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J G Patton

Publications and source records attributed to J G Patton.

36 records · Page 2Linked to original sources
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

Creative alliances between nursing service and education in times of economic constraint.

Health care and higher education in America are both in economic crisis and undergoing reform. Forming collaborative relationships between health care providers and nursing educational institutions is a savvy, economical, and mutually beneficial strategy in these changing times. Creative alliances between service and education are addressed in terms of clinical educational preceptorships for senior nursing students and joint appointments between service and education. The educational and service perspectives are discussed, from development to implementation.

Clinical Competence↗
PubMed1994

A collaborative model for evaluation of clinical preceptorships.

Nurses engaged in practice, recruitment, and staff development were asked to evaluate an educational clinical preceptorship jointly developed by nurse managers and nurse educators. An open-ended six-item questionnaire and a guided telephone interview were used to gather evaluative data. Nursing service respondents valued the precepted clinical experience, not only for the students but for the professional development of the staff participants. The educational clinical preceptorship provided the opportunity for collaboration and the development of collegial relationships between academia and service. An evaluation model evolved that emphasizes the importance of feedback from all participants.

Clinical Competence↗
PubMed1993

The involvement of a spliceosome component in internal initiation of human rhinovirus RNA translation.

Human rhinoviruses (HRVs) and encephalomyocarditis virus (EMCV) belong to different genera of the picornavirus family, but the translation of the RNAs of both viruses is by the same mechanism, that is, internal ribosome entry. In rabbit reticulocyte lysates this translation initiation is efficient for mRNAs bearing the EMCV 5' untranslated region (5' UTR), but very inefficient for mRNAs bearing the HRV 5' UTR, unless factors from HeLa cells are added. The copurification of the HeLa cell translation stimulatory activity with proteins which can be specifically cross-linked to the HRV 5' UTR by u.v. irradiation has been examined. Both the EMCV and HRV 5' UTRs can be cross-linked to a 58/60K protein doublet present in HeLa cell extracts in higher amounts than in reticulocyte lysates, which is shown to be very similar, if not identical to the polypyrimidine tract binding protein (PTB) previously identified as a component of a multi-subunit complex necessary for pre-mRNA splicing. However, the activity in HeLa cell extracts that specifically stimulates translation initiation on mRNAs with the HRV 5' UTR does not copurify with the majority of the 58/60K protein present in these extracts, but copurifies with a minor fraction of these proteins and with a 97K protein which can be cross-linked to the HRV 5' UTR but not to the EMCV 5' UTR, and which is absent from reticulocyte lysates. It is proposed that the specific translation initiation stimulatory activity found in HeLa cells is due to a high M(r) complex containing the 97K polypeptide and PTB.

Animals↗
PubMed1993

Cloning and characterization of PSF, a novel pre-mRNA splicing factor.

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.

Amino Acid Sequence↗
PubMed1992

Cloning and domain structure of the mammalian splicing factor U2AF.

A complementary DNA clone encoding the large subunit of the essential mammalian pre-messenger RNA splicing component U2 snRNP auxiliary factor (U2AF65) has been isolated and expressed in vitro. It contains two functional domains: a sequence-specific RNA-binding region composed of three ribonucleoprotein-consensus sequence domains, and an arginine/serine-rich motif necessary for splicing but not for binding to pre-mRNA.

Amino Acid Sequence↗
PubMed1991

Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system.

Alternative splicing has emerged in recent years as a widespread device for regulating gene expression and generating protein diversity. Its analysis has provided some mechanistic understanding of this form of gene regulation and, in addition, has provided new insights into some fundamental aspects of splicing. This mode of regulation is particularly prevalent in muscle cells, where genes such as troponin T are able to generate up to 64 different isoforms from a single transcriptional unit. Alternative splicing has the potential to raise the coding capacity of the small multigene families that code for the contractile proteins so that several million structurally different sarcomeres can be generated. The mammalian alpha-tropomyosin gene has proved particularly useful for the analysis of the mechanisms involved in this type of regulation. In particular, the mutually exclusive splicing of exons 2 and 3 has provided answers about the processes involved in the three main regulatory steps: (a) establishment of mutually exclusive behavior; (b) the elements involved in setting up the default pattern of splicing, and (c) the switch from the default to the regulated splicing pattern in some cell types.

Animals↗
PubMed1991

Alpha-tropomyosin mutually exclusive exon selection: competition between branchpoint/polypyrimidine tracts determines default exon choice.

