RESUMO
BACKGROUND: Staff working in intensive care units (ICUs) have faced significant challenges during the COVID-19 pandemic which have the potential to adversely affect their mental health. AIMS: To identify the rates of probable mental health disorder in staff working in ICUs in nine English hospitals during June and July 2020. METHODS: An anonymized brief web-based survey comprising standardized questionnaires examining depression, anxiety symptoms, symptoms of post-traumatic stress disorder (PTSD), well-being and alcohol use was administered to staff. RESULTS: Seven hundred and nine participants completed the surveys comprising 291 (41%) doctors, 344 (49%) nurses and 74 (10%) other healthcare staff. Over half (59%) reported good well-being; however, 45% met the threshold for probable clinical significance on at least one of the following measures: severe depression (6%), PTSD (40%), severe anxiety (11%) or problem drinking (7%). Thirteen per cent of respondents reported frequent thoughts of being better off dead, or of hurting themselves in the past 2 weeks. Within the sample used in this study, we found that doctors reported better mental health than nurses across a range of measures. CONCLUSIONS: We found substantial rates of probable mental health disorders, and thoughts of self-harm, amongst ICU staff; these difficulties were especially prevalent in nurses. Whilst further work is needed to better understand the real level of clinical need amongst ICU staff, these results indicate the need for a national strategy to protect the mental health, and decrease the risk of functional impairment, of ICU staff whilst they carry out their essential work during COVID-19.
Assuntos
COVID-19/psicologia , Cuidados Críticos/psicologia , Unidades de Terapia Intensiva , Transtornos Mentais/etiologia , Saúde Mental , Pandemias , Recursos Humanos em Hospital/psicologia , Adulto , Transtornos Relacionados ao Uso de Álcool/epidemiologia , Transtornos Relacionados ao Uso de Álcool/etiologia , Ansiedade/etiologia , Transtornos de Ansiedade/epidemiologia , Transtornos de Ansiedade/etiologia , Depressão/etiologia , Transtorno Depressivo/epidemiologia , Transtorno Depressivo/etiologia , Inglaterra/epidemiologia , Feminino , Humanos , Masculino , Transtornos Mentais/epidemiologia , Enfermeiras e Enfermeiros/psicologia , Exposição Ocupacional , Médicos/psicologia , Prevalência , SARS-CoV-2 , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Transtornos de Estresse Pós-Traumáticos/etiologia , Inquéritos e QuestionáriosRESUMO
BACKGROUND: While it is known that some UK Armed Forces (UK AF) personnel and veterans experience physical and mental health problems, the possible future healthcare needs of military veterans are unknown. AIMS: To estimate the number of military personnel who may experience physical and/or psychological health problems associated with their military service. METHODS: Data were obtained via Freedom of Information requests to several sources, including Defence Statistics. Raw data from research studies were also used where available. Data were analysed using meta-analytic methods to determine the rate of physical, mental or comorbid health problems in AF personnel. RESULTS: Musculoskeletal problems were the predominant reason for medical discharge from service. In terms of mental health, meta-analyses estimated that veteran reservists (part-time military members) previously deployed to operational areas had the highest proportion of general health problems (35%), previously deployed veteran regulars (those in full time military employment) and veteran reservists had the highest proportion of post-traumatic stress disorder (9%), and regular personnel with a deployment history had the highest proportion of alcohol problems (14%). Overall, our findings suggest that at least 67515 veterans are likely to suffer from mental and/or physical health problems at some point as a result of their service between 2001 and 2014. CONCLUSIONS: The results of this study highlight that the difficulties personnel may face are largely musculoskeletal or mental health-related. These findings may help with planning the provision of future physical and mental health care and support for those who serve in the UK AF.
Assuntos
Transtornos Mentais/epidemiologia , Militares/estatística & dados numéricos , Doenças Musculoesqueléticas/epidemiologia , Saúde dos Veteranos/estatística & dados numéricos , Alcoolismo/epidemiologia , Humanos , Militares/psicologia , Fatores de Risco , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Reino Unido/epidemiologia , Exposição à Guerra/estatística & dados numéricosRESUMO
BACKGROUND: Military service can be a traumatic experience and cause mental health problems in a minority of personnel, such as post-traumatic stress disorder (PTSD), which is linked to negative long-term outcomes. As a result, PTSD has received significant research attention. However, post-traumatic growth (PTG) is a newer construct, with comparatively little known about its presentation and development. AIMS: To qualitatively examine the experience of (PTG) in military and ex-military personnel. METHODS: A qualitative systematic search of electronic databases was conducted, with studies assessed for methodological quality and data analysed using thematic analysis. Nine qualitative studies, carried out between 2011 and 2016, met the inclusion criteria with 195 participants in total, including both military and ex-military personnel. RESULTS: Six themes were identified: appreciation for life, re-evaluating sense of purpose, improvement of personal human traits, bonding and connecting with others, integrating into society, and being proud of heritage and feeling valuable to society. CONCLUSIONS: The results of this review illustrate that military personnel may experience PTG due to deployment-related trauma exposure, and the presentation of PTG in this population is not dissimilar to that of civilians. This study highlights the need for additional research to quantify the long-term psychological impact of PTG and whether a focus on PTG may be helpful in psychological treatment for (ex-) military personnel.
Assuntos
Distúrbios de Guerra/complicações , Transtornos Mentais/etiologia , Crescimento Psicológico Pós-Traumático , Adaptação Psicológica , Adulto , Distúrbios de Guerra/psicologia , Feminino , Humanos , Masculino , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Militares/psicologia , Militares/estatística & dados numéricos , Pesquisa Qualitativa , Veteranos/psicologia , Veteranos/estatística & dados numéricosRESUMO
Background: Remote area medics (RAMs) may be at increased risk of mental health difficulties. Aims: To explore the occupational experiences of RAMs to identify stressors and the mental health impact. Methods: Semi-structured interviews were conducted with six RAMs working in Iraq to gather data, which was explored using interpretative phenomenological analysis. Results: Three key themes emerged from the data (i) the experience of being remote, (ii) cultural shock and (iii) social support. A number of key stressors were identified, including loneliness and boredom, associated with being remote, and the loss of professional identity due to the occupational role. Three out of the six participants reported substantial depressive symptoms. A number of positive coping strategies were identified, particularly relationships with other RAMs, via instant messaging forums. Conclusions: RAMs experience a number of particular stressors that could put them at risk of depression. Adaptive coping strategies were identified; in particular, virtual social support. These findings should be of interest to companies which employ RAMs.
Assuntos
Auxiliares de Emergência/psicologia , Guerra do Iraque 2003-2011 , Estresse Psicológico/complicações , Guerra , Adaptação Psicológica , Adulto , Auxiliares de Emergência/organização & administração , Feminino , Humanos , Iraque , Masculino , Pessoa de Meia-Idade , Apoio Social , Estresse Psicológico/etiologia , Estresse Psicológico/psicologiaRESUMO
For music and language processing, memory for relative pitches is highly important. Functional imaging studies have shown activation of a complex neural system for pitch memory. One region that has been shown to be causally involved in the process for nonmusicians is the supramarginal gyrus (SMG). The present study aims at replicating this finding and at further examining the role of the SMG for pitch memory in musicians. Nonmusicians and musicians received cathodal transcranial direct current stimulation (tDCS) over the left SMG, right SMG, or sham stimulation, while completing a pitch recognition, pitch recall, and visual memory task. Cathodal tDCS over the left SMG led to a significant decrease in performance on both pitch memory tasks in nonmusicians. In musicians, cathodal stimulation over the left SMG had no effect, but stimulation over the right SMG impaired performance on the recognition task only. Furthermore, the results show a more pronounced deterioration effect for longer pitch sequences indicating that the SMG is involved in maintaining higher memory load. No stimulation effect was found in both groups on the visual control task. These findings provide evidence for a causal distinction of the left and right SMG function in musicians and nonmusicians.
Assuntos
Memória/fisiologia , Música , Lobo Parietal/fisiologia , Percepção da Altura Sonora/fisiologia , Estimulação Transcraniana por Corrente Contínua , Estimulação Acústica , Adolescente , Adulto , Análise de Variância , Feminino , Lateralidade Funcional , Humanos , Imageamento por Ressonância Magnética , Masculino , Estimulação Luminosa , Competência Profissional , Tempo de Reação/fisiologia , Adulto JovemRESUMO
BACKGROUND: Schizophrenia (SZ) and bipolar disorder (BD) have substantial negative impact on the quality of human life. Both, microRNA (miRNA) expression profiling in SZ and BD postmortem brains [and genome-wide association studies (GWAS)] have implicated miRNAs in disease etiology. Here, we aim to determine whether significant GWAS signals observed in the Psychiatric Genetic Consortium (PGC) are enriched for miRNAs. METHOD: A two-stage approach was used to determine whether association signals from PGC affect miRNAs: (i) statistical assessment of enrichment using a Simes test and sum of squares test (SST) and (ii) biological evidence that quantitative trait loci (eQTL) mapping to known miRNA genes affect their expression in an independent sample of 78 postmortem brains from the Stanley Medical Research Institute. RESULTS: A total of 2567 independent single nucleotide polymorphisms (SNPs) (R2 > 0.8) were mapped locally, within 1 Mb, to all known miRNAs (miRBase v. 21). We show robust enrichment for SZ- and BD-related SNPs with miRNAs using Simes (SZ: p ≤ 0.0023, BD: p ≤ 0.038), which remained significant after adjusting for background inflation in SZ (empirical p = 0.018) and approached significance in BD (empirical p = 0.07). At a false discovery rate of 10%, we identified a total of 32 eQTLs to influence miRNA expression; 11 of these overlapped with BD. CONCLUSIONS: Our approach of integrating PGC findings with eQTL results can be used to generate specific hypotheses regarding the role of miRNAs in SZ and BD.
Assuntos
Transtorno Bipolar/genética , Esquizofrenia/genética , Autopsia , Encéfalo , Mapeamento Cromossômico , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , MicroRNAs/genética , Polimorfismo Genético , Locos de Características QuantitativasRESUMO
INTRODUCTION: Despite making up about 11% of the UK military, there remains limited investigation on the impact of adversity women experience during their service in the UK military. Military adversity can result in a range of well-being difficulties that may persist following transition out of military. The present study therefore examined the prevalence and correlates of different types of military adversity (defined as sexual harassment, sexual assault, emotional bullying and physical assault) within a community sample of UK women veterans. METHODS: Participants were recruited from a UK charity supporting women veterans. 750 women veterans completed an online survey collecting information on sociodemographic and military factors, military adversity, as well as mental health and well-being difficulties. Associations between variables were explored using multivariate logistic regressions. RESULTS: The findings indicate a high prevalence of military adversity (22.5% sexual harassment, 5.1% sexual assault, 22.7% emotional bullying and 3.3% physical assault). Younger women, those who held an officer rank during service and those who reported having a combat or combat support role during service were most at risk of military adversity. All types of adversity were significantly associated with probable post-traumatic stress disorder. Sexual harassment was additionally significantly associated with physical somatisation; sexual assault with alcohol difficulties; and emotional bullying with common mental health difficulties, low social support and loneliness. CONCLUSIONS: This study indicates that UK women veterans are at risk of a range of adverse experiences during military service and provides evidence of the impact of such adversities on mental health and well-being. Further research is required to better understand these relationships.
Assuntos
Bullying , Militares , Veteranos , Humanos , Feminino , Veteranos/psicologia , Saúde Mental , Trauma Sexual MilitarRESUMO
INTRODUCTION: Moral injury (MI) significantly impacts the lives of many UK military veterans however, there is a lack of manualised treatment to address the needs of this population. To develop future treatments that are acceptable and well tolerated, veterans should be consulted on their experiences of existing psychological treatments and suggestions for future treatments. METHODS: 10 UK military veterans were interviewed about their experiences of receiving treatment for psychological difficulties after MI, and beliefs about core components of future treatments. Thematic analysis of these interviews were conducted. RESULTS: 2 superordinate themes were identified: experiences of previous mental health treatment and perceptions of the proposed treatments. Reflections on cognitive behavioural therapy were mixed, with some describing that it did not ameliorate their guilt or shame. In future treatments, focusing on values, using written letters and including therapy sessions with close companions were considered beneficial. Veterans reported that a strong rapport with therapist was key for MI treatment. CONCLUSION: Findings provide a useful account of how current post-trauma treatments may be experienced by patients with MI. Although limited by sample size, the results highlight therapeutic approaches that may be helpful in future and provide important considerations for therapists treating MI.
RESUMO
INTRODUCTION: Individuals who have been exposed to a traumatic event can develop profound feelings of guilt, shame and anger. Yet, studies of treatments for post-traumatic stress disorder (PTSD) have largely investigated changes in PTSD symptoms relating to a sense of ongoing fear or threat and the effectiveness of such treatments for post-trauma related guilt, shame or anger symptom reduction is comparatively not well understood. METHODS: This review systematically examined the effectiveness of existing treatment approaches for three symptoms associated with exposure to traumatic events: guilt, shame and anger. Studies included had to be published after 2010 with a sample size of n=50 or greater to ensure stable treatment outcome estimates. RESULTS: 15 studies were included, consisting of both civilian and (ex-) military population samples exposed to a wide range of traumatic events (eg, combat-related, sexual abuse). Findings indicated a moderate strength of evidence that both cognitive-based and exposure-based treatments are similarly effective in reducing symptoms. Cognitive-based treatments were found to effectively reduce post-trauma related guilt and anger, while exposure-based treatments appeared effective for post-trauma related guilt, shame and anger. CONCLUSIONS: The findings suggest the importance of confronting and discussing the traumatic event during therapy, rather than using less directive treatments (eg, supportive counselling).Nonetheless, while these results are promising, firm conclusions regarding the comparative effectiveness and long-term impact of these treatments could not be drawn due to insufficient evidence. Further empirical research is needed to examine populations exposed to traumatic events and investigate which treatment approaches (or combination thereof) are more effective in the long-term.
RESUMO
Moral injury is a relatively new, but increasingly studied, construct in the field of mental health, particularly in relation to current and ex-serving military personnel. Moral injury refers to the enduring psychosocial, spiritual or ethical harms that can result from exposure to high-stakes events that strongly clash with one's moral beliefs. There is a pressing need for further research to advance understanding of the nature of moral injury; its relationship to mental disorders such as posttraumatic stress disorder and depression; triggering events and underpinning mechanisms; and prevalence, prevention and treatment. In the meantime, military leaders have an immediate need for guidance on how moral injury should be addressed and, where possible, prevented. Such guidance should be theoretically sound, evidence-informed and ethically responsible. Further, the implementation of any practice change based on the guidance should contribute to the advancement of science through robust evaluation. This paper draws together current research on moral injury, best-practice approaches in the adjacent field of psychological resilience, and principles of effective implementation and evaluation. This research is combined with the military and veteran mental health expertise of the authors to provide guidance on the design, implementation and evaluation of moral injury interventions in the military. The paper discusses relevant training in military ethical practice, as well as the key roles leaders have in creating cohesive teams and having frank discussions about the moral and ethical challenges that military personnel face.
RESUMO
An assay has been developed to measure the rate of transposition of the transposable element Ty in Saccharomyces cerevisiae. The assay is based on the altered expression of the glucose-repressible alcohol dehydrogenase gene of yeast upon insertion of a Ty in front of this gene. By this assay the transposition rate of Ty elements was found to increase approximately 100-fold at temperatures lower than 30 degrees C, the optimum growