-acting elements involved in regulating the expression of these two closely related Ly49 genes within NK cells. Luciferase reporter assays in EL-4 cells suggested that the 5' regions of Ly49c and j contain promoter elements and repressor sequences. In addition, luciferase assays suggest that Ly49j also contains an active promoter in the first intron, although the transcripts produced from this promoter appear to be severely truncated. Finally, comparisons of the 3' noncoding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c 130 bp downstream of the termination codon. The polyadenylation signal for Ly49j is located downstream of the Ly49c poly(A) site, which results in a much longer 3' untranslated region (UTR). When the Ly49j 3'UTR was used to provide the polyadenylation signal for the green fluorescent protein (GFP) reporter gene, GFP expression was reduced twofold. These results suggest that both internal promoters, repressors, and 3' regions play a role in regulating Ly49 gene expression.
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PMID:Functional analysis of 5' and 3' regions of the closely related Ly49c and j genes. 1122 Jun 23

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.
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PMID:Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. 1129 39

The sequence and gene organization of the ribosomal RNA (rRNA) genes of Leishmania major Friedlin (LmjF) were determined. Interestingly, the rDNA repeat unit contained a duplicated 526 bp fragment at the 3' end of the unit with two copies of the LSUepsilon rRNA gene. Our results suggested the presence of only approximately 24 copies of the rRNA unit per diploid genome in LmjF. Repetitive elements (IGSRE) of 63 bp occurred in the intergenic spacer (IGS) between the LSUepsilon and the SSU rRNA genes. Among the different rDNA units, the region containing the IGSRE fluctuated in length from approximately 1.3 to approximately 18 kb. The transcription initiation site (TIS) of the rRNA unit was localized by primer extension to 1043 bp upstream of the SSU gene and 184 bp downstream of the IGSRE. Sequence comparison among several species of Leishmania showed a high degree of conservation around the TIS. Moreover, the IGSRE also showed considerable similarity between Leishmania species. In transient transfection assays, a fragment containing the TIS directed a 164- to 178-fold increase in luciferase activity over the no-insert control, indicating the presence of a promoter within this 391 bp fragment. The LmjF promoter region was also functional in other species of Leishmania. Nuclear run-on analyses demonstrated that only the rRNA-coding strand is transcribed, downstream of this RNA polymerase I (pol I) promoter. These experiments also suggested that transcription terminates upstream of the IGSRE.
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PMID:Genomic organization and functional characterization of the Leishmania major Friedlin ribosomal RNA gene locus. 1152 48

The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that required a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implicated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a -1 ribosomal frameshift in the GLV transcript.
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PMID:Structural analysis of the -1 ribosomal frameshift elements in giardiavirus mRNA. 1160 3

Replication of hepatitis A virus (HAV) in cultured cells is inefficient and difficult to study due to its protracted and generally noncytopathic cycle. To gain a better understanding of the mechanisms involved, we constructed a subgenomic HAV replicon by replacing most of the P1 capsid-coding sequence from an infectious cDNA copy of the cell culture-adapted HM175/18f virus genome with sequence encoding firefly luciferase. Replication of this RNA in transfected Huh-7 cells (derived from a human hepatocellular carcinoma) led to increased expression of luciferase relative to that in cells transfected with similar RNA transcripts containing a lethal premature termination mutation in 3D(pol) (RNA polymerase). However, replication could not be confirmed in either FrhK4 cells or BSC-1 cells, cells that are typically used for propagation of HAV. Replication was substantially slower than that observed with replicons derived from other picornaviruses, as the basal luciferase activity produced by translation of input RNA did not begin to increase until 24 to 48 h after transfection. Replication of the RNA was reversibly inhibited by guanidine. The inclusion of VP4 sequence downstream of the viral internal ribosomal entry site had no effect on the basal level of luciferase or subsequent increases in luciferase related to its amplification. Thus, in this system this sequence does not contribute to viral translation or replication, as suggested previously. Amplification of the replicon RNA was profoundly enhanced by the inclusion of P2 (but not 5' noncoding sequence or P3) segment mutations associated with adaptation of wild-type virus to growth in cell culture. These results provide a simple reporter system for monitoring the translation and replication of HAV RNA and show that critical mutations that enhance the growth of virus in cultured cells do so by promoting replication of viral RNA in the absence of encapsidation, packaging, and cellular export of the viral genome.
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PMID:Replication of subgenomic hepatitis A virus RNAs expressing firefly luciferase is enhanced by mutations associated with adaptation of virus to growth in cultured cells. 1177 93

