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Mitochondrial small subunit (mtSSU) rDNA variable (V1, V2, V4, V6, V8 and V9) domain sequences and rRNA secondary structures evidenced eight molecular groups within 32 strains of the Agrocybe aegerita multispecies complex from different continents. mtSSU-rRNA secondary structure evolution occurred mainly by insertion/deletion of sequences from 8 to 57nt long. Preferential insertion/deletion sites correlated with loops of the mtSSU-rRNA secondary structures, and suggested that these events occurred in regions without interactions in the ribosomal-protein assembly. Indels modified the stem length (V1 and V4 domains) or the size and loop number (V6 and V9 domains). Three indels inserted in the V1 and V4 domains had 76.5% to 94.7% identity with short sequences of the mitochondrial cytochrome c oxidase gene; this fact and the presence of inverted repeated motifs within indel sequences suggested a mechanism of evolution based on insertion/deletion of sequences from another region of the mitochondrial genome. Phylogenetic relationships inferred using both ribosomal DNA sequences and rRNA secondary structures were congruent and evidenced three clades within the A. aegerita complex: European, Argentinean, and a more distant Asian-American clade including A. aegerita and A. chaxingu strains. These results suggested that numerous genetic exchanges occurred between Asian-American strains after isolation of the European clade. V4-V6-V9 concatenated sequences of European and Argentinean clades had 86.1% identity, similar to the value calculated between two Agrocybe closely related species, suggesting that these clades could represent different species. A cleaved amplified polymorphic sequence test for rapid characterization of strains was developed.

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.

Registers of the management of infarction can complement information obtained from randomised trials evaluating the methods and practice of treatment. In order to do this, the quality of the registers must be assured, and in particular the accuracy of the recorded cases. The objective of this study was to evaluate the accuracy of a register for the in-hospital and pre-hospital management of acute coronary syndromes with ST segment elevation of less than 12 hours' duration. Using a capture-recapture method, the study compared cases in the register with eligible cases present in the hospital and emergency ambulance service databases at two establishments, giving a recruitment rate of 61%. The rate of accuracy was estimated at 84% (95% CI [82 ; 86]). The independent factors associated with failure of notification were female sex (ORa=6.65 [2.04-21.69]), presentation at nights, weekends or bank holidays (ORa=4.13 [1.33-12.85]), direct admission to hospital without passing by the emergency ambulance service (ORa=2.85 [1.03-7.69]), primary angioplasty (ORa=6.18 [1.60-23.79]) and the absence of reperfusion (ORa=40.38 [6.21-262.40]). With more than 80% accuracy, the results produced by the register are robust. The selection bias linked to the under-representation of certain subgroups, while real, has only a marginal impact on estimates derived from the register. Factors associated with failure of notification should be taken into account when operating such a register.

The Agrocybe chaxingu and Agrocybe aegerita mitochondrial apocytochrome b coding sequences are highly similar (97% of nt identity), but have highly different sizes (2312 and 4867nt, respectively), due to the presence of three large group IB introns: two (iAae1 and iAae2) in A. aegerita, one (iAch1) in A. chaxingu. All these introns encode a homing endonuclease (HE) similar to those described in introns of mitochondrial genes (cob, cox1, and nad5) from various organisms. Phylogenetic trees were built with these HE sequences. From these trees, the Agrocybe coding introns argue for recent lateral transfers, i.e., occurring after the separation of the two Agrocybe species, involving phylogenetically distant fungi such as members of the Ascomycota phylum (for iAch1 and iAae2) and, for the first time to our knowledge, a member of the Chytridiomycota phylum (for iAae1). The grouping of the HE gene (HEG) sequences according to the mitochondrial gene (cob, cox1, and nad5) where they are inserted, suggests modifications of the interactions between the HE and the recognized sequences, leading to new target genes. The largest distribution of the iAch1 HE, shared by several cob and cox1 mitochondrial genes from Ascomycota, Basidiomycota, and Chytridiomycota phyla, suggests a higher target flexibility of this HE, perhaps related to the presence of two different LAGLIDADG motifs in the catalytic site of the enzyme.

Objective: The Grenoble clinical research centre carried out a prospective satisfaction survey of subjects taking part in clinical studies undertaken in its buildings., Materials and Methods: A questionnaire adapted from Grenoble teaching hospital's inpatient satisfaction survey was sent to 198 subjects. It comprised 24 items pooled in categories on which were calculated average scores. The participation rate of the survey was 82.6%. On the whole, 94.4% of the questioned subjects would accept to take part in a new study if we requested them, and 72.2% were satisfied or very satisfied with their stay; 24% had no opinion. The mean overall satisfaction score was 8.6 (95% confidence interval: 7.6-9.6) out of 10., Conclusion: This investigation shows that the assumption of responsibility of the subjects by specific personnel and in an adapted place, within the framework of clinical protocol of research, is associated with a high satisfaction of the subjects. However, we still have to improve the transmission of the clinical study results to the patients.

