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R Reed

Publications and source records attributed to R Reed.

At least 19 recordsLinked to original sources
PubMed1996

Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A.

A critical step in the pre-mRNA splicing reaction is the stable binding of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are not known. Saccharomyces cerevisiae homologs of all of the SF3a, but none of the SF3b, subunits have been identified. Here we report the isolation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the identification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex proteins can be extended to SF3b adds strong new evidence that the mechanism of A complex assembly is highly conserved. To investigate this mechanism in the mammalian system we analyzed proteins that cross-link to 32P-site-specifically labeled pre-mRNA in the A complex. This analysis revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS. Mutation of this region, which we have designated the anchoring site, has no apparent effect on U2 snRNP binding. In contrast, when a 2'O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-linking of the SF3a/SF3b subunits are blocked. These data indicate that sequence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pre-mRNA.

Amino Acid Sequence↗
PubMed1996

Skeletal myogenesis: the preferred pathway of chick embryo epiblast cells in vitro.

The epiblast layer of the chick embryo gives rise to all embryonic tissues. In vitro analyses were carried out to determine whether epiblast cells could form skeletal muscle prior to entry into the primitive streak. Epiblasts were separated from the mesoderm, hypoblast, and primitive streak, dissociated to produce a single cell suspension, and plated at high density. Myogenesis began on the first day in culture, and by the fifth day most cells had differentiated into skeletal muscle. Some cells differentiated without replicating. MyoD messenger RNA was present in epiblast tissue and translated in practically all cells in culture. Cells from regions of the epiblast which do not form muscle later in the embryo did so in vitro. Epiblasts cultured for 2 days as an intact epithelium, or in the presence of the mesoderm and hypoblast, did not undergo myogenesis. These findings demonstrate that myogenic potential is wide-spread within the primitive streak stage epiblast, and that muscle differentiation, which occurs relatively autonomously in culture, can be prevented by cell and tissue interactions.

Animals↗
PubMed1995

Members of a family of proteins (the RD family) detected by a U1 70K monoclonal antibody are present in spliceosomal complexes.

We have characterized a monoclonal antibody (mAb) to the U1 snRNP component U1 70K. We find that this antibody recognizes several proteins, in addition to U1 70K, in purified spliceosomal complexes and in total HeLa cell nuclear extract preparations. The novel mAb U1 70K antigens can also be specifically immunoprecipitated by the antibody. Similarly to U1 70K, many of the mAb U1 70K antigens can be phosphorylated by a co-purifying kinase activity. The epitope recognized by mAb U1 70K was previously shown to be a repeating arginine/aspartate (RD) dipeptide. Thus we have designated the novel mAb U1 70K antigens the RD family. Comparison of mAb U1 70K with a recently characterized antibody, mAb 16H3, whose epitope is a repeating R/D or R/E motif, showed that a large subset of the antigens are common. In contrast, most of the mAb U1 70K antigens are distinct from the proteins detected by mAb 104, an antibody to the SR family of splicing factors.

Amino Acid Sequence↗
PubMed1995

A two-step mechanism for 5' and 3' splice-site pairing.

A fundamental question in the splicing of precursor messenger RNA is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. It has been proposed that spliceosome assembly in metazoan pre-mRNAs can be initiated through interaction between the 3' splice site and specific sequence elements on the downstream exon (an exonic enhancer or a 5' splice site). Pairing of the intronic 5' and 3' splice sites occurs subsequently. We report here that 5' and 3' splice sites located on separate synthetic pre-mRNA substrates can be efficiently trans-spliced if the 3' trans-splicing substrate contains these downstream sequence elements. Moreover, selection of the trans 5' splice site can occur after the second pre-spliceosomal complex A has assembled on the 3' trans-splicing substrate. Thus our data demonstrate that 5' and 3' splice-site pairing in metazoans can occur in two distinct steps.

Animals↗
PubMed1995

Ethanol elimination rates in an ED population.

Knowledge of the rate of ethanol elimination is essential in the assessment of the intoxicated patient. Surprisingly little literature is available regarding ethanol elimination rates in emergency department (ED) patients; prior studies almost exclusively examined populations of alcoholics or normal controls. Consequently, this prospective observational study was undertaken to assess the rate of ethanol elimination in an ED population. Twenty-four consecutive adult ED patients clinically suspected of intoxication who had serum ethanol determinations drawn were enrolled. Patients underwent serial ethanol determinations via breathalyzer (Intoxilyzer 1400, CMI Inc., Owensboro, KY). Linear regression analysis of the plot of decrease in ethanol level over time was performed to determine the rate of ethanol elimination. Initial ethanol levels in the 24 patients ranged from 58 to 447 mg/dL (mean, 249 +/- 109 [SD] mg/dL). Patients were observed for a minimum of 2 and a maximum of 9 observations (mean, 3.9 +/- 1.7), over a period of 0.5 to 12.1 hours (mean, 4.4 +/-3.5 h). Clinical features of intoxication were poorly correlated with ethanol level (r < .5). The rate of ethanol elimination in the ED population was 19.6 mg/dL/h (r = .83; 95% confidence interval [CI], 16.9 to 22.3 mg/dL/h). Subgroup analysis found differences that were statistically significant but small. Multiple regression analysis showed that time was the major variable useful in predicting changes in ethanol level (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1995

Literacy and performance on the Mini-Mental State Examination.

OBJECTIVE: Mini-Mental State Examination (MMSE) scores are associated with age, education, and ethnicity. The objective of this research was to determine the relationship between MMSE and literacy. DESIGN: Cross-sectional. SETTING: A Community population in Tucson, Arizona. PARTICIPANTS: Senior citizens in public assistance housing. MEASUREMENTS: MMSE score, reading level, and sociodemographic variables. MAIN RESULTS: A total of 214 subjects were tested. The mean age was 72.3 (+/- 8.01), and the mean education level was 10.3 years (range 0-20 years). Reading levels ranged from grade 0 (non-reader) to 8 (maximum value for the test used). with a mean of 4.8 (+/- 2.8). MMSE scores ranged from a mean of 21.9 (+/- 4.6) for non-readers to a mean of 28.0 (+/- 2.0) for subjects with > or = 8th grade reading skills. Multiple regression using reading level, education, age, and ethnicity as independent variables, and MMSE score as the dependent variable, showed the highest correlation was between MMSE and reading level (R2 = .265). Education level made only a small additional contribution; age and ethnicity did not enter the regression equation. Correlations between reading level and individual MMSE subsections were highest between reading level and MMSE language items (r = .499). CONCLUSIONS: The correlation between MMSE score and reading skills is stronger than the correlation with other sociodemographic variables. Proper interpretation of MMSE scores requires knowledge of patients' reading levels.

Aged↗
PubMed1995

Accumulation of a novel spliceosomal complex on pre-mRNAs containing branch site mutations.

Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

Base Sequence↗
PubMed1995

Ethical and clinical issues with Native-American elders. End-of-life decision making.

This article offers guidance to clinicians for approaching and conducting end-of-life decision-making conversations with Native American elders. The guidelines emphasize the need for flexibility and clarity in communication, avoidance of insistence on formal structures and rigid time frames for decision-making, sensitivity to the cultural and family situation of the elder, and recognition that cultural as well as language interpretation may be necessary. Given the great diversity of the tribes and bands as well as languages among native people and the paucity of empirical work on this topic, the tentative nature of these guidelines is stressed.

Advance Directives↗
PubMed1994

The prespliceosome components SAP 49 and SAP 145 interact in a complex implicated in tethering U2 snRNP to the branch site.

The mammalian spliceosome-associated protein, SAP 49, is associated specifically with U2 snRNP and is the most efficiently UV cross-linked protein in the spliceosomal complexes A, B, and C. We show here that SAP 49 cross-links to a region in the pre-mRNA immediately upstream of the branchpoint sequence in the prespliceosomal complex A. In addition to the RNA-binding activity of SAP 49, we show that this protein interacts directly and highly specifically with another U2 snRNP-associated spliceosomal protein, SAP 145. We have isolated a cDNA-encoding SAP 49 and find that it contains two amino-terminal RNA-recognition motifs (RRMs), consistent with the observation that SAP 49 binds directly to pre-mRNA. The remainder of the protein is highly proline-glycine rich (39% proline and 17% glycine). Unexpectedly, the SAP 49-SAP 145 protein-protein interaction requires the amino-terminus of SAP 49 that contains the two RRMs. The observation that SAP 49 and SAP 145 interact directly with both U2 snRNP and the pre-mRNA suggests that this protein complex plays a role in tethering U2 snRNP to the branch site.

Amino Acid Sequence↗
PubMed1994

A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction.

We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.

Base Sequence↗
PubMed1994

Specific protein-protein interactions between the essential mammalian spliceosome-associated proteins SAP 61 and SAP 114.

Spliceosome-associated proteins (SAPs) 61, 62, and 114 can be UV-crosslinked to pre-mRNA in purified spliceosomal complexes and are associated with U2 small nuclear ribonucleoproteins (snRNP). These proteins also compose the essential heterotrimeric splicing factor SF3a, and products of yeast pre-mRNA processing genes PRP9, PRP11, and PRP21 are their likely yeast counterparts. We report the isolation of a cDNA encoding SAP 61 and find that it is 30% identical in amino acid sequence to PRP9. A C-terminal Cys2His2 zinc-finger-like motif, which could be involved in the pre-mRNA binding, is the most highly conserved region of the protein. We also demonstrate specific protein-protein interactions between SAPs 61 and 114 and show that the N terminus of SAP 61 is required for this interaction. Significantly, the corresponding proteins are also known to interact in yeast: PRP9 interacts with PRP21, and the N-terminal portion of PRP9 is required. Previous work showed that direct interactions also occur between SAPs 62 and 114 and between the corresponding PRPs 11 and 21. These observations indicate that the specific protein-protein interactions that occur between the three prespliceosomal factors have been conserved between yeast and mammals.