The basic helix-loop-helix (bHLH) transcription factor, Hand1, plays an important role in the development of the murine extra-embryonic trophoblast cell lineage. In the present study, we have analysed the expression of Hand1 in human extra-embryonic cell types and determined its binding specificity and transcriptional activity upon interaction with different class A bHLH factors. Northern blotting and in situ hybridization showed that Hand1 mRNA is specifically expressed in amnion cells at different stages of gestation. Accordingly, we demonstrate that the protein is exclusively produced in the amniotic epithelium in vivo and in purified amnion cells in vitro using a novel polyclonal Hand1 antiserum. Reverse transcriptase-PCR and immunohistochemical staining of blastocysts revealed the production of Hand1 mRNA and polypeptide in the trophectodermal cell layer. In the presence of E12/E47, Hand1 stimulated the transcription of luciferase reporters harbouring degenerate E-boxes, suggesting that E-proteins are potential dimerization partners in trophoblastic tumour and amnion cells. In contrast, Hand1 diminished E12/E47-dependent transcription of reporters containing perfect E-boxes by inhibiting the interaction of Hand1/E-protein heterodimers with the palindromic cognate sequence. Furthermore, we show that Hand1 down-regulated GAL-E12-dependent reporter expression, indicating that the protein can also act directly as a transcriptional repressor. Mutational analyses of GAL-Hand1 suggested that two protein regions located within its N-terminal portion mainly confer the repressing activity. In conclusion, human Hand1 may play an important role in the differentiation of the amniotic membrane and the pre-implanting trophoblast. Furthermore, the data suggest that Hand1 can act as a repressor by two independent mechanisms; sequestration of class A bHLH factors from E-boxes and inhibition of their transcriptional activity.
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PMID:Human Hand1 basic helix-loop-helix (bHLH) protein: extra-embryonic expression pattern, interaction partners and identification of its transcriptional repressor domains. 1180 95

Herpesvirus entry into cells and herpesvirus-induced cell fusion are related processes in that virus penetration proceeds by fusion of the viral envelope and cell membrane. To characterize the human herpesvirus 8 (HHV-8) glycoproteins that can mediate cell fusion, a luciferase reporter gene activation assay was used. Chinese hamster ovary (CHO) cells expressing the HHV-8 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with human cells transfected with T7 RNA polymerase. Because HHV-8 glycoprotein B (gB) expressed in CHO cells localizes to the perinuclear region, a truncated form of gB (designated gB(MUT)) that lacks putative endocytosis signals was constructed by deletion of the distal 58 amino acids of the cytoplasmic tail. HHV-8 gB(MUT) was expressed efficiently on the surface of CHO cells. HHV-8 gB, gH, and gL could mediate the fusion of CHO cells with two different human cell types, embryonic kidney cells and B lymphocytes. Substituting gB(MUT) for gB significantly enhanced the fusion of CHO cells with human embryonic kidney cells but not B lymphocytes. Thus, two human cell types known to be susceptible to HHV-8 entry were also suitable targets for cell fusion induced by HHV-8 gB, gH, and gL. For human embryonic kidney cells and B cells at least, optimal fusion was noted with the expression of all three HHV-8 glycoproteins.
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PMID:Human herpesvirus 8 glycoprotein B (gB), gH, and gL can mediate cell fusion. 1193 6


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