Unlabelled: Failure of the French health care services to diagnose and manage problem drinkers has been established on many occasions. This results from a relative lack of knowledge of the basics of alcoholism and the low level of involvement of health care professionals in the management of these patients. In response to this inadequacy, the French Public Health Ministry promoted the development of managed care and coordination of care for these patients. Teams in charge of coordinating care for problem drinkers have been implanted in 96 French hospitals since 1996. These teams aim to provide support to health care providers caring for problem drinkers. For this purpose, they have delivered continuing medical education designed to prepare health care providers to identify and manage these patients in various settings. However, no formal assessment of the impact of these interventions on the knowledge, perceptions, and practices of health care professionals has been planned. The assessment of these interventions can rely on qualitative methods such as observation, focus groups, or individual interviews. However, qualitative methods require specific skills, are time-consuming, and cannot be implemented on a large scale. In contrast, quantitative methods using survey questionnaires or standardized instruments appear to be more appropriate for large scale or repeated evaluations. However, the accuracy of ratings provided by these instruments can be affected by many methodological factors, including the quality of the instrument in terms of validity, reliability, and sensitivity to changes. The aim of this paper is to describe the properties of French and English language questionnaires designed to assess the knowledge, perceptions, and practices of health care professionals with regards to alcoholic patients., Methods: The instruments were retrieved by searching the Medline, Pascal, and Sudoc computerized bibliographic databases from January 1964 to December 2002. The following medical Subject Headings (MeSH) and text words were used: "*alcoholism/psychology/therapy", "questionnaire", "healthy care surveys", "attitude of health personnel", and "*knowledge/attitude/practice". In addition, the table of contents of the French journals devoted to alcoholism that were not indexed in electronic bibliographic databases were examined (Alcoologie et addictologie, Alcoologie). The bibliographies of relevant articles were also examined for additional citations. Finally, a phone survey of 34 professionals caring for alcoholic patients located in French university hospitals was carried out to retrieve unpublished questionnaires. Two authors independently extracted data from each study using a standard date abstract form. Disagreements were resolved through consensus. They extracted information on the development process and properties of validity, reliability, and sensitivity to changes. Validity is a concept concerned with the extent to which an instrument actually measures what it is supposed to measure. It is assessed through different facets (content, construct, criterion, discriminant, and predictive validity). Reliability reflects the amount of error inherent in any measurement. Sensitivity to change corresponds to the property of an instrument to identify small but clinically significant changes in attitude or practice., Results: A total of 57 relevant publications involving 39 original instruments were identified. Eighty questionnaires were not available, despite the solicitation of their developers. Overall, the study included 21 instruments. Of these, 20 were English-language questionnaires and one was in French. The conceptual frameword was specified for only two questionnaires: "the Addiction Belief Scale" and "the Alcohol and Alcohol Problems Perception Questionnaire". The number of items ranged from 9 to 122. Items were derived from qualitative surveys in only two cases. In the other cases, they were derived from the literature, expert panels, or form unknown sources. The internal construct validity of eight questionnaires was assessed using principal component analysis, factor analysis, or cluster analysis. We considered that external construct validity was analyzed by testing empirical hypotheses derived from the literature for 15 of the instruments studied. Predictive validity cas evidenced for only one questionnaire. Internal consistency was documented for seven instruments, using Cronbach's coefficient. Test-retest or inter-rater reliability was assessed for none of the instruments. The sensitivity to change of two instruments was analyzed. The response rate to the instruments ranged from 46% to 93%., Discussion: The main finding of this study is that properties of validity, reliability, and sensitivity of questionnaires designed to assess the knowledge, perceptions, and practices of health care professionals with regards to alcoholic patients are neglected. Moreover, these questionnaires generally lack a theoretical background. Hence, the interpretation of responses to these questionnaires may be misleading. The present study has several limitations. The search strategy could be criticized for searching only a small number of databases, and selecting French and English language questionnaires. However, given the large range of journals included, it was unlikely that the strategy has limited the generality of the results. In addition, we identified no additional relevant articles by contacting professionals working with alcoholic patients in French university hospitals. Articles reporting instruments designed to assess the knowledge, perceptions, and practices of health care professionals with regards to alcoholic patients are incomplete. Journals should require minimum evidence of validity, reliability, and sensitivity when reporting results of surveys, development of questionnaires or standardized instruments., Conclusion: The psychometric characteristics of French language questionnaires were given very little attention. In order to evaluate the effectiveness of their continuing medical education programs, the teams that train French medical staff caring for alcoholic patients should either develop a standardized survey questionnaire following a strict methodology or translate an English language instrument, which would also require a cross-cultural validation.

Objective: To assess the degree of agreement between four competing guidelines regarding the recommendation for prophylactic heparin therapy and to report to what extent actual practice agreed with or differed from the recommendations made before these guidelines were disseminated., Design: Four French guidelines were applied to data from a cross-sectional study conducted before their dissemination., Setting: Twenty-six medical units of a teaching and a nonteaching hospital., Patients: One thousand thirty-two medical inpatients., Main Measurements: Interguideline agreement rated by the kappa coefficient and percentage of patients receiving prophylactic heparin treatment., Results: The percentage of patients requiring prophylactic treatment ranged from 35.4% to 54.6% (overall kappa coefficient, 0.65 [0.63 to 0.68]), depending on the guideline. The four guidelines agreed in recommending prophylactic heparin treatment in 330 patients (32.0%). The corresponding rate of prophylactic treatment use was 57.0% (188/330). None of the guidelines recommended prophylactic heparin treatment in 385 patients (37.3%). The physicians did not order prophylactic treatment in 80.3% of these patients (309/385). The guidelines disagreed in recommending prophylactic treatment in 317 patients (30.7%). The corresponding rate of prophylactic treatment use was 32.8% (104/317)., Conclusion: The four guidelines agreed in 69.3% of patients but physician practices were already quite appropriate in these patients before the guidelines were disseminated. Active dissemination of the guidelines can be expected to improve physician practices in the treatment of these patients, but likely with limited impact. In contrast, the four guidelines disagreed in 30.7% of patients. Further clinical trials are needed in this subgroup of patients.

