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Biomedical subjects

J Abelson

Publications and source records attributed to J Abelson.

At least 37 records · Page 2Linked to original sources
PubMed1996

18S rRNA processing requires the RNA helicase-like protein Rrp3.

We report the identification of a new gene, RRP3 (rRNA processing), which is required for pre-rRNA processing. Rrp3 is a 60.9 kDa protein that is required for maturation of the 35S primary transcript of pre-rRNA and is required for cleavages leading to mature 18S RNA. RRP3 was identified in a PCR screen for DEAD box genes. DEAD box genes are part of a large family of proteins homologous to the eukaryotic transcription factor elF-4a. Most of these proteins are RNA-dependent ATPases and some of them have RNA helicase activity. This is the third yeast DEAD box protein that has been shown to be involved in rRNA assembly, but the only one required for the processing of 18S RNA. Mutants of the two other putative helicases, Spb4 and Drsl, both show processing defects in 25S rRNA maturation. In strains where Rrp3 is depleted, 35S precursor RNA is improperly processed. Cleavage normally occurs at sites A0O, Al and A2, but in the Rrp3 depletion stain cleavage occurs between A2 and B1. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity which is not specific for rRNA.

Adenosine Triphosphatases↗
PubMed1996

In search of informed input: a systematic approach to involving the public in community decision making.

Given the task of distributing scarce resources, decision makers are faced with the question of how to involve an increasingly threatened and disenfranchised public in decisions affecting their communities. This article introduces a systematic approach to public involvement in community decision-making and identifies key elements in the design of institutional driven public participation exercises. Examples are drawn from the health care system restructuring experiences of three Ontario communities.

Community Health Planning↗
PubMed1996

Shifting boundaries in professional care.

The nature of the work undertaken by different health professionals and inter-professional boundaries are constantly shifting. The greater knowledge of users of health care, and the increasing technical and organizational complexity of modern medicine, have partly eroded the control of health professionals over the substance of their work. The definition of a field of work as lying within the province of any one profession is culturally rather than scientifically determined. It is evident that care of good quality should be delivered at the lowest possible cost. This might include delivery of care by a less trained person than heretofore, or by someone with limited but focused training. Sharing of skills is a more sensible subject for discussion than transfer of tasks. We review a number of studies which show the effectiveness of inter-professional substitution in various care settings, and also the effectiveness of substitution by those other than health professionals. The views of users of health services on inter-professional substitution need to be considered. Health professionals and others need to work together to devise innovative ways of delivering effective health care. The legal issues need clarification.

Delivery of Health Care↗
PubMed1996

Preventive care and barriers to effective prevention. How do family physicians see it?

OBJECTIVES: To assess how adequately family physicians think they are delivering preventive care and to examine barriers to providing preventive care. DESIGN: Cross-sectional survey. SETTING: Primary care medical practices in south-central Ontario. PARTICIPANTS: Four hundred eighty family physicians and general practitioners who graduated from medical school between 1972 and 1988. MAIN OUTCOME MEASURES: Satisfactory preventive care delivery versus self-assessed coverage of patients for 15 preventive maneuvers. Perceived reasons for lack of success in providing recommended preventive care. RESULTS: For 10 of the 15 maneuvers, the proportion of physicians who regarded 90% or higher as satisfactory coverage was twice as great as the proportion who thought they provided that level of coverage. For 11 of the 15 maneuvers, most respondents reported coverage lower than the level they regarded as satisfactory. For six maneuvers, more than two thirds thought they provided less than satisfactory coverage. More than two thirds of respondents suggested these barriers to providing recommended preventive care: patient is healthy and does not visit; patient refuses, is not interested, or does not comply; no effective systems to remind patients to come in for preventive care; and priority given to presenting problem. CONCLUSION: Many family physicians and general practitioners in south-central Ontario provide preventive care to their patients at lower levels than they consider satisfactory. They identified barriers to providing preventive services successfully; these barriers suggest approaches for improving care.

