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Biomedical subjects

P A Sharp

Publications and source records attributed to P A Sharp.

At least 19 recordsLinked to original sources
PubMed2007

DNA-PK phosphorylation sites on Oct-1 promote cell survival following DNA damage.

Octamer transcription factor-1 (Oct-1) has recently been shown to function as a stress sensor that promotes cell survival subsequent to DNA damage. Here, we show that the survival signal imparted by Oct-1 following exposure to ionizing radiation (IR) is dependent upon DNA-dependent protein kinase (DNA-PK)-dependent phosphorylation of a cluster of 13 specific ser/thr residues within the N-terminal transcriptional regulatory domain of Oct-1. Although IR treatment did not affect the recruitment of Oct-1 to the histone H2B promoter, the recruitment of RNA polymerase II, TATA-binding protein and histone H4 acetylation were strongly reduced, consistent with a decrease in Oct-1 transcriptional regulatory potential following IR exposure. Ser/Thr-Ala substitution of 13 sites present in Oct-1 transcriptional regulatory domain eliminated Oct-1 phosphorylation subsequent to IR exposure. Further, these substitutions prevented Oct-1 from rescuing the survival of IR-treated Oct-1-/- murine embryonic fibroblasts, providing a direct link between DNA-PK-dependent phosphorylation and the contribution of Oct-1 to cell survival. These results implicate Oct-1 as a primary effector in a DNA-PK-dependent cell survival pathway that is activated by double-stranded DNA breaks.

3T3 Cells↗
PubMed2006

Non-haem iron transport in the rat proximal colon.

BACKGROUND: Only 10% of dietary iron is absorbed in the duodenum which implies that 90% (approximately 9 mg day(-1)) reaches the lower small intestine and colon. Therefore the purpose of this study was to assess the iron transport capacity of the rat proximal colon and to determine whether iron absorption is regulated by changes in dietary iron content. MATERIALS AND METHODS: Rats were fed for 14 days on either iron adequate (44 mg Fe kg(-1) diet) or iron-deficient (< 0.5 mg Fe kg(-1) diet) diets. The 59Fe transport across the colonic epithelium and its subsequent appearance in the blood were measured in vivo. In separate studies the colon was excised and used to measure divalent metal transporter expression. RESULTS: Divalent metal transporter (DMT1) was expressed at the apical membrane of the surface epithelium in rat proximal colon. In animals fed an iron-deficient diet, DMT1 mRNA and protein expression were increased. This was accompanied by a significant increase in tissue 59Fe uptake. CONCLUSIONS: The proximal colon can absorb non-haem iron from the intestinal lumen. The purpose of this mechanism remains to be elucidated.

Animals↗
PubMed2001

Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production.

Lactate and ammonia are the two major waste products formed during mammalian cell growth. Accumulation of these side products can have a negative effect on cell growth, and has drawn recent attention because of their inhibitory effects on the specific product synthesis rate. Our aim is to reduce lactate formation in the cell culture by genetically manipulating of the pathway of lactate synthesis with an aim to achieve high monoclonal antibody production. We have partially disrupted the LDH-A gene by homologous recombination in hybridoma cells (ATCC-CRL-1606). The cells that received the newly introduced DNA were selected by G418, and an LDH-deficient cell was identified by a screening method based on medium color changing in 96-well plates. A variant cell, LDH-neo21, was identified through this screening method and was characterized. The specific productivity of lactate by LDH-neo21 cells was 50% lower than that of parental cells. Intracellular LDH enzyme activity was significantly reduced. The cell growth was improved both in terms of cell density and cell viability. Total cell density potentially reached 5 x 10(6) cells/mL while the parental hybridoma cells had a cell density of 3.5 x 10(6) cells/mL, which represented a 30% increase. The antibody production of LDH-neo21 cells was threefold greater than that of parental cells during 5-day batch culture. Polymerase chain reaction (PCR) results showed that at least one copy of the LDH-A gene was disrupted in the LDH-neo21 cells. The variant of the hybridoma cell exhibited a significant advantage of reduced lactate formation in the cell culture with a high concentration of glucose, which led to a higher production of monoclonal antibody. 2001 John Wiley & Sons, Inc.

Ammonia↗
PubMed2001

Positive transcription elongation factor B phosphorylates hSPT5 and RNA polymerase II carboxyl-terminal domain independently of cyclin-dependent kinase-activating kinase.