Geosmin (trans-1,10-dimethyl-trans-9-decalol), an earthy-musty compound, has been identified in wines and in grape juice, in which its presence is highly detrimental to the aromatic quality. Geosmin has a biological origin, and the analysis of rotten grape microflora has been done on two grape varieties (Semillon, Cabernet Sauvignon) from six parcels of the Bordeaux region over 3 years (1999, 2000, 2001). Forty-three Penicillium-related species have been analyzed by gas chromatography-mass spectrometry (GC-MS) for their geosmin production. GC-MS analysis has demonstrated that the earthy odor was always correlated with the presence of geosmin. Phenotypic characterization of Penicillium spp. being ambiguous, a molecular characterization by rDNA internal transcribed spacer (ITS1) sequencing was performed for all strains. The results evidenced that all strains producing geosmin belonged to only one species, P. expansum, and that the other strains, not producing geosmin, belonged to three species: P. purpurogenum, P. thomii, and Talaromyces wortmanii.

Objective: To assess compliance of anesthesiologist practices in antibiotic prophylaxis during total hip replacement (THR) surgery with the French Society of Anesthesiology and Intensive Care consensus-based guidelines., Design: Retrospective review of medical records. Compliance of anesthesiologist practices with the guidelines was assessed according to antibiotic prophylaxis use, antimicrobial agent, dosage of first injection, time from first dose to incision, and total duration of antibiotic prophylaxis., Setting: Orthopedic surgery wards in a 2,200-bed French teaching hospital., Patients: A random sample of 416 patients undergoing THR from January 1999 to December 2000., Results: Three hundred eighty-six (93%) of the sampled medical records were usable. Antibiotic prophylaxis was used for 366 (95%) of the patients. Total duration of prophylaxis did not exceed 48 hours in 98% (359 of 366) of the patients. Drug selection complied with national guidelines in 259 (71%) of the patients. Dosage and timing of the first injection were appropriate in 98% (290 of 296) and 80% (236 of 296) of the patients, respectively, who received one of the recommended antibiotics. Overall, 53% (203 of 386) of the patients met all five criteria. In multivariate analysis, there was a significant anesthesiologist effect on overall compliance with the guidelines (likelihood ratio chi-square with 9 degrees of freedom, 25.7; P < .01). Undergoing surgery during 2000 was the only patient characteristic associated with an increased rate of appropriate practices (adjusted OR, 1.56; CI95 1.02-2.38)., Conclusion: The overall compliance rate should be improved by disseminating the guidelines and the results of this study following audit and feedback.

Several states, of whom France, have been alarmed about suicide in the military. An observational longitudinal cohort study was conducted to check whether the suicide risk in the French army was higher than in the general population and to find out the more prone to self-harm destruction personnel categories. From 1997 to 2000 inclusive, 230 suicides occurred among 315,934 person.years; i.e., the overall annual crude suicide rate was 18.2 per 100,000 active-duty personnel. With the National data for 1999 as reference, army men had a lower suicide risk than men in the overall population of similar age categories (standardised mortality ratio = 67). A Poisson regression model showed that the incidence rate in the Gendarmerie was twice as high as in the Land Forces (incidence rate ratio = 2.15), that the incidence relative risk increased threefold over 4 years, and that the incidence rate ratio amongst under 25 and from 40 to 44-year-old personnel was almost twice as high as in the 25-29-year-old category. The main suicide methods were use of a firearm (51%) and hanging (28%). Despite a global lower risk than in the general population, certain army categories, i.e., Gendarmerie personnel and young men, deserve specific surveillance and preventive measures.

The objective of this study was to check psychometric properties of a French-language in-patient experience questionnaire in a test sample different from the development sample. The questionnaire was sent out to 5,736 in-patients, within two to four weeks of discharge from a teaching hospital of 2,200 beds. Overall 4,095 questionnaires (71.4 per cent) were returned. Of these, 3,879 questionnaires were analyzed. In principal component analysis, seven principal components accounted for 62.4 per cent of the total variance. Cronbach's alpha coefficient ranged from 0.62 to 0.90, with the exception of the seventh scale (convenience scale, two items, Cronbach = 0.39). The overall patient experience score increased with increasing patient age (except for patients older than 65), male sex, low education level, use of a single room, and prior stay in the department. It also differed with respect to patients' behavioral intentions, answers to an overall satisfaction item, and open-ended comments.

Objective: To assess the conformity of practitioners' practices in the management of community acquired pneumonia with the French Agence Nationale d'Accréditation et d'Evaluation en Santé (Anaes) guidelines., Methods: We retrospectively reviewed a random sample of 210 medical records which included a principal or associated diagnosis of pneumonia in a French university hospital., Results: A hundred and one medical records were assessable. Sixty-two patients were high risk (Pneumonia Severity Index class IV or V of the prediction rule of Fine et al.), and 10 patients were admitted into an intensive care unit. The overall in-hospital mortality was 14 patients [8-22]. The level of care was appropriate according to the guidelines in 40 cases ([30-50)]. Seven patients did not require hospitalisation, 31 patients required admission into a medical department, 56 patients into an intensive care unit and 7 patients were managed in non specified conditions. Eighteen patients ([11-27]) had appropriate microbiologic investigations. Forty-three patients (([33-53]) received antibiotics within 8 hours of arrival. Empirical antibiotic treatment (dosage and molecule) was appropriate in 38 patients ([28-48]). There was no significant relationship between compliance with the guidelines and in-hospital mortality., Conclusion: The rate of conformity of practitioners' practices with the Anaes guidelines for management of community-acquired pneumonia is low in our hospital. It could be improved by active implementation of these guidelines.

Introduction: Management guidelines for acute community acquired pneumonia vary considerably. The objective is to estimate by a retrospective study the uniformity of the recommendations for the management of patients and the choice of initial empirical antibiotic therapy., Methods: Eight English and French language guidelines published between 1998 and 2001 were identified by a search of the literature. They were applied retrospectively to a sample of 101 patients admitted to a university hospital in 2000 with a diagnosis of pneumonia., Results: Hospital admission was advocated for between 61% and 95% and admission to intensive care for between 8% and 35% of the patients, depending on the guidelines under consideration. The actual management conformed to that advocated for between 34% and 94% of the patients (kappa=0.27 [0,19; 0,34]). Compliance of the empirical antibiotic therapy (drug, dose, mode of administration) with the recommendations varied from 0% to 68% of the patients depending on the guidelines considered (kappa=0.01 [-0,10; 0,12])., Conclusions: The heterogeneity of the guidelines is manifest by important variations in the recommendations for management and initial empirical therapy. These differences are due, in part, to a paucity of evidence based data upon which to base the guidelines. It would appear essential to harmonise the guidelines in a way that is appropriate for the country of their intended use.

