PubMed Health⌕ Search

Biomedical subjects

S McCracken

Publications and source records attributed to S McCracken.

At least 19 recordsLinked to original sources
PubMed2001

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans.

BACKGROUND: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs. RESULTS: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother. CONCLUSIONS: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

Amino Acid Sequence↗
PubMed2001

Maori work-related fatal injury, 1985-1994.

AIMS: To document the rate of work-related fatal injury for Maori; to establish whether a difference exists between Maori and non-Maori; and to examine possible explanations in the event that differences did occur. METHODS: Coronial files collected as part of the examination of work-related fatal injuries occurring between 1985 and 1994, excluding motor vehicle fatalities on public roads, were reviewed. Maori were identified by either the classification recorded upon death certificates or if they were identified as Maori within coroner's files. RESULTS: 89 Maori were identified within the 741 worker fatalities. Agreement between the data sources used to identify ethnic status was approximately 52%. The crude rate for the decade was significantly higher for Maori than non-Maori. A significant linear decline across years was evident for the non-Maori rates but not for Maori rates. CONCLUSIONS: This study, the first to specifically investigate work-related injury for Maori, confirms that an overall disparity exists between Maori and non-Maori, and that it is probably due to differences in employment patterns.

Accidents, Occupational↗
PubMed2000

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes.

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

Antigens, Nuclear↗
PubMed1999

MCM proteins are associated with RNA polymerase II holoenzyme.

MCMs are a family of proteins related to ATP-dependent helicases that bind to origin recognition complexes and are required for initiation of DNA replication. We report that antibodies against MCM2(BM28) specifically inhibited transcription by RNA polymerase II (Pol II) in microinjected Xenopus oocytes. Consistent with this observation, MCM2 and other MCMs copurified with Pol II and general transcription factors (GTFs) in high-molecular-weight holoenzyme complexes isolated from Xenopus oocytes and HeLa cells. Pol II and GTFs also copurified with MCMs isolated by anti-MCM3 immunoaffinity chromatography. MCMs were specifically displaced from the holoenzyme complex by antibody against the C-terminal domain (CTD) of Pol II. In addition, MCMs bound to a CTD affinity column, suggesting that their association with holoenzyme depends in part on this domain of Pol II. These results suggest a new function for MCM proteins as components of the Pol II transcriptional apparatus.

Animals↗
PubMed1999

SR-related proteins and the processing of messenger RNA precursors.

The processing of messenger RNA precursors (pre-mRNA) to mRNA in metazoans requires a large number of proteins that contain domains rich in alternating arginine and serine residues (RS domains). These include members of the SR family of splicing factors and proteins that are structurally and functionally distinct from the SR family, collectively referred to below as SR-related proteins. Both groups of RS domain proteins function in constitutive and regulated pre-mRNA splicing. Recently, several SR-related proteins have been identified that are associated with the transcriptional machinery. Other SR-related proteins are associated with mRNA 3' end formation and have been implicated in export. We review these findings and evidence that proteins containing RS domains may play a fundamental role in coordinating different steps in the synthesis and processing of pre-mRNA.

Animals↗
PubMed1998

The guanylyltransferase domain of mammalian mRNA capping enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II.

We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.

Amino Acid Sequence↗
PubMed1998

Optimization of capillary chromatography ion trap-mass spectrometry for identification of gel-separated proteins.

The current paradigm for protein identification using mass spectrometric derived peptide-mass and fragment-ion data employs computer algorithms which match uninterpreted or partially interpreted fragment-ion data to sequence databases, both protein and translated nucleotide sequence databases. Nucleotide sequence databases continue to grow at a rapid rate for some species, providing an unsurpassed resource for protein identification in those species. Ion-trap mass spectrometers with their ability to rapidly generate fragment-ion spectra in a data-dependent manner with high sensitivity and accuracy has led to their increased use for protein identification. We have investigated various parameters on a commercial ion trap-mass spectrometer to enhance our ability to identify peptides separated by capillary reversed phase-high performance liquid chromatography (RP-HPLC) coupled on-line to the mass spectrometer. By systematically evaluating the standard parameters (ion injection time and number of microscans) together with selection of multiple ions from the full mass range, improved tandem mass spectrometry (MS/MS) spectra were generated, facilitating identification of proteins at a low pmol level. Application of this technology to the identification of a standard protein and an unknown from an affinity-enriched mixture are shown.

Amino Acid Sequence↗
PubMed1998

The late expression factors 8 and 9 and possibly the phosphoprotein p78/83 of Autographa californica multicapsid nucleopolyhedrovirus are components of the virus-induced RNA polymerase.

Attempts were made to identify some of the subunits of the baculovirus-induced RNA polymerase following purification of its enzymatic activity by conventional chromatography. Polymerase activity was extracted from lysates of insect cells infected with Autographa californica multicapsid nucleopolyhedrovirus by polyethylenimine precipitation and subsequently purified by phosphocellulose, anion exchange, poly(A) Sepharose affinity, and gel filtration chromatography. The presence of the polymerase was monitored by its alpha-amanitin-resistant activity in in vitro transcription assays. A number of polypeptides associated with the enzymatic activity were identified. Peptide-specific antibodies were generated against a variety of late-expression factors (LEFs) and these antibodies, along with antisera directed against several other baculovirus proteins, were used in an immunoblot analysis of the purified polymerase. The results revealed that both the viral helicase (p143) and the virogenic stroma protein, pp31, copurify with the baculovirus-induced RNA polymerase activity through several chromatographic steps and may be loosely associated with the RNA polymerase. LEF8, LEF9 and p78/83, a nucleocapsid-associated phosphoprotein, were found to associate with the viral-induced polymerase activity. LEF8 and LEF9 contain regions of sequence homology with components of other DNA-directed RNA polymerases, while a portion of p78/83 exhibits some homology to the sigma factor of bacterial RNA polymerase, the RAP30 protein found in the mammalian transcription complex TFIIF, and the RAP94 polypeptide associated with vaccinia virus RNA polymerase. The p78/83 protein has previously been shown by our laboratory to be a capsid protein, but it may also play some role with the RNA polymerase. These results represent a first attempt to identify specific components of the RNA polymerase associated with infections of insect cells by A. californica nucleopolyhedrovirus.

Amino Acid Sequence↗
PubMed1997

5'-Capping enzymes are targeted to pre-mRNA by binding to the phosphorylated carboxy-terminal domain of RNA polymerase II.

We have investigated the role of the RNA Polymerase II (Pol II) carboxy-terminal domain (CTD) in mRNA 5' capping. Transcripts made in vivo by Pol II with a truncated CTD had a lower proportion of capped 5' ends than those made by Pol II with a full-length CTD. In addition, the enzymes responsible for cap synthesis, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase bound directly to the phosphorylated, but not to the nonphosphorylated, form of the CTD in vitro. These results suggest that: (1) Pol II-specific capping of nascent transcripts in vivo is enhanced by recruitment of the capping enzymes to the CTD and (2) capping is co-ordinated with CTD phosphorylation.

Amino Acid Sequence↗
PubMed1997

An alternative pathway for expression of p56lck from type I promoter transcripts in colon carcinoma.

The lymphoid-specific protein tyrosine kinase, p56lck which is essential for both T cell development and function, is aberrantly expressed in colon and small lung carcinoma lines. In this paper, we demonstrate p56lck is also expressed in colon tumour biopsies due predominantly or exclusively to the use of the lck type I promoter. In T leukaemia lines, the lck type I promoter requires binding sites for both Ets- and Myb-related transcription factors. In contrast, in colon tumour lines the activation of the lck type I promoter requires the Ets but not the Myb binding site. In these lines, a consensus binding site for HMG-related transcription factors, AACAAAG, is required for efficient lck type I promoter activity. Sox-4 is a candidate transcription factor for binding and activating the lck type l promoter in colon carcinoma cells. Co-expression of Ets-1 and Sox-4, but neither protein alone, was sufficient to activate the lck type l promoter in HeLa cells which do not normally express lck transcripts. These results suggest that aberrant expression of p56lck from the lck type l promoter in colon carcinoma arises from transcriptional activation mediated by Ets- and HMG-related transcription factors.

Alternative Splicing↗
PubMed1997

The C-terminal domain of RNA polymerase II couples mRNA processing to transcription.

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

Animals↗
PubMed1997

Collegial support and barriers to behavioral programs for severe mental illness.

Previous investigations have identified staff beliefs about barriers to implementing behavioral interventions in programs for persons with severe mental illness. One of these barriers, institutional constraints, was found to be associated with collegial support; i.e., staff who report more collegial support were less likely to endorse institutional constraints. The purpose of this study was to determine how the components of collegial support were associated with beliefs about institutional constraints. Fifty-six staff members completed measures of staff opinions about barriers to implementing behavior therapy, satisfaction with collegial support, source of support, and functions of support. Results suggested that collegial support is significantly associated with co-worker and supervisor support, but not the support of family and friends. Endorsing institutional constraints was inversely associated with the support of co-workers and supervisors; institutional constraints were positively associated with the support of family and friends. Endorsing institutional constraints was also inversely associated with the sense that others rely upon the individual for their well-being. Implications of these findings for diminishing barriers to behavioral interventions are discussed.

Behavior Therapy↗
PubMed1996

Can severely mentally ill adults reliably report their needs?

