Biomedical subjects
J Abelson
Publications and source records attributed to J Abelson.
Crystal structure and evolution of a transfer RNA splicing enzyme.
The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.
Evolution of a transfer RNA gene through a point mutation in the anticodon.
The transfer RNA (tRNA) multigene family comprises 20 amino acid-accepting groups, many of which contain isoacceptors. The addition of isoacceptors to the tRNA repertoire was critical to establishing the genetic code, yet the origin of isoacceptors remains largely unexplored. A model of tRNA evolution, termed "tRNA gene recruitment," was formulated. It proposes that a tRNA gene can be recruited from one isoaccepting group to another by a point mutation that concurrently changes tRNA amino acid identity and messenger RNA coupling capacity. A test of the model showed that an Escherichia coli strain, in which the essential tRNAUGUThr gene was inactivated, was rendered viable when a tRNAArg with a point mutation that changed its anticodon from UCU to UGU (threonine) was expressed. Insertion of threonine at threonine codons by the "recruited" tRNAArg was corroborated by in vitro aminoacylation assays showing that its specificity had been changed from arginine to threonine. Therefore, the recruitment model may account for the evolution of some tRNA genes.
Provision of preventive care to unannounced standardized patients.
OBJECTIVE: To examine the relation between physician, training and practice characteristics and the provision of preventive care as described in the guidelines of the Canadian Task Force on the Periodic Health Examination. DESIGN: Cross-sectional study. SETTING: Family practices open to new patients within 1 hour's drive of Hamilton, Ont. PARTICIPANTS: A total of 125 family physicians were randomly selected from respondents to an earlier preventive care survey. Of the 125, 44 (35.2%) declined to participate, and an additional 19 (15.2%) initially consented but later withdrew when they closed their practices to new patients. Sixty-two physicians thus participated in the study. INTERVENTION: Unannounced standardized patients posing as new patients to the practice visited study physicians' practices between September 1994 and August 1995, portraying 4 scenarios: 48-year-old man, 70-year-old man, 28-year-old woman and 52-year-old woman. OUTCOME MEASURES: Proportion of preventive care manoeuvres carrying grade A, B, C, D and E recommendations from the Canadian Task Force on the Periodic Health Examination that were performed, offered or advised. A standard score was computed based on the performance of grade A and B manoeuvres (good or fair evidence for inclusion in the periodic health examination) and the non-performance of grade D and E manoeuvres (fair or good evidence for exclusion from the periodic health examination). RESULTS: Study physicians performed or offered 65.6% of applicable grade A manoeuvres, 31.0% of grade B manoeuvres, 22.4% of grade C manoeuvres, 21.8% of grade D manoeuvres and 4.9% of grade E manoeuvres. The provision of evidence-based preventive care was associated with solo (v. group) practice and capitation or salary (v. fee-for-service) payment method. Preventive care performance was unrelated to physician's sex, certification in family medicine or problem-based (v. traditional) medical school curriculum. CONCLUSIONS: Preventive care guidelines of the Canadian Task Force on the Periodic Health Examination have been incompletely integrated into clinical practice. Research is needed to identify and reduce barriers to the provision of preventive care and to develop and apply effective processes for the creation, dissemination and implementation of clinical practice guidelines.
What factors influence primary care physicians' charges for their services? An exploratory study using standardized patients.
OBJECTIVE: To determine the extent of variation in physicians' charges for health care encounters with unannounced standardized patients and factors associated with the variation. DESIGN: Cross-sectional study. SETTING: Family practices open to new patients within 1 hour's drive of Hamilton, Ont. PARTICIPANTS: A stratified random sample of 125 physicians who had responded to an earlier survey regarding preventive care were invited to participate. Of the 125, 44 (35.2%) declined to participate, and an additional 19 (15.2%) initially consented but later withdrew because they closed their practices to new patients. Sixty-two physicians thus participated in the study. INTERVENTION: Unannounced standardized patients posing as new patients to the practice visited study physicians' practices between September 1994 and August 1995, portraying 4 scenarios: 28-year-old woman, 52-year-old woman, 48-year-old man and 70-year-old man. OUTCOME MEASURES: Physician characteristics, encounter characteristics and charges made for services. RESULTS: The 62 physicians had 246 encounters with the standardized patients. Charges were made to the health insurance plan for services by 59 physicians for up to 4 encounters (215 encounters in all). Charges varied considerably both within and across patient scenarios. Time spent with the patient was an important factor predicting charges made (p < 0.01), although the effect of time spent on charges varied across scenarios (p < 0.01). Fee-for-service physicians charged more for their services than physicians who usually had alternative billing arrangements (p < 0.01). Female physicians charged more for their services than their male colleagues (p = 0.03). No relation was found between quality of preventive care and charges made (p = 0.15). CONCLUSIONS: Physician-related factors are better able to account for the variability in charges for their services than patient-related factors. Physicians seeing comparable patients may earn much more or less than their colleagues because of differences in the services they provide and the way they apply the fee schedule. Quality-assurance techniques are likely needed to reduce the variability in charges seen and increase value for money spent in health care.