The Aa1-Pri4 gene was cloned from the edible mushroom Agrocybe aegerita. The gene, specifically expressed during fruiting initiation, encodes a glycine-rich protein of 116 amino acids, with no homology to already known proteins. Homologous genes were amplified from two other strains belonging to the Agr. aegerita complex and originating from South-East Asia; and a comparison of the three genes revealed a high conservation of the coding sequences (72.8-97.8%). The PRI4 putative protein sequences were highly similar (87.5-100.0%); and all of them contained two protein kinase C sites, suggesting a potential supplementary regulation by phosphorylation at the protein level. The 5' uncoding regions all presented a leader intron, very variable in sequence (45.7% identity), but with a high C+T content (74.5-79.0%). The presence of such CT-rich sequences previously described in the promoter of highly expressed fungal genes suggests that the leader intron of the Aa1-Pri4 gene could be involved in the high-level, stage-specific expression.

The complete sequences and secondary structures of the mitochondrial small subunit (SSU) ribosomal RNAs of both mostly cultivated mushrooms Agaricus bisporus (1930 nt) and Lentinula edodes (2164 nt) were achieved. These secondary structures and that of Schizophyllum commune (1872 nt) were compared to that previously established for Agrocybe aegerita. The four structures are near the model established for Archae, Bacteria, plastids, and mitochondria; particularly the helices 23 and 37, described as specific to bacteria, are present. Within the four Agaricales (Homobasidiomycota), the SSU-rRNA "core" is conserved in size (966 to 1009 nt) with the exception of an unusual extension of 40 nt in the H17 helix of S. commune. The four core sequences possess 76% of conserved positions and a cluster of C in their 3' end, which could constitute a signal involved in the RNA maturation process. Among the nine putative variable domains, three (V3, V5, V7) do not show significant length variations and possess similar percentages of conserved positions (69%) than the core. The other six variable domains show important length variations, due to independent large size inserted/deleted sequences, and higher rates of nucleotide substitutions than the core (only 31% of conserved positions between the four species). Interestingly, the inserted/deleted sequences are located in few preferential sites (hot spots for insertion/deletion) where they seem to arise or disappear haphazardly during evolution. These sites are located on the surface of the tertiary structure of the 30S ribosomal subunit, at the beginning of hairpin loops; the insertions lead to a lengthening of existing hairpins or to branching loops bearing up to five additional helices.

Data on compliance with medical advice given by telephone consultation services are currently lacking. The aim of this study was to assess patient compliance with medical advice given by a call center. A cross-sectional telephone survey was carried out on a random sample of 463 callers 72 hours after contacting the Grenoble Dial 15 center in France. Four hundred nine subjects (88.3%) participated in the study. Of these, 286 callers (69.9%) complied with the medical advice given. Compliance was 61.4% among patients who were advised to treat themselves, 83.9% among patients who were advised to consult a general practitioner during business hours, and 64.0% among patients who were advised to go to an accident and ED (P <.01). The survey pointed out adverse events resulting from the service. Assessing patient compliance can be an important source of information for improving aspects of patient management provided by telephone consultation services.

Objective: To assess the impact of a continuing medical education program based on the WHO's 10 steps to successful breast-feeding., Study Design: An observational before-and-after study at a teaching hospital. Data for two random samples of 50 women before and 50 after the intervention were collected from medical records and completed by a mail questionnaire., Results: Seventy-six percent of mothers initiated breast-feeding in the two samples. The median duration of breast-feeding was 12 weeks and did not differ between the before and after groups. The percentage of newborns separated from their mother more than 4h a night decreased substantially after the intervention (13% versus 52% before the intervention, P<0.01). In-hospital formula provision also decreased after the intervention (63% of newborns versus 82% before intervention, P=0.07)., Conclusion: This pilot study has enabled authors to document the feasibility of evaluating the impact of continuing education of maternity ward staff.

Objectives: This study evaluated a strategy for implementing continuous quality improvement based on a decentralized quality management system in the clinical departments of a hospital., Setting: The institution is a 2000-bed teaching hospital of tertiary health care employing 8000 people., Methods: The quality management intervention was tested in six volunteer departments. This intervention comprised an instructional seminar, methodological assistance, and the dissemination of guidelines. The program was evaluated 1 year after the intervention and included a quality audit, interviews with department staff, and analysis of the written documents produced by the departments., Results: The quality management systems are functioning in all the departments. Quality teams meet regularly and multidisciplinary work groups are in place. The topics most often addressed are patient reception and communication between department staff members. The level of compliance with the guidelines has increased, from 39% before the seminar to 54% 1 year later (P < 0.05). All of the staff members interviewed judged the process useful for them and for the department, while waiting for the concrete results. Among the difficulties the staff members encountered were changing their work habits, lack of time, and the tedious aspect of writing procedures., Conclusion: Implementing continuous quality improvement in hospital departments seems to be an interesting alternative to organization-wide implementation strategies. However, these results need to be confirmed by long-term evaluations and by deploying the program i n other departments.