Attitude of Health Personnel↗
PubMed1996

Site-specific crosslinks of yeast U6 snRNA to the pre-mRNA near the 5' splice site.

We have introduced a single photochemical crosslinking reagent into specific sites in the central domain of U6 to identify the sites that are in close proximity to the pre-mRNA substrate. Four distinct U6 snRNAs were synthesized with a single 4-thiouridine (4-thioU) at positions 46, 51, 54, and 57, respectively. Synthetic U6 RNA containing the 4-thioU modifications can functionally reconstitute splicing activity in cell-free yeast splicing extracts depleted of endogenous U6 snRNA. Upon photoactivation with UV (>300 nm), 4-thioU at position 46 forms crosslinks to pre-mRNA near the 5' splice site at nt +4, +5, +6, and +7 in the intron, whereas 4-thioU at position 51 crosslinks to the pre-mRNA at positions -2, -1, +1, +2, +3, and at the invariant G in the lariat intermediate. All crosslinks are dependent on the presence of ATP and the splicing substrate. The two crosslinks to the pre-mRNA from position 46 and 51 of U6 can also occur in prp2 heat-inactivated yeast splicing extracts blocked immediately prior to the first chemical step. Significantly, the crosslink from position 51 can undergo subsequent splicing when the mutant extract is complemented with functional Prp2 protein in a chase experiment, indicating that the crosslink reflects a functional interaction that is maintained during the first step. The crosslink to lariat intermediate appears when the mutant spliceosomes are complemented with functional Prp2 protein added exogenously. This experiment is a paradigm for future studies in which different mutant extracts are used to establish the stage in assembly at which particular RNA-RNA interactions defined by unique crosslinks occur.

Actins↗
PubMed1996

Do female primary care physicians practise preventive care differently from their male colleagues?

OBJECTIVE: To assess whether female primary care physicians' reported coverage of patients eligible for certain preventive care strategies differs from male physicians' reported coverage. DESIGN: A mailed survey. SETTING: Primary care practices in southern Ontario. PARTICIPANTS: All primary care physicians who graduated between 1972 and 1988 and practised in a defined geographic area of Ontario were selected from the Canadian Medical Association's physician resource database. Response rate was 50%. MAIN OUTCOME MEASURES: Answers to questions on sociodemographic and practice characteristics, attitudes toward preventive care, and perceptions about preventive care behaviour and practices. RESULTS: In general, reported coverage for Canadian Task Force on the Periodic Health Examination's (CTFPHE) A and B class recommendations was low. However, more female than male physicians reported high coverage of women patients for female-specific preventive care measures (i.e., Pap smears, breast examinations, and mammography) and for blood pressure measurement. Female physicians appeared to question more patients about a greater number of health risks. Often, sex of physician was the most salient factor affecting whether preventive care services thought effective by the CTFPHE were offered. However, when evidence for effectiveness of preventive services was equivocal or lacking, male and female physicians reported similar levels of coverage. CONCLUSION: Female primary care physicians are more likely than their male colleagues to report that their patients eligible for preventive health measures as recommended by the CTFPHE take advantage of these measures.

Adult↗
PubMed1995

Does the community want devolved authority? Results of deliberative polling in Ontario.

OBJECTIVE: To obtain and contrast the informed opinions of people in five decision-making groups that could have a role in devolved governance of health care and social services. DESIGN: Deliberative polling. SETTING: Three rural and three urban communities selected from the 32 areas covered by a district health council in Ontario. PARTICIPANTS: A total of 280 citizens from five potential decision-making groups: randomly selected citizens, attendees at town-hall meetings, appointees to district health councils, elected officials and experts in health care and social services. INTERVENTION: Participants' opinions were polled during 29 structured 2-hour meetings. MAIN OUTCOME MEASURES: Participants' opinions on their personal willingness and their group's suitability to be involved in devolved decision making, desired type of decision-making involvement, information preferences, preferred areas of decision-making involvement and preferred composition of decision-making bodies. RESULTS: Mean attendance at each meeting was 9.6 citizens. Although there were some significant differences in opinion among the five potential decision-making groups, there were few differences among citizens from different geographic areas. A total of 189 (72%) of people polled were personally willing to take on a role involving responsibility for overall decision-making, but far fewer thought that their group was suited to taking on responsibility (30%) or a consulting role (55%). Elected officials were the most willing (85% personally willing, 50% thought their group was suitable) and randomly selected citizens the least willing (60% personally willing, 17% thought their group was suitable) to take responsibility for overall decision making. Most citizens polled indicated less interest in involvement in specific types of decisions, except for planning and setting priorities, than in overall decision making. Only 24 participants (9%) rated their own group as suitable to take responsibility for raising revenue, 91 (33%) deemed their group suited to distribution of funds and 108 (39%) felt their group was suitable for management of services. People in all five groups ranked health care needs (mean rank 1.5 out of four options) as the most important and preferences (mean rank 3.6) as the least important information. They rated a combination body involving several community groups as the most suitable overall decision-making body (8.8 on 10-point scale). Participants favoured the representation of elected officials, the provincial government and experts on combination bodies responsible for the specific types of decisions. Overall, as the complexity of devolved decision making became clear, participants tended to assign authority to traditional decision makers such as elected officials, experts and the provincial government, but also favoured a consulting role for attendees at town-hall meetings (i.e., interested citizens). CONCLUSION: There are significant differences among groups in the community in their willingness to be involved, desired roles and representation in devolved decision making on health care and social services in Ontario.

Adult↗
PubMed1995

Specificity of Prp24 binding to RNA: a role for Prp24 in the dynamic interaction of U4 and U6 snRNAs.

Prp24 was previously isolated as a suppressor of a cold-sensitive U4 mutation and is required for at least the first step of splicing in vitro. Our investigation of the in vitro RNA binding properties of the purified Prp24 protein shows that it binds preferentially to the U4/U6 hybrid snRNAs compared to other snRNAs. The interaction between Prp24 and the U4/U6 hybrid appears to involve two regions in the RNA: the 39-57 region of U6 and stem II of the U4/U6 hybrid. Interestingly, some U4 mutations, which destabilize stem II, increase the affinity of Prp24 for the U4/U6 RNAs compared to the wild type. This suggests that the binding of Prp24 to the U4/U6 RNAs may involve some destabilization of the RNA duplex. We also found that Prp24 can stimulate the annealing of U4 and U6, suggesting that Prp24 participates in both the formation and disassembly of the U4/U6 hybrid during splicing.

Base Sequence↗
PubMed1994

Novel substrate specificity engineered in the arabinose binding protein.

The L-arabinose binding protein (ABP) of Escherichia coli naturally binds L-arabinose and D-galactose with very high affinity and, with reduced affinity, a variety of other sugars that differ only at the C5 position of the pyranose ring. However, there are stringent specificity requirements at the 1, 2, 3 and 4 positions. Based on the high resolution crystallographic structure of the ligand-protein complex, remodelling of the binding pocket was attempted to shift the specificity towards C1-substituted galactosides. To create space in the vicinity of the reducing end of bound galactose, four residues, Lys10, Asp90, Thr147 and Leu145, have been mutated for residues with smaller side chains. Forty-seven mutants containing different combinations of these mutations were tested by fluorometry for their ability to bind methyl-beta-D-galactoside (met-beta-Gal) or iso-propyl-beta-D-thio-galactoside (IPTG). Two double-residue mutants carrying Ser at position 147 and Ala or Gly at position 90 appeared of particular interest for being able to bind met-beta-Gal or IPTG, respectively, and no longer galactose. Fluorescence experiments and molecular modelling indicate that the mode of binding of the new substrates to the mutant proteins might be similar to that of the natural ligands to wild-type ABP.