Amino Acid Sequence↗
PubMed1994

Occupational asthma in a latex doll manufacturing plant.

BACKGROUND: Occupational asthma caused by latex has been reported in health care workers and workers in glove manufacturing plants. OBJECTIVE: We report occupational asthma from latex in a newly identified occupational setting, a latex doll manufacturing plant. METHODS: We evaluated an index case of asthma associated with work in a latex doll manufacturing plant by performing a workplace challenge and evaluating the work environment. We then performed an occupational survey and skin testing of 22 workers in the doll manufacturing plant. RESULTS: The patient, a 21-year-old woman, had severe immediate bronchospasm within minutes of beginning a workplace challenge where sanding of latex parts was performed. Two of 22 workers surveyed (including the patient) reported flushing, rhinoconjunctivitis, and wheezing on exposure to sanded doll parts. These two workers were the only subjects surveyed to have a history of atopy and positive immediate-type skin test responses to a raw latex extract and to common aeroallergens. CONCLUSIONS: Sanding or grinding of solid latex during the manufacturing process may result in a significant incidence of occupational asthma and rhinoconjunctivitis from latex sensitization. Atopic workers appear to be most susceptible to developing latex sensitivity in this setting.

Adult↗
PubMed1994

Analysis of cerbB2 expression using a panel of 6 commercially available antibodies.

Results are presented of a study comparing cerbB2 (neu or Her2) expression as assessed immunohistochemically in breast neoplasia using a panel of 6 commercially available antibodies. The antibodies were examined utilizing conventional formalin fixed paraffin embedded tissue, and compared with molecular analysis of gene amplification. The aim was to determine the practical utility of each antibody, assessing ease of use, specific and non-specific staining characteristics, and expense, thus allowing a specific recommendation as to antibody of choice for immunohistochemical assessment of cerbB2 expression. Reassuringly, amongst the 38 breast lesions (36 carcinomas, 2 fibroadenomas) subjected to immunohistochemically (IHC) with the panel of 6 antibodies (Ab), no gross discrepancy of staining pattern was seen. Of the 38 cases, 10 were positive (26%), where at least one Ab demonstrated clear cytoplasmic membrane staining. Of a total of 45 breast lesions (43 carcinomas, 2 fibroadenomas), including all those examined by IHC, the total number of cases showing cerbB2 amplification by DNA analysis was 14 (31%). Using the DNA amplification as a base line for comparison, one Ab (No. 4) was found to stain 6 of the 14 cases of breast carcinoma that were assessed as showing amplification at the DNA level. Four Abs (1,3,5,6) stained 5 of these cases. However, Abs 3,4 and 6 displayed artefactual cytoplasmic staining (in the absence of membrane staining) that precluded the practical use of these reagents. Therefore, based on additional considerations of cost and ease of use, Ab No. 1 was finally chosen for recommendation from the 6 Ab panel.

Adult↗
PubMed1994

Patterns of speech disorders in schizophrenia and mania.

Formal thought disorder (FTD), defined as abnormal speech, has been associated with schizophrenia and likened to fluent aphasia. Whether FTD differentiates subtypes of schizophrenics and discriminates schizophrenics from other patients is unclear. We studied this issue by analyzing ratings of FTD of 170 schizophrenics and 62 manics. Eighty percent of emotionally blunted schizophrenics had FTD compared with 6.5% of manics. Factor analysis revealed verbiage disturbance and disorganized speech factors (44% of the variance). We assessed the discriminating ability of these factors, and compared these results to those from factors derived from Andreasen's positive/negative FTD construct, and to factors derived from speech and language diagnostic criteria for schizophrenia and mania in the proposed DSM-IV. Overall classifications were similar (91%, 91%, and 88%, respectively). We also found that FTD was related to emotional blunting, but not to other psychopathology.

Adolescent↗
PubMed1994

SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex.

We show that addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble on RNAs containing only 5' and 3' splice sites, respectively. We conclude that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting for this function in HeLa nuclear extracts. Using UV cross-linking, we demonstrate specific, splice site-dependent RNA-protein interactions of SR proteins in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. We also show that a discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which we have designated the Enhancer complex, contains U1 small nuclear RNP (snRNP) and is associated with different SR protein family members, depending on the sequence of the enhancer. We propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex.

Base Sequence↗