The aim of this study was to assess whether clinical guidelines complied with the instructions for writing structured care management tools in a French university hospital. A cross-sectional study of guidelines for appropriate antimicrobial agent use in the authors' institution was carried out. A total of 221 guidelines were retrieved in 62 hospital units. The number of guidelines by unit ranged from one to 22 and 198 guidelines (90 per cent) had been developed at the local level. None of the guidelines fully complied with the ten criteria of the instructions. Each guideline met, on average, 4.2 criteria (3.9-4.5). The partial compliance rate was 75 per cent (68-80). In two-level multivariate analysis, factors associated with partial compliance were: dissemination of guidelines after implementation of the instructions (odds ratio = 6.25 (2.41-16.21)), existence of more than one storage site for guidelines in each unit (OR = 3.26 (1.03-10.32)), and hospital unit (variance of the intercept = 1.54).

Coding and regulating sequences of Pri3 (a novel gene specifically expressed in both nucleus types of dikaryons during fruiting initiation) were compared within nine strains of the Agrocybe aegerita complex originating from Europe, Asia and Latin America. Highly homologous coding sequences were found; and the 95-amino-acid-long PRI3 deduced proteins all shared eight cysteines and eight glycines, a putative signal peptide suggesting an extra-cellular localization, a protein kinase C site (T(70)) and a cAMP-dependent kinase site (S(74)). The PRI3 proteins presented no significant homologies with already known proteins and constitute a new class of small cysteine-rich proteins. Within the 5' uncoding regions, two leader introns and a putative upstream regulating sequence resembling the QA1-F activator site were highly conserved. Comparison of the gene sequences within the A. aegerita complex suggested a divergent evolution of the European and Asian/Latin American groups of genes.

The aim of the study was to assess the impact of an individual patient order form which concerned the 21 most costly antibiotics in a university hospital. Antibiotics expenditures were monitored from 1995 to 1999 and were expressed in 1999 French Francs per 100 patient days (p.d.). The time series were analyzed by auto-regressive models. The trend of antibiotics expenditures which were concerned by the individual patient order form was a yearly increase of 50 FF/100 p.d. (p < 0.01). The individual patient order form had no significant impact on global antibiotics expenditures but there were some differences across departments: antibiotics costs decreased 1.293 FF/100 p.d. (p = 0.02) in intensive care departments. Monitoring antibiotics consumption should be continued in order to increase power of analysis and to assess the impact of the implementation of guidelines.

The Agrocybe aegerita mitochondrial genome contains a truncated family B DNA polymerase gene (Aa-polB P1) whose nucleotide sequence is 86% identical to the previously described and potentially functional Aa-polB gene. A tRNA(Met) gene occurs at the 3' end of the Aa-polB P1 gene. The Aa-polB P1 gene could result from reverse transcription of an Aa-polB mRNA primed by a tRNA(Met) followed by the integration of the cDNA after recombination at the mitochondrial tRNA locus. Two naturally occurring alleles of Aa-polB P1 carry one or two copies of the disrupted sequence. In strains with two copies of Aa-polB P1, these copies are inverted relative to one another and separated by a short sequence carrying the tRNA(Met) gene. Both A. aegerita mitochondrial family B DNA polymerases were found to be related to other family B DNA polymerases (36 to 53% amino acid similarity), including the three enzymes of the archaebacterium Sulfolobus solfataricus. If mitochondria originated from a fusion between a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium, then the A. aegerita family B DNA polymerase genes could be remnants of the archaebacterial genes.

In Quebec, the evaluation of health care providers' practices is organized for each professional group. It is an official part of the health care regulation policy and is defined by legal texts. Evaluation of medical practices is controlled by physicians through their professional order and consultative medical councils in hospitals. The role of the Collège des Médecins du Québec is to warrant the quality of medical acts. The College watches over the quality of the physician's initial education using a process of agreement for all training departments and for the medical school curriculum. Young graduates must pass the College's examination before being licensed to practice. The College watches over improvements in professional practices via inspections of individual practices and hospitals and continuous education programs. There are three levels of physician supervision: screening for poor practices, inspection, and in-depth investigation. In hospitals, a consultative medical council is in charge of assuring quality medial practice. Mortality and morbidity conferences, claims analysis, audits with explicit evaluation criteria and indicator monitoring are the basic tools used for assessing the quality of medical practice. The Quebec experience demonstrating how a medical profession can organize a full system for evaluating medical practice can be a model for other countries.

An Agaricus bisporus microsatellite with the tetranucleotide motif TATG tandemly repeated was isolated from an A. bisporus library enriched in repeated sequences. The use of the 16-mer oligonucleotide (TATG)4 indicates that many loci contain nearby copies of the microsatellite in opposite orientations. The wide distribution of the microsatellite in the A. bisporus genome was assessed (i) by polyacrylamide gel electrophoresis of the products generated by directed amplification of microsatellite-region DNA (DAMD) and (ii) by hybridization of these products with A. bisporus chromosomes separated by pulsed-field gel electrophoresis. This is, to our knowledge, the first microsatellite reported in the cultivated edible mushrooms. DAMD-PCR products were generated using DNA of three Pleurotus species (P. pulmonarius, P. sajor-caju, and P. florida), indicating that (TATG)4 repeats are also present in these cultivated species. The variability found within closely related strains indicates that such microsatellites are useful in fingerprinting and studying genetic variability in wild and commercial mushrooms.

Objectives: Acute acoustic trauma (AAT) refers to damage to sensitive cochlear structures caused by excessive exposure to impulse noise. The aim of this study was to 1) assess the incidence of ATT in French soldiers and 2) to clarify the circumstances under which AAT occurs., Patients and Methods: We carried out a prospective study of AAT events reported to medical officers in 1998., Results: The incidence of AAT was 156 per 100,000 person-years (150-161). It was higher in conscripts and in soldiers aged under 31 years. Fifty-seven percent were wearing hearing protectors when the accident took place., Conclusion: According to these results, specific preventive programs were planned and should be assessed by epidemiological surveillance of exposed populations.