Amino Acid Sequence↗
PubMed1993

Recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase.

A study of the recognition of tRNA(Cys) by Escherichia coli cysteinyl-tRNA synthetase using in vivo and in vitro methods was performed. All three anticodon nucleotides, the discriminator nucleotide (73), and some elements within the tertiary domain (the D stem/loop, the T psi C stem/loop, and the variable loop) are important for recognition; the anticodon stem and acceptor stem appear to contain no essential elements. A T7 RNA polymerase transcript corresponding to tRNA(Cys) is only a 5.5-fold worse substrate than native tRNA(Cys) (in terms of the specificity constant, kcat/Km), mainly due to an increase in the value of Km for the transcript. The greatest loss of specificity caused by mutation of a single nucleotide occurs when the discriminator U73 is changed; kcat/Km declines 3-4 orders of magnitude depending on the substitution. Mutations in the wobble nucleotide of the anticodon also cause reductions in the specificity constant of 3 orders of magnitude, while mutations in the other anticodon nucleotides caused lesser effects. Interestingly, a C35A mutation (with the phenylalanine anticodon GAA) had no effect on aminoacylation by the cysteinyl-tRNA synthetase. Several amber suppressor tRNAs were constructed whose in vivo identity did not correlate with their in vitro specificity, indicating the need for both types of experiments to understand the factors which maintain tRNA specificity.

Acylation↗
PubMed1993

Novel activity of a yeast ligase deletion polypeptide. Evidence for GTP-dependent tRNA splicing.

Yeast tRNA ligase possesses multiple activities which are required for the joining of tRNA halves during the tRNA splicing process: cyclic phosphodiesterase, kinase, adenylylate synthetase, and ligase. A deletion polypeptide of a dihydrofolate reductase-ligase fusion protein, designated DAC, was previously shown to join tRNA halves although ATP-dependent kinase activity was not measurable in the assay used. We describe here a characterization of the mechanism of joining used by DAC and the structure of the tRNA product. DAC produces a joined tRNA and a splice junction with a structure identical to that produced by DAKC, the full-length dihydrofolate reductase-ligase fusion. Furthermore, DAC can use GTP as the sole cofactor in the joining reaction, in contrast to DAKC, which can only complete splicing in the presence of ATP. Both enzymes exhibit GTP-dependent kinase activity at 100-fold greater efficiency than with ATP. These results suggest that a potential function for the center domain of tRNA ligase (missing in DAC) is to provide structural integrity and aid in substrate interactions and specificity. They also support the hypothesis that ligase may prefer to use two different cofactors during tRNA splicing.

Base Sequence↗
PubMed1993

Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites.

We have examined multiple cofactor usage by yeast tRNA ligase in splicing in vitro. The ligase mechanism of action requires expenditure of two molar equivalents of nucleotide cofactor per mole of tRNA product. Recent evidence (Westaway, S.K., Belford, H.G., Apostol, B.L., Abelson, J., and Greer, C.L. (1993) J. Biol. Chem. 268, 2435-2443) demonstrated that the ligase-associated kinase activity is more efficient with GTP as cofactor than with ATP. Employing a ligase fusion construct with dihydrofolate reductase (Apostol, B.L., Westaway, S.K., Abelson, J., and Greer, C.L. (1991) J. Biol. Chem. 266, 7445-7455) for purposes of enzyme purification, we performed joining assays demonstrating that ATP and GTP are the most effective combination of cofactors. ATP was essential to the joining reaction, while UTP, CTP, or ATP replaced GTP inefficiently. Specific and functionally independent binding sites were confirmed for ATP and GTP by direct binding measurement. A third site was implicated in UTP- and CTP-ligase interactions. Comparison of binding constants with Kapp values determined for nucleotide-dependent joining suggested both that nucleotide triphosphate binding may be limiting in tRNA joining and that tRNA ligation occurs most efficiently using GTP for the kinase reaction and ATP as the adenylylate synthetase cofactor.

Adenosine Triphosphate↗
PubMed1993

Yeast tRNA-splicing endonuclease cleaves precursor tRNA in a random pathway.

Introns interrupt many of the tRNA genes of Saccharomyces cerevisiae at a constant position in the anticodon loop. Pre-tRNA transcripts must be accurately cleaved at 3' and 5' splice sites by tRNA endonuclease to release these introns. In order to study splice site cleavage order, substrates were prepared in which the ribose 2'-OH at each of the splice sites was phosphorylated. This modification blocked cleavage by the endonuclease. We found that whichever splice site was blocked the endonuclease can cleave the other site, indicating that the two splice sites were cleaved independently. The endonuclease also cleaved both 3'- and 5'-nicked pre-tRNA(Phe). In addition, both kinds of "2/3 molecules" (exon+intron) were observed in kinetic studies, indicating that they were true biochemical intermediates. The rates of cleavage at the 3' and 5' splice sites of pre-tRNA were compared in several ways. The results showed that the endonuclease cleaves 3' and 5' sites at almost the same rate in the first cleavage, whereas in the second cleavage the 3' site was cleaved faster, indicating that the rates of the two routes for cleavage were unequal. These results demonstrated that the endonuclease cleaved pre-tRNA in a random order, creating two routes for removal of introns from pre-tRNA.

Bacteriophage T7↗
PubMed1993

Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA.

We have analyzed the functions of several pre-mRNA processing (PRP) proteins in yeast spliceosome formation. Here, we show that PRP5 (a DEAD box helicase-like protein), PRP9, and PRP11 are each required for the U2 snRNP to bind to the pre-spliceosome during spliceosome assembly in vitro. Genetic analyses of their functions suggest that they and another protein, PRP21, act concertedly and/or interact physically with each other and with the stem-loop IIa of U2 snRNA to bind U2 snRNP to the pre-mRNA. Biochemical complementation experiments also indicate that the PRP9 and PRP11 proteins interact. The PRP9 and PRP11 proteins may be functioning similarly in yeast and mammalian cells. The requirement for ATP and the helicase-like PRP5 protein suggests that these factors might promote a conformational change (involving either the U1 or U2 snRNP) that is required for the association of U2 snRNP with the pre-mRNA.

Adenosine Triphosphate↗
PubMed1993

Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent.

We have analyzed pre-mRNA splicing in yeast extracts immunodepleted of the PRP18 protein. We find that while the first step of splicing (cleavage at the 5' splice site, and generation of the exon 1 and lariat intermediates) is unaffected by the absence of PRP18, the second step of splicing (excision of the lariat intron and formation of mRNA) is substantially slower in the absence of PRP18. The splicing intermediates that are formed in the absence of PRP18 can be rapidly chased into products by the addition of purified PRP18 protein. This chasing is not dependent on ATP, implying that ATP is not required during the second cleavage-and-ligation reaction. This result suggests that there are ordered stages within the second step of splicing and that PRP18 acts late in the second step, perhaps during the catalytic step. The ATP independence also supports the idea that this reaction proceeds by a transesterification mechanism.

Adenosine Triphosphate↗
PubMed1993

PRP19: a novel spliceosomal component.

We have isolated the gene of a splicing factor, PRP19, by complementation of the temperature-sensitive growth defect of the prp19 mutant of Saccharomyces cerevisiae. The gene encodes a protein of 502 amino acid residues of molecular weight 56,500, with no homology to sequences in the data base. Unlike other PRP proteins or mammalian splicing factors, the sequence of PRP19 has no discernible motif. Immunoprecipitation studies showed that PRP19 is associated with the spliceosome during the splicing reaction. Although the exact function of PRP19 remains unknown, PRP19 appears to be distinct from the other PRP proteins or other spliceosomal components.

Amino Acid Sequence↗