Fielding standardized patients in primary care settings: lessons from a study using unannounced standardized patients to assess preventive care practices.
OBJECTIVES: To document detection and suspicion rates of unannounced standardized patients visiting community-based practices. DESIGN: Primary care physicians were recruited to participate in a study using standardized patients. Four standardized patient scenarios were used. SETTING: Community-based primary care physicians' practices in southern Ontario between September 1994 and August 1995. STUDY PARTICIPANTS: Sixty-two primary care physicians. MAIN OUTCOME MEASURES: A 'believability' questionnaire completed after all four standardized patients had visited the practices. RESULTS: Of the primary care physicians approached, 50% (62) agreed to participate. Twenty-one per cent of all visits were suspected as standardized patient encounters. Forty-six per cent suspected one or more standardized patients. Only five physicians (8%) suspected all four standardized patients. Reasons for suspecting standardized patients were associated with the characteristics of the physician's practices, the physician's practice profile and the standardized patient cover story. CONCLUSION: The portrayal of asymptomatic patients seeking a new primary care physician presents unique challenges. Carefully constructed cover stories, and detailed knowledge of the local area and of the practices of the participating physicians is required to allow standardized patients cases to be tailored to fit into primary care settings without arousing suspicion.
Do professions have a future?
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Site-specific deoxynucleotide substitutions in yeast U6 snRNA block splicing of pre-mRNA in vitro.
We have identified 2'-hydroxyl groups of the U6 phosphate-ribose backbone which are required for reconstitution of splicing activity in U6-depleted yeast extract. To screen the 2'-hydroxyls of yeast U6 at nucleotides 39-88, spanning the conserved central domain, synthetic U6 RNAs were constructed with deoxyribonucleotides incorporated site specifically. Only four individual deoxynucleotide substitutions blocked splicing activity: dA51 (in the ACAGAG sequence), dA62 (next to the AGC triad), and dU70 and dC72 (both in the loop of the 3' intramolecular stem-loop). Native gel analysis revealed that these deoxy-substituted U6 RNAs were competent for assembly of spliceosomes. Interestingly, a 2'-O-methyl substituent at A51, A62, U70 or C72 did not inhibit splicing activity, indicating that the essential 2'-OH groups at these positions in U6 act as hydrogen bond acceptors or neutral coordinated ligands. The requisite 2'-hydroxyls at A62, U70 and C72 show both similarities and differences relative to the positions of essential 2'-hydroxyls of catalytic domain V of group II ribozymes. The identification of the essential 2'-hydroxyls at positions 62, 70 and 72 corroborates that the 3' intramolecular stem-loop in U6 plays an important role in pre-mRNA splicing.
The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA.
The Saccharomyces cerevisiae protein Prp5 is a member of the "DEAD box" family of putative RNA-dependent ATPases and helicases. The protein was purified from Escherichia coli and determined to be an RNA-dependent ATPase. The ATPase activity is 7-fold more specific for full-length U2 than for any of the other small nuclear RNAs or nonspecific RNAs tested. An RNaseH assay in extracts was used to demonstrate that Prp5 mediates an ATP-dependent conformational change in the intact U2 small nuclear ribonucleoprotein. We propose that this conformational change makes the branch point pairing sequence of U2 RNA accessible for pairing with the intron allowing formation of the pre-spliceosome.
In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation.
Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.
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.
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.
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.
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.
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.
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.
Registering patients and paying capitation in family practice.
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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.