Objectives: To assess the impact of patient satisfaction survey method on response rate, data quality and satisfaction., Patients and Methods: Four modes of data collection were assessed during a randomized trial that included 400 inpatients discharged from a teaching hospital., Results: The response rate was 58% within the mail survey group (72% with follow-up letter) versus 73% and 81% within the home and telephone interview group (p < 0.01). 69% of the mailed questionnaires contained no missing values versus 94% and 96% for home and telephone interview modes (p < 0.01). The global satisfaction scale score was greater within the mail survey groups (8.1/10 without follow-up letter and 7.9 with follow-up letter) than within the telephone interview group (7.8) and the face to face interview group (7.3), (p < 0.05)., Conclusion: Mail survey with follow-up letter constitutes an operational method despite lower data quality and overestimation of patients' satisfaction scores.

A comparative study of the V4, V6 and V9 domains of the mitochondrial small-subunit (SSU) rRNA was conducted to evaluate the use of these sequences to investigate phylogenetic relatedness within the genus Pleurotus. The PCR products encompassing these regions from 48 isolates belonging to 16 Pleurotus species were sequenced and compared. From this comparison, the length and sequence of the three domains were found to be constant within a species. Significant inter-species variations due to insertion/deletion events were found, in most cases occurring in regions not directly involved in the maintainance of the standard SSU rRNA secondary structure. Phylogenetic analysis based upon these mitochondrial sequences was in agreement with relationships previously established by morphological descriptions and with previous studies based upon the nuclear genome or isozymes; moreover such analysis resolved some ambiguities in earlier analyses. It was confirmed that P. ostreatus and P. florida represent a single species, as well as P. pulmonarius and P. sajor-caju. The phylogenetic analysis also made it possible to assess the relative positions of P. rattenburyi, P. lampas, P. sapidus, P. colombinus and P. eryngii. The results clearly showed that sequences of the V4, V6 and V9 domains of the mitochondrial SSU rRNA could provide good markers for use in the taxonomy and phylogeny of species of Basidiomycota. Because of their nucleotide conservation, the major advantage of these species-specific markers was the possibility to study only one isolate from each species to determine phylogenetic relatedness.

Objectives: Following 1996 legislation requiring French hospitals to assess patient satisfaction, this study developed and validated a brief French-language multidimensional questionnaire designed to measure outpatient satisfaction with hospital visits and compared data quality for two patient-satisfaction survey methods., Design: Authors developed a 19-item questionnaire following a strict procedure (identification of dimensions to explore, formulation, and selection of items)., Setting: Validation data were obtained from patients of six outpatient clinics in a teaching hospital., Participants: 586 consenting eligible patients were randomized to receive the questionnaire 2 weeks after their visit with one of two survey methods: a mailed self-administered questionnaire or a telephone interview., Results: The response rate (79%) was not significantly different between the two survey methods. The risk of having one or more missing values was higher in the mail survey group (odds ratio, 1.65; 95% confidence interval, 1.03-2.63), but mail respondents were less likely to use the "extremely positive" response category. Principal component analysis identified four factors that accounted for 56% of the variance: interpersonal skills and information transfer, physical surroundings, convenience, and appointment delay. Patients' comments on open-ended questions validated the semantic content of the factorial construct. The internal consistency coefficient was greater than 0.70 for three of four subscales. Patient background characteristics accounted for less than 10% of the factorial score variance. Patient satisfaction was correlated with age, type of visit, and, to a lesser extent, gender and education level., Conclusion: This easily administered, multidimensional out-patient-satisfaction questionnaire provided encouraging preliminary psychometric characteristics.

A comparative study of variable domains V4, V6, and V9 of the mitochondrial small-subunit (SSU) rRNA was carried out with the genus Agrocybe by PCR amplification of 42 wild isolates belonging to 10 species, Agrocybe aegerita, Agrocybe dura, Agrocybe chaxingu, Agrocybe erebia, Agrocybe firma, Agrocybe praecox, Agrocybe paludosa, Agrocybe pediades, Agrocybe alnetorum, and Agrocybe vervacti. Sequencing of the PCR products showed that the three domains in the isolates belonging to the same species were the same length and had the same sequence, while variations were found among the 10 species. Alignment of the sequences showed that nucleotide motifs encountered in the smallest sequence of each variable domain were also found in the largest sequence, indicating that the sequences evolved by insertion-deletion events. Determination of the secondary structure of each domain revealed that the insertion-deletion events commonly occurred in regions not directly involved in the secondary structure (i.e., the loops). Moreover, conserved sequences ranging from 4 to 25 nucleotides long were found at the beginning and end of each domain and could constitute genus-specific sequences. Comparisons of the V4, V6, and V9 secondary structures resulted in identification of the following four groups: (i) group I, which was characterized by the presence of additional P23-1 and P23-3 helices in the V4 domain and the lack of the P49-1 helix in V9 and included A. aegerita, A. chaxingu, and A. erebia; (ii) group II, which had the P23-3 helix in V4 and the P49-1 helix in V9 and included A. pediades; (iii) group III, which did not have additional helices in V4, had the P49-1 helix in V9 and included A. paludosa, A. firma, A. alnetorum, and A. praecox; and (iv) group IV, which lacked both the V4 additional helices and the P49-1 helix in V9 and included A. vervacti and A. dura. This grouping of species was supported by the structure of a consensus tree based on the variable domain sequences. The conservation of the sequences of the V4, V6, and V9 domains of the mitochondrial SSU rRNA within species and the high degree of interspecific variation found in the Agrocybe species studied open the way for these sequences to be used as specific molecular markers of the Basidiomycota.

The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527aa (58377Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility. When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc). This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions. When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes. The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences. The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences. These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF. The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e. the highest values reported to date between introns of two phylogenetically distant species. This low divergence argues for a recent lateral transfer between the two species. On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences. The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns. This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.