We have used exons 2 and 3 of the rat alpha-tropomyosin gene to analyze the basis of mutually exclusive exon selection. The basis of the strict mutually exclusive behavior of this exon pair is enforced by the proximity of the exon 3 branchpoint to the 5' splice site of exon 2. With the exception of smooth muscle cells, exon 3 rather than exon 2 is incorporated into mRNA in all cell types. We show here, using both in vivo and in vitro cell-free systems, that this alternative exon selection is a consequence of general principles that govern 3' splice site selection. In the absence of exon 3, exon 2 is utilized efficiently in all cells. Selection of exon 3 is therefore the default result of a competition between exons 2 and 3 for the flanking constitutive splice sites. The basis of this competition is the relative strength of the polypyrimidine tract/branchpoint elements of the two exons. The major determinant of this splice site strength is the pyrimidine content adjacent to the branchpoint, and this involves no other sequence specificity. The branchpoint elements play an important but secondary role. The functional strengths of the different polypyrimidine tract/branchpoint combinations, as determined in cis competition assays, showed a perfect correlation with their binding affinities to a spliceosome component that interacts with the pre-mRNA in an ATP-independent manner. Selection of exon 3 in most cell types therefore reflects the preferential interaction of these splice site elements with constitutive splicing factors early in spliceosome assembly. The aspects of splice site selection analyzed here are likely to be of general applicability to constitutive and alternative pre-mRNA splicing.

Animals↗
PubMed1991

Characterization and molecular cloning of polypyrimidine tract-binding protein: a component of a complex necessary for pre-mRNA splicing.

alpha-Tropomyosin exons 2 and 3 are spliced in a mutually exclusive manner. Exon 3 is included as the default exon in the mRNA of most cell types, whereas exon 2 is only included in the mRNA of smooth muscle cells. The primary determinant for the default selection of exon 3 is the branchpoint/polypyrimidine tract. This element upstream of exon 3 clearly and effectively outcompetes the corresponding element upstream of exon 2. To identify trans-acting factors that bind to this important cis element, we used UV cross-linking to identify a 57-kD protein whose binding characteristics directly correlate with 3'-splice-site selection in cis-competition splicing assays. This protein appears to be identical to polypyrimidine tract-binding protein. In this report we have used oligonucleotides derived from peptide sequences to isolate and sequence cDNA clones encoding this 57.2-kD protein. The primary sequence reveals a novel protein with significant homology to other RNA-binding proteins. Expression of the mRNA is detected in all tissues and cells examined, although its levels exhibit tissue-specific and developmental regulation. Using a biochemical complementation assay, we have found that this protein, along with a 100-kD protein, exists as part of a large complex that is required to rescue splicing from depleted nuclear extracts.

Amino Acid Sequence↗
PubMed1989

Scanning from an independently specified branch point defines the 3' splice site of mammalian introns.

During pre-messenger RNA splicing the lariat branch point in mammalian introns is specified independently of the 3' splice site by the sequence surrounding the branch point and by an adjacent downstream polypyrimidine tract. The 3' splice site is dispensable for spliceosome assembly and cleavage at the 5' splice site, and is itself determined by a scanning process that recognizes the first AG located 3' of the branch point/polypyrimidine tract, irrespective of distance or sequence environment.

Animals↗
PubMed1987

U1 precursors: variant 3' flanking sequences are transcribed in human cells.

Using RNase protection and oligonucleotide hybridization experiments, we have shown that U1 precursors are derived by transcription of 3' flanking sequences. A labeled SP6 transcript of one of the true U1 genes (pD2) was able to protect a subset of the 3' flanking sequences present in HeLa cytoplasmic U1 RNA. However, not all U1 precursors were protected using this probe, suggesting that variant U1 precursor 3' tail sequences are expressed in HeLa cells. This conclusion has been confirmed by hybridization of HeLa RNA samples with specific oligonucleotide probes representing variant U1 3' flanking sequences. Interestingly, these variant tail sequences contain the putative Sm antigen binding site, A(U)3-6G. The conservation of this flanking sequence through evolution suggests a possible functional role for these precursor tails in ordering protein binding to U1 RNA.

Animals↗
PubMed1986

Plasma lipids and apolipoprotein A-I and A-II levels in alcoholic patients.

This prospective study compared total plasma lipids, high-density lipoprotein cholesterol (HDL-C), and apolipoproteins A-I (apo A-I) and A-II (apo A-II) in 72 alcoholic patients and in 285 nonalcoholic controls. The HDL-C in the alcoholic group was not significantly different from that in the nonalcoholic controls. Alcoholic men had significantly higher levels of cholesterol and triglycerides and lower levels of apo A-I when compared with nonalcoholic controls. Alcoholic women had significantly higher levels of cholesterol and apo A-II when compared with nonalcoholic controls. Serial measurements in 25 alcoholic patients showed a significant decline in HDL-C, apo A-I, and apo A-II levels during the 4-wk hospital stay. HDL-C demonstrated its expected inverse relationship with plasma triglyceride level and its direct relationship with apo A-I, apo A-II, and the hepatic enzyme aspartate aminotransferase.

Adult↗
PubMed1983

Monoclonal antibodies to human plasma low-density lipoproteins. I. Enhanced binding of 125I-labeled low-density lipoproteins by combined use of two monoclonal antibodies.

