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

Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidence that this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-beta-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, were present at equivalent levels in transcription complexes prepared in the presence or absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated prior to elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.
Mol Cell Biol 2002 Jul
PMID:Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. 1205 71

Treatment of small airway epithelial (SAEC) cells or lung epithelial (Beas2B) cells with TNF-alpha or PMA induces urokinase-type plasminogen activator (uPA) expression. Treatment of these cells with TNF-alpha, PMA or cycloheximide but not TGF-beta increased steady-state expression of uPAmRNA. TNF-alpha, PMA or cycloheximide caused 8-10 fold extensions of the uPAmRNA half-life in SAEC or Beas2B cells treated with DRB, a transcriptional inhibitor. These findings suggest that uPA gene expression involves a post-transcriptional regulatory mechanism. Using gel mobility shift and UV cross-linking assays, we identified a 30 kDa uPA mRNA binding protein (uPA mRNABp) that selectively binds to a 66 nt protein binding fragment of uPA mRNA containing regulatory information for message stabilization. Binding of cytoplasmic uPA mRNABp to uPA mRNA was abolished after treatment with TNF-alpha but not TGF-beta. In addition, we found the accumulation of 30 kDa uPAmRNABp in the nuclear extracts of TNF-alpha but not TGF-beta treated cells. The uPA mRNABp starts moving to the nucleus from the cytoplasmic compartment as early as three hours after TNF-alpha treatment. Complete translocation is achieved between 12-24 h, which coincides with the maximal expression of uPA protein effected by cytokine stimulation. Treatment of Beas2B cells with NaF inhibited TNF-alpha-mediated translocation of uPA mRNABp from the cytoplasm to the nucleus and concomitant inhibition of uPA expression. TNF-alpha stabilizes uPA mRNA by translocating the uPA mRNABp from the cytoplasm to the nucleus. Our results demonstrate a novel mechanism governing uPA mRNA stability through shuttling of uPA mRNABp between the nucleus and cytoplasm. This newly identified pathway may have evolved to regulate uPA-mediated functions of the lung epithelium in inflamation or neoplasia.
Mol Cell Biochem 2002 Aug
PMID:Cytoplasmic-nuclear shuttling of the urokinase mRNA binding protein regulates message stability. 1223 87

The multisubunit transcription elongation factor NELF (for negative elongation factor) acts together with DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) sensitivity-inducing factor (DSIF)/human Spt4-Spt5 to cause transcriptional pausing of RNA polymerase II (RNAPII). NELF activity is associated with five polypeptides, A to E. NELF-A has sequence similarity to hepatitis delta antigen (HDAg), the viral protein that binds to and activates RNAPII, whereas NELF-E is an RNA-binding protein whose RNA-binding activity is critical for NELF function. To understand the interactions of DSIF, NELF, and RNAPII at a molecular level, we identified the B, C, and D proteins of human NELF. NELF-B is identical to COBRA1, recently reported to associate with the product of breast cancer susceptibility gene BRCA1. NELF-C and NELF-D are highly related or identical to the protein called TH1, of unknown function. NELF-B and NELF-C or NELF-D are integral subunits that bring NELF-A and NELF-E together, and coexpression of these four proteins in insect cells resulted in the reconstitution of functionally active NELF. Detailed analyses using mutated recombinant complexes indicated that the small region of NELF-A with similarity to HDAg is critical for RNAPII binding and for transcriptional pausing. This study defines several important protein-protein interactions and opens the way for understanding the mechanism of DSIF- and NELF-induced transcriptional pausing.
Mol Cell Biol 2003 Mar
PMID:Human transcription elongation factor NELF: identification of novel subunits and reconstitution of the functionally active complex. 1261 62

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of a chimeric MLL-ELL fusion protein. ELL is an RNA Polymerase II (Pol II) transcriptional elongation factor that interacts with the recently identified EAF1 protein. Here, we show that ELL and EAF1 are components of Cajal bodies (CBs). Although ELL and EAF1 colocalize with p80 coilin, the signature protein of CBs, ELL and EAF1 do not exhibit a direct physical interaction with p80 coilin. Treatment of cells with actinomycin D, DRB, or alpha-amanitin, specific inhibitors of Pol II, disperses ELL and EAF1 from CBs, indicating that localization of ELL and EAF1 in CBs is dependent on active transcription by Pol II. The concentration of ELL and EAF1 in CBs links the transcriptional elongation activity of ELL to the RNA processing functions previously identified in CBs. Strikingly, CBs are disrupted in MLL-ELL leukemia. EAF1 and p80 coilin are delocalized from CBs in murine MLL-ELL leukemia cells and in HeLa cells transiently transfected with MLL-ELL. Nuclear and cytoplasmic fractionation revealed diminished expression of p80 coilin and EAF1 in the nuclei of MLL-ELL leukemia cells [corrected]. These studies are the first demonstration of a direct role of CB components in leukemogenesis.
Mol Biol Cell 2003 Apr
PMID:ELL and EAF1 are Cajal body components that are disrupted in MLL-ELL leukemia. 1268 6

While interactions among demography, behaviour and genetic structure are well-documented for neutral genetic markers, the role of these parameters and the effects of genetic drift and selection are considerably less well understood in functional genes, such as the major histocompatibility complex (MHC). In this study, the consequences of habitat fragmentation and the effects of a current population decline on noncoding (mitochondrial DNA) and two coding MHC loci (DQA, DRB) with different functional importance were investigated in the small remnant subdivided population of the endangered Malagasy giant jumping rat (Hypogeomys antimena). Both neutral and selective markers revealed a significant genetic differentiation between the two remnant subpopulations. The FST values were much lower in the MHC DQA and DRB genes than in the mitochondrial data. The MHC DRB loci display the effects of both balancing selection (high sequence diversity, four times higher nonsynonymous than synonymous substitutions in the functionally important antigen-binding site positions, twice the average heterozygosity of individual amino acids at the positions identified as part of the antigen-binding site (ABS) than those outside the ABS and nonselective forces including genetic drift. Simultaneously with a current population decline offspring reduced their dispersal distances. No substantial effects were detected within the first 6 years of reduced gene flow in either mitochondrial or MHC markers.
Mol Ecol 2003 Oct
PMID:Effects of habitat fragmentation and changes of dispersal behaviour after a recent population decline on the genetic variability of noncoding and coding DNA of a monogamous Malagasy rodent. 1296 86

There is now considerable evidence to suggest the cheetah (Acinonyx jubatus) has limited genetic diversity. However, the extent of this and its significance to the fitness of the cheetah population, both in the wild and captivity, is the subject of some debate. This reflects the difficulty associated with establishing a direct link between low variability at biologically significant loci and deleterious aspects of phenotype in this, and other, species. Attempts to study one such region, the feline leucocyte antigen (FLA), are hampered by a general reliance on cloning and sequencing which is expensive, labour-intensive, subject to PCR artefact and always likely to underestimate true variability. In this study we have applied reference strand-mediated conformational analysis (RSCA) to determine the FLA-DRB phenotypes of 25 cheetahs. This technique was rapid, repeatable and less prone to polymerase chain reaction (PCR)-induced sequence artefacts associated with cloning. Individual cheetahs were shown to have up to three FLA-DRB genes. A total of five alleles were identified (DRB*ha14-17 and DRB*gd01) distributed among four genotypes. Fifteen cheetahs were DRB*ha14/ha15/ha16/ha17, three were DRB*ha15/ha16/ha17, six were DRB*ha14/ha16/ha17 and one was DRB*ha14/ha15/ha16/ha17/gd01. Sequence analysis of DRB*gd01 suggested it was a recombinant of DRB*ha16 and DRB*ha17. Generation of new alleles is difficult to document, and the clear demonstration of such an event is unusual. This study confirms further the limited genetic variability of the cheetah at a biologically significant region. RSCA will facilitate large-scale studies that will be needed to correlate genetic diversity at such loci with population fitness in the cheetah and other species.
Mol Ecol 2004 Jan
PMID:The use of reference strand-mediated conformational analysis for the study of cheetah (Acinonyx jubatus) feline leucocyte antigen class II DRB polymorphisms. 1465 2

Northern elephant seals were hunted to near extinction in the 19th century, yet have recovered remarkably and now number around 175,000. We surveyed 110 seals for single-strand conformation polymorphism (SSCP) and sequence variation at three major histocompatibility (MHC) class II loci (DQA, DQB and DRB) to evaluate the genetic consequences of the population bottleneck at these loci vs. other well-studied genes. We found very few alleles at each MHC locus, significant variation among breeding sites for the DQA locus, and linkage disequilibrium between the DQB and DRB loci. Northern elephant seals are evidently inbred, although there is as yet no evidence of correlative reductions in fitness.
Mol Ecol 2004 Mar
PMID:Major histocompatibility complex variation at three class II loci in the northern elephant seal. 1487 73

A20 is an immediate-early NF-kappaB target gene. Prior to NF-kappaB stimulation, the A20 promoter is bound by the polymerase II machinery to allow rapid transcription activation. Here we show that the basal A20 transcription is repressed at the level of elongation in a promoter-specific fashion. Immunodepletion in vitro and RNA interference in cultured cells suggest that the basal elongation inhibition is conferred by DRB sensitivity-inducing factor (DSIF). We have identified a negative upstream promoter element called ELIE that controls DSIF activity. Remarkably, following NF-kappaB stimulation, inhibition of the A20 promoter by DSIF persists, but it is now regulated by NF-kappaB rather than ELIE. Similar regulation by DSIF is shown for another NF-kappaB-responsive gene, the IkappaBalpha gene. These findings reveal an intimate and dynamic relationship between DSIF inhibition of elongation and promoter-bound transcription factors. The potential significance of the differential regulation of DSIF activity by cis-acting elements is discussed.
Mol Cell Biol 2004 Mar
PMID:Elongation inhibition by DRB sensitivity-inducing factor is regulated by the A20 promoter via a novel negative element and NF-kappaB. 1499 82

Preimplantation HLA matching has recently emerged as a tool for couples desiring to conceive a potential donor progeny for transplantation in a sibling with a life-threatening disorder. In this paper we describe a strategy optimized for preimplantation genetic diagnosis (PGD) of haemoglobinopathies combined with HLA matching. This procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The above assay can also be used for preimplantation HLA matching as a primary indication. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassaemia, one for Wiscott-Aldrich syndrome and two for leukaemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. Minisequencing-based HLA typing combined with HLA STR haplotyping has been shown to be a reliable strategy for preimplantation HLA matching. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case.
Mol Hum Reprod 2004 Jun
PMID:Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching. 1504 7

Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery.
Mol Cell Biol 2004 Apr
PMID:Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro. 1506 Jan 54


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