The CDK9-cyclin T kinase complex, positive transcription elongation factor b (P-TEFb), stimulates the process of elongation of RNA polymerase (Pol) II during transcription of human immunodeficiency virus. P-TEFb associates with the human immunodeficiency virus Tat protein and with the transactivation response element to form a specific complex, thereby mediating efficient elongation. Here, we show that P-TEFb preferentially phosphorylates hSPT5 as compared with the carboxyl-terminal domain of RNA Pol II in vitro. Phosphorylation of hSPT5 by P-TEFb occurred on threonine and serine residues in its carboxyl-terminal repeat domains. In addition, we provide several lines of evidence that P-TEFb is a CDK-activating kinase (CAK)-independent kinase. For example, CDK9 was not phosphorylated by CAK, whereas CDK2-cyclin A kinase activity was dramatically enhanced by CAK. Therefore, it is likely that P-TEFb participates in regulation of elongation by RNA Pol II by phosphorylation of its substrates, hSPT5 and the CTD of RNA Pol II, in a CAK-independent manner.

Base Sequence↗
PubMed2001

A ribozyme selected from variants of U6 snRNA promotes 2',5'-branch formation.

In vitro selection was used to sample SnRNA-related sequences for ribozyme activities, and several 2',5'-branch-forming ribozymes were isolated. One such ribozyme is highly dependent upon an 11-nt motif that contains a conserved U6 snRNA sequence (ACAGAGA-box) known to be important for pre-mRNA splicing. The ribozyme reaction is similar to the first step of splicing in that an internal 2'-hydroxyl of an unpaired adenosine attacks at the 5'-phosphate of a guanosine. It differs in that the leaving group is diphosphate rather than a 5' exon. The finding that lariat formation can be accomplished by a small RNA with sequences related to U6 snRNA indicates that the RNA available in the spliceosome may be involved in RNA-catalyzed branch formation.

Animals↗
PubMed2000

XIST RNA associates with specific regions of the inactive X chromatin.

Microscopy studies have shown that XIST RNA colocalizes with the inactive X chromosome (Xi). However, the molecular basis for this colocalization is unknown. Here we provide two lines of evidence from chromatin immunoprecipitation experiments that XIST RNA physically associates with the Xi chromatin. First, XIST RNA can be co-precipitated by antiserum against macroH2A, a histone H2A variant enriched in the Xi. Second, XIST RNA can be co-precipitated by antisera that recognize unacetylated, but not acetylated, isoforms of histones H3 and H4. The specificity of XIST RNA association with hypoacetylated chromatin, together with the previous finding that hypoacetylated histone H4 is enriched at promoters of X-inactivated genes, raises the possibility that XIST RNA may contribute to the hypoacetylation of specific regions of the Xi so as to alter the expression of X-linked genes.

Animals↗
PubMed2000

RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

Double-stranded RNA (dsRNA) directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). Using a recently developed Drosophila in vitro system, we examined the molecular mechanism underlying RNAi. We find that RNAi is ATP dependent yet uncoupled from mRNA translation. During the RNAi reaction, both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length. Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA. The mRNA is cleaved only within the region of identity with the dsRNA. Cleavage occurs at sites 21-23 nucleotides apart, the same interval observed for the dsRNA itself, suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavage.

Adenosine Triphosphate↗
PubMed2000

View of life sciences in the 21st century.

As we contemplate the nature of life sciences in the 21st century, we should briefly consider the changes that have occurred in the past century. Surely, the sources of progress of this science in the next century are the advances emerging now. Furthermore, the likely pace of discovery and change in life sciences in the next century can best be estimated by a reflection on its history.

Animals↗
PubMed2000

The SRm160/300 splicing coactivator subunits.

The SRm160/300 splicing coactivator, which consists of the serine/arginine (SR)-related nuclear matrix protein of 160 kDa and a 300-kDa nuclear matrix antigen, functions in splicing by promoting critical interactions between splicing factors bound to pre-mRNA, including snRNPs and SR family proteins. In this article we report the isolation of a cDNA encoding the 300-kDa antigen and investigate the activity of it and SRm160 in splicing. Like SRm160, the 300-kDa antigen contains domains rich in alternating S and R residues but lacks an RNA recognition motif; the protein is accordingly named "SRm300." SRm300 also contains a novel and highly conserved N-terminal domain, several unique repeated motifs rich in S, R, and proline residues, and two very long polyserine tracts. Surprisingly, specific depletion of SRm300 does not prevent the splicing of pre-mRNAs shown previously to require SRm160/300. Addition of recombinant SRm160 alone to SRm160/300-depleted reactions specifically activates splicing. The results indicate that SRm160 may be the more critical component of the SRm160/300 coactivator in the splicing of SRm160/300-dependent pre-mRNAs.

Amino Acid Sequence↗
PubMed2000

A novel 50-kilodalton fragment of host cell factor 1 (C1) in G(0) cells.

Host cell factor 1 (HCF-1; also called C1) is a 230-kDa protein which is cleaved posttranslationally into separate but associated N- and C-terminal polypeptides. These polypeptides are components of the C1 complex, along with Oct-1 and the viral protein VP16. The C1 complex is formed when herpes simplex virus (HSV) infects a cell and is responsible for transcription of the HSV immediate-early genes. A temperature-sensitive mutation in the N-terminal kelch domain of HCF-1 reversibly arrests cells in a G(0)-like state when grown at the nonpermissive temperature, and the same domain interacts with VP16 in the formation of the C1 complex. The form of HCF-1 in primary G(0) cells was investigated by using peripheral blood mononucleocytes and serum-arrested human primary fibroblasts. A novel 50-kDa N-terminal fragment of HCF-1 encompassing the kelch domain was identified in the cytoplasm of these cells. This fragment arises by proteolysis of the full-length HCF-1 protein and is able to associate with VP16.

Carrier Proteins↗
PubMed1999

Targeted mRNA degradation by double-stranded RNA in vitro.

Double-stranded RNA (dsRNA) directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. The biochemical mechanisms underlying this dsRNA interference (RNAi) are unknown. Here we report the development of a cell-free system from syncytial blastoderm Drosophila embryos that recapitulates many of the features of RNAi. The interference observed in this reaction is sequence specific, is promoted by dsRNA but not single-stranded RNA, functions by specific mRNA degradation, and requires a minimum length of dsRNA. Furthermore, preincubation of dsRNA potentiates its activity. These results demonstrate that RNAi can be mediated by sequence-specific processes in soluble reactions.

3' Untranslated Regions↗
PubMed1999

Promoter-specific hypoacetylation of X-inactivated genes.

The histone H4 acetylation status of the active X (Xa) and inactive X (Xi) chromosomes was investigated at the level of individual genes. A moderate level of acetylation was observed along the lengths of genes on both the Xi and Xa, regardless of their X inactivation status. However, this moderate level of acetylation was modified specifically in promoter regions. Transcriptionally active genes showed elevated levels of acetylation at their promoters on both the Xi and Xa. In contrast, promoters of X-inactivated genes were markedly hypoacetylated, which coincided with the methylation of adjacent CG dinucleotides. This promoter-specific hypoacetylation may be a key component of an X inactivation machinery that operates at the level of individual genes.

Acetylation↗
PubMed1999

Crystal structure of an OCA-B peptide bound to an Oct-1 POU domain/octamer DNA complex: specific recognition of a protein-DNA interface.

We have determined the crystal structure, at 3.2 A, of a ternary complex containing an OCA-B peptide, the Oct-1 POU domain, and an octamer DNA site. The OCA-B peptide binds in the major groove near the center of the octamer site, and its polypeptide backbone forms a pair of hydrogen bonds with the adenine base at position 5 of the octamer DNA. Numerous protein-protein contacts between the OCA-B peptide and the POU domain are also involved in the ternary complex. In particular, the hydrophobic surface from a short alpha-helix of OCA-B helps to stabilize the complex by binding to a hydrophobic pocket on the POU-specific domain. The structure of this ternary complex is consistent with previous biochemical studies and shows how peptide-DNA and peptide-protein contacts from OCA-B provide structural and functional specificity in the regulation of immunoglobulin transcription.

Amino Acid Sequence↗
PubMed1999

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain.

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Amanitins↗
PubMed1999

The SRm160/300 splicing coactivator is required for exon-enhancer function.

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing "coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound "activators" and the snRNP machinery of the spliceosome.

Animals↗