Four monoclonal antibodies (IgG2b) to human plasma low-density lipoproteins (LDL) have been characterized. The binding affinities of each monoclonal antibody to 125I-labeled LDL were moderately high, ranging from 10(8) to 10(10) L/mol at 4 degrees C, but were reduced by at least 50-70% at 37 degrees C. The maximum binding of each monoclonal antibody was unique, ranging from 20 to 95% of total 125I-labeled LDL, suggesting that LDL particles were immunochemically heterogeneous. One antibody, LP-34, had both high and low binding affinities to LDL. Another, LP-47, exhibited high affinity for isolated LDL, yet reacted poorly with native LDL in plasma, indicating that the conformation of isolated LDL differs from that of native LDL in plasma. Unlike polyclonal serum antibodies, a mixture of four monoclonal antibodies failed to precipitate LDL, but did show a drastic increase in binding to LDL. We found that only two of our monoclonal antibodies were necessary for such synergistic enhancement. We propose that one of the monoclonal antibodies may serve as a catalytic reagent, and discuss the clinical significance of this finding.

Antibodies, Monoclonal↗
PubMed1983

Monoclonal antibodies to human plasma low-density lipoproteins. II. Evaluation for use in radioimmunoassay for apolipoprotein B in patients with coronary artery disease.

We describe the importance of the low-density lipoproteins (LDL) used in preparing radioimmunoassay standard curves and the clinical application of monoclonal antibodies to LDL. LDL isolated from five normal men produced five parallel displacement curves with conventional mouse antiserum but there was a significant difference of immunoreactivity among the LDL. None of our four monoclonal antibodies could eliminate the heterogeneous immunoreactivity of different LDL. Thus, the determination of plasma apolipoprotein (apo) B will vary depending on the selection of LDL standards, and the comparison of absolute apo B values between laboratories will be of questionable value unless they use the same LDL standard. Nonetheless, in a radioimmunoassay our monoclonal antibody, LP-22, detected a more significant (p less than 0.0001) increase of plasma apo B in patients with angiographically documented coronary artery disease than did conventional antiserum (p less than 0.001). In addition, the overlap of apo B concentrations for patients with and without disease was less when monoclonal antibody LP-22 was used. We conclude that patients with coronary artery disease have a significant increase in the form of plasma apo B that is specifically recognized by LP-22 monoclonal antibody. Perhaps monoclonal antibodies will be able to sort out the various components of apo B, delineate their possible atherogenic roles, and offer us a predictive value for diagnosing such patients.

Antibodies, Monoclonal↗
PubMed1982

Evaluation of monoclonal antibodies to human plasma low density lipoproteins. A requirement for lipids to maintain antigenic structure.

Human plasma low density lipoproteins (LDL) are composed of approximately 25% apoproteins and 75% lipids (w/w). Immunochemical properties of LDL were studied using monoclonal antibodies. BALB/c mice were immunized with LDL and the spleen cells from these mice were then fused with a non-immunoglobulin secreting myeloma cell line (F0). The clones producing desirable antibodies were selected to study the antigenic properties of LDL by enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay. First, it was found that the maximal binding of 125I-labeled LDL to polyvinyl chloride microtiter dishes was not temperature dependent. The binding affinity was high with a Ka value of approximately 1.9 X 10(10) M-1 while the monoclonal antibodies possessed an affinity to LDL of 5 X 10(8) M-1 which was 2 orders less than the affinity of LDL to the dishes. The former binding, once established, was irreversible as judged by a subsequent incubation with an excess of unlabeled LDL. The latter binding could be displaced by unlabeled LDL. Therefore, the ELISA technique offered a satisfactory approach to study the interaction between LDL and monoclonal antibodies. Removal of lipids from bound LDL by organic extraction resulted in a 50% loss of immunoreactivity, suggesting that the lipids of LDL are important in maintaining the antigenic structure of LDL. Since the apoprotein of LDL also constitutes approximately 40% of the mass (w/w) of very low density lipoproteins (VLDL), the immunoreactivity of VLDL assessed by LDL-monoclonal antibodies was also carried out. Removal of triglycerides from VLDL by lipoprotein lipase resulted in a substantial loss of immunoreactivity as determined by radioimmunoassay. These findings are consistent with the concept that lipids play a role in maintaining the integrity of the antigenic structure of LDL.

Animals↗
PubMed1980

Occupational therapy for the limb-deficient child: a developmental approach to treatment planning and selection of prostheses for infants and young children with unilateral upper extremity limb deficiencies.

Habilitation of the upper extremity limb-deficient child is enhanced when the treatment program is based on normal growth and development guidelines. Specific developmental tasks have been identified as key indicators of a child's readiness to benefit from prosthetic fitting, activation of major components, use training, and changes in prosthetic design such as modular prostheses and electric components. Use of developmental guidelines also enhances family support because their role in the program can be realistically defined and supported.

Arm↗