Due to their structural complexity and their evolutionary dimension, rRNAs are the most investigated nucleic acids in prokaryotes, eukaryotes and organelles. However, no complete sequence of a mitochondrial small subunit (SSU) rRNA was available in the basidiomycotina subdivision. The mitochondrial gene encoding the SSU rRNA of the cultivated basidiomycete Agrocybe aegerita was cloned and its complete nucleotide sequence achieved; the 5'- and 3'-ends were localized by nuclease SI mapping, leading to a size of 3277 nt. The secondary structure of the SSU rRNA (1906 nt in size) possessed all the helices and loops of the prokaryotic model; a unique modification was found in a conserved nucleotide predicted by the model: the nt 487 was A instead of C. The same modification, has been found in all the partial basidiomycete mitochondrial sequences available in databases. The Agrocybe aegerita SSU rRNA was characterized by large and unusual extensions leading to additional helices in the variable domains V4, V6 and V9, which were the longest of the known prokaryotic or mitochondrial SSU rRNAs. Nucleotide sequence analysis indicated a 1371-bp intron, belonging to subgroup-IC2, located in a conserved loop in the 3'-part of the SSU rRNA. This intron, which is the second example reported in a fungal mitochondrial SSU rDNA, encoded a putative protein (407 aa) sharing homologies with endonucleases involved in group-I intron mobility. This report constitutes the first complete mitochondrial SSU rRNA sequence and secondary structure of any member of the basidiomycotina subdivision.

Development and implementation of guidelines constitutes the basis of quality management systems for any organization. The authors have studied the internal documentation produced by professionals on 88 functional units of a university hospital. Reveals the existence of many documents concerning quality of care with an average of 102 available procedures or protocols per unit. However, this documentation is badly organized, making it difficult to consult and to put into practice. The results of this study were provided to other professionals at our hospital in order to make them aware of the necessity of rigorous document management. We have also written and sent recommendations for drawing up procedures and implementing an efficient documentary management system. This effort complements development of the hospital quality assurance plan.

DNA sequences of the basidiomycete Agrocybe aegerita were cloned in E. coli based on their ability to drive the expression of the bacterial promoterless tetracycline (Tc)-resistance gene. A 0.48% frequency of the cloned sequences promoted antibiotic-resistance. The sequence conferring the highest Tc resistance (40 μg/ml) was selected to drive the expression in E. coli of two other promoterless genes encoding chloramphenicol and neomycin resistance. One of the derivative vectors, pN13-A2, carrying a chimeric neomycin-resistance gene, was used to transform an A. aegerita neomycin-sensitive strain by protoplast electroporation. Transformation frequencies ranged from 1 to 2.8 transformants per μg of DNA per 10(3) viable cells, in a relatively high background of spontaneous-resistant colonies (2% of the surviving protoplasts). Molecular analyses showed that transformation had occurred by the integration of pN13-A2 sequences, either ectopically or at the resident locus carrying the A. aegerita promoter-like sequence, with probable molecular rearrangements. The nucleotide sequence of the promoter-like fragment revealed the presence of a CT motif that is known to be involved in a promoter function in some highly expressed genes of filamentous fungi.

In the ribosomal DNA unit ofPleurotus cornucopiae, the rDNA coding regions are in the order 5', 5S-18S-5.8S-25S, 3', with the 5' location of the 5S gene differing from its 3' location found in other basidiomycetes. The most discriminating probe used to study the rDNA polymorphism consisted of a fragment that included the 5S, 18S and part of the 5.8S and 25S genes flanking three intergenic sequences. A high degree of rDNA polymorphism was observed in the sevenP. cornucopiae dikaryons studied. For the first time within a basidiomycete species, the restrictions maps distinguished two types of rDNA units (I and II). In each rDNA type, length variations in the external intergenic sequence IGS 1 located between the 25S and 5S genes allowed characterization of two different rDNA units in type I and four rDNA units in type II. This suggested that theP. cornucopiae rDNA units were derived from two kinds of ancestors (type I and II) by insertion or deletion events (100-700 bp) in the IGS 1. In four dikaryotic strains, two rDNA units of the same type (I or II) differing only by the IGS 1 length, were found in a similar number of copies, and presented a meiotic segregation in homokaryotic progeny. In one progeny, some homokaryotic strains possessed two different rDNA units: one with a high copy number and another with a lower one, showing that two different rDNA units could coexist in a single nucleus.

The URA1 gene, encoding dihydroorotate dehydrogenase of the pyrimidine pathway, cloned into pUC18 (pUra1-1) was used to develop an homologous transformation system for the cultivated mushroom Agrocybe aegerita. Protoplasts of a ura1 auxotrophic strain were transformed by electroporation with efficiencies ranging from 1 to 26 transformants per micrograms of DNA. The phenotype of the stable Ura+ transformants suggested a strong nuclear heterogeneity further confirmed by Southern-blot analysis. All transformants acquired extrachromosomal forms derived from pUra1-1. Integration of pUra1-1 into chromosomal DNA occurred for some transformants. Plasmids containing the integrant of pUC18 recombined to different parts of the URA1 gene were rescued from A. aegerita transformants through transformation of E. coli. Their molecular analysis indicated that they represent products of the continuous excision of primary-integrated vector sequences rather than ARS-dependent autoreplicative forms.

The URA1 gene encoding dihydroorotate dehydrogenase (DHOdehase) from the edible basidiomycete, Agrocybe aegerita, has been cloned by complementation of the Escherichia coli pyrD mutation. The nucleotide sequence of a 1531-bp genomic fragment carrying URA1 revealed two uninterrupted open reading frames (ORFs) separated by 61 bp. The larger ORF can encode a 328-amino acid (aa) DHOdehase that has 53% homology with the corresponding protein from E. coli. Comparison with other DHOdehase aa sequences showed essentially conservation of the cofactor-binding site of flavoproteins.

Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two single-copy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.

The study of fruiting in the basidiomycete Agrocybe aegerita has shown that some haploid homokaryotic strains can spontaneously switch their mating specificities at the two unlinked A and B mating type factors. This event causes the dikaryotisation of primary homokaryons without plasmogamy and leads to the differentiation of sporulating fruit-bodies (pseudo-homokaryotic fruiting). For each mating type factor, the genetic analyses have revealed that: (1) parental and switched mating types segregate meiotically as Mendelian markers, (2) a total of six switched mating type factors (two parental and four nonparental) were obtained from a wild strain, (3) most of the nonparental factors have specificities differing from those of a large series of wild factors, (4) strains with the same expressed mating type can generate different specificities, (5) switching is always restricted to the same mating type in a homokaryon, (6) nonparental types can switch again, and (7) meiosis fixes the specificities to which switching can occur. This suggests, for the first time in filamentous fungi, the existence of a mechanism analogous to the mating type switching in yeasts. We hypothese that both A and B mating type regions in A. aegerita are constituted of three loci, one specialized in expression and two other carrying silent information. Mating type switching in homokaryotic strains would occur by copy transposition of silent A and B information into the expression loci. Moreover, we propose that during meiosis the silent loci are substituted by copies of the expressed loci.

The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80,500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.

An Agrocybe aegerita cDNA library, constructed from fruit body primordia poly(A)+ RNAs, was screened by differential colony hybridization. Clones which preferentially hybridized to poly(A)+ RNA sequences from fruit body primordia, versus poly(A)+ RNAs from mycelium, were isolated. Eight of these clones (EMAa-1 to EMAa-8) encoded eight different poly(A)+ RNAs which were demonstrated to be undetectable in the four stages preceding primordia formation and to be concomitantly accumulated when primordia differentiate, suggesting that EMAa gene products are closely involved in the morphogenesis of primordia. The eight EMAa cDNAs hybridize to at least seven unique regions distributed randomly in the A. aegerita genome. The expression of two EMAa cDNA sequences in E. coli led to the isolation of their gene products as fusion proteins.

The variability of the sexual incompatibility genes of Agrocybe aegerita was investigated in the homokaryotic progeny of 13 wild dikaryotic strains from five distinct European geographic origins. Results of mating tests allowed identification of 18 A alleles and 16 B alleles out of a possible 26 different alleles for each in the sample. The determination and the comparison by a contingency χ (2) test of the frequencies of allele replications between intra- and interregional matings showed no departure from a random distribution of incompatibility alleles. The allelic series estimated for the incompatibility genes of the entire population of A. aegerita, 30 A and 25 B aleles, are significantly less extensive than those already hypothesized for other tetrapolar hymenomycetes. However, the low variability of incompatibility genes has little effect on the outbreeding efficiency (92.6%) of this mushroom. The low variability of the incompatibility alleles and the apparent absence of intrafactorial recombination could relate to a single-locus structure of the incompatibility genes in A. aegerita.

A mutant (modx) was selected on the basis of the suppression of self-lysis due to a recessive mutation (modB). modx, a dominant mutation, reduced hyphal branching from nonapical cells, abolished protoperithecium formation, and induced the death of stationary cells only when these were isolated to obtain further development. Mutant ascospores, formed in the fruiting bodies which occasionally occur under specific conditions (32 degrees C on starved medium), showed a delay in the germination process (up to 3 months instead of about 5 h for wild-type ascospores) when submitted to incubation under standard conditions (26 degrees C on germination medium) and failed to germinate at 18 degrees C. Revertants from modx strains, selected on the basis of the suppression of the nonrenewal of growth from stationary cells, were wild type for all the other three defects. Indirect arguments suggested that the modx mutant strain might be defective in the control of a specific class of stable messenger ribonucleic acids which would be essential for the physiology of ascospores and stationary cells.

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.

A procedure suitable for the extraction and mapping of total proteins from the basidiomycete, Agrocybe aegerita, was developed. A. aegerita mycelia were fragmented either with a Dangoumeau grinder, an X-press bomb or a sonicator and the efficiency of these three disruption methods were compared. The extraction buffer composition was optimized to avoid proteolytic activities. 2D-SDS-PAGE analysis of protein extracts showed that the rate of reproducibility depending on extractions and electrophoretic separations was always greater than 96% for all strains. The differences in efficiency observed between the breaking procedures indicate that the A. aegerita cell wall is more mechanically resistant than that of other basidiomycetes. The efficient action of protease inhibitors (PMSF and SDS) showed that A. aegerita mycelia contains numerous and/or highly active proteases. Reproducibility of protein extraction and separation methods allowed the establishment and the comparison of standard maps. Qualitative and quantitative variations in gene products between a wild dikaryotic strain and 11 homokaryotic strains from its progeny were examined. The genetic diversity, determined by comparing the distribution of proteic variations in 11 homokaryons from the same progeny, was comparable to that observed between co-isogenic homokaryons of another basidiomycete.

The study of resistance marker rearrangement in Spiroplasma citri mutants provides evidence of transfer of chromosomal information followed by recombination. This is the first report of such a transfer in vivo in the mollicutes--that is, in the smallest self-replicating organisms. The double-resistant phenotypes obtained are stable even without selection pressure. The mechanism of gene transfer is insensitive to deoxyribonuclease, requires contact, and possibly, areas of fusion of the cell membranes; it shares properties with the transfer by protoplast fusion in Gram-positive bacteria. The extensive degenerative evolution of mollicutes has retained, in S. citri, bacterial functions of chromosomal transfer and recombination.

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.