Proponents of psychiatric rehabilitation believe that active patient participation in rehabilitation planning is essential. Participation assumes, however, that severely mentally ill patients can reliably report their needs. Unfortunately, reliably responding to a needs assessment may be diminished by cognitive deficits that are associated with psychosis and low intelligence. To address this issue, 35 severely mentally ill outpatients completed the Needs and Resources Assessment twice, with 1 week intervening. Their reliability on this task was compared with that of 16 control subjects. Results showed that subjects in both groups performed more reliably (measured as raw agreement) on the more highly structured, standardized items. Patients were less reliable than controls on individualized and standardized items. Further analyses suggested that the patients' raw agreement was found to be significantly associated with thought disturbance. Assessment methods must be sufficiently structured to help cognitively disordered patients provide reliable information for rehabilitation planning.

Adult↗
PubMed1996

Three functional classes of transcriptional activation domain.

We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.

Base Sequence↗
PubMed1996

TFIIH functions in regulating transcriptional elongation by RNA polymerase II in Xenopus oocytes.

We investigated the role of TFIIH in transcription by RNA polymerase II (pol II) in vivo by microinjection of antibodies against this factor into Xenopus oocytes. Five different antibodies directed against four subunits of TFIIH were tested for effects on transcription of coinjected human immunodeficiency virus type 2 and c-myc templates. Each of these antibodies severely reduced the efficiency of elongation through human immunodeficiency virus type 2 and c-myc terminator elements. In contrast, an anti-TFIIB antibody did not inhibit elongation. Anti-TFIIH antibodies also had a much smaller inhibitory effect on total transcription than did anti-TFIIB or anti-pol II large subunit. Three inhibitors of TFIIH kinase activity, H-7, H-8, and dichlororibofuranosylbenzimidazole (DRB), inhibited elongation similarly to anti-TFIIH antibodies. These results strongly suggest a role for TFIIH in the stimulation of transcriptional elongation in vivo.

Animals↗
PubMed1996

Expression of granulocyte-colony stimulating factor and its receptor is regulated during the development of the human placenta.

The development of the placenta is dependent upon the regulated proliferation, invasion and differentiation of trophoblast. Expression of cytokines at the feto-maternal interface suggests that these molecules may participate in placentation. The expression of granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) during the development of the human placenta was studied by immunohistochemistry using an anti-G-CSF monoclonal antibody (mAb) and two novel anti-G-CSFR mAbs. G-CSF was present in the stroma of fetal chorionic villi and maternal decidual tissues throughout pregnancy. G-CSFR was detected at high levels in fetal first and third, but not second trimester placental tissues. Staining for G-CSFR was undetectable in maternal decidual tissue from all gestational stages. In first trimester tissues, staining for placental G-CSFR was strongest in differentiated syncytiotrophoblast and invasive, extravillous cytotrophoblast, and weak staining was evident in undifferentiated cytotrophoblast. Immunohistochemical data suggesting temporal regulation of G-CSFR were corroborated by Western blotting and amplification by reverse transcription and PCR of G-CSFR mRNA. These data suggested that expression of G-CSFR in the human placenta is regulated both temporally and spatially, and that placental G-CSF is involved in paracrine regulation, and indicate a role for G-CSF and G-CSFR in trophoblast growth or function during placentation.

Base Sequence↗
PubMed1994

Myb and Ets related transcription factors are required for activity of the human lck type I promoter.

The lck gene, which encodes a lymphoid-specific Src family tyrosine kinase, is transcribed from two promoters that are differentially utilized during T cell development. We have shown previously that the human lck type I promoter, which is preferentially expressed in immature thymocytes, requires a binding site (-97 to -90) for the Ets family of transcription factors for its activity in Jurkat T leukemia cells. Three putative Myb binding sites (-86 to -82, -77 to -72 and -59 to -54) were analysed for their ability to activate the lck type I promoter. In vitro assays demonstrated specific binding of purified, bacterially expressed c-Myb DNA binding domain to the Myb (-59 to -54) site. Transient transfection assays using the site-directed mutants of the lck type I promoter in Jurkat cells revealed that mutation of the Myb (-59 to -54) site abolished transcriptional activity. In transiently transfected HeLa cells, the lck type I promoter was activated by co-transfection with a vector that expresses c-Myb. This c-Myb dependent activation required the presence of intact Myb and Ets binding sites, indicating that the expressed c-Myb functions with endogenous Ets related transcription factors to activate the lck type I promoter. This effect was further enhanced by co-transfection with vectors that express either Ets1 or Ets2. These results demonstrate that Myb and Ets related transcription factors synergistically activate the human lck type I promoter.

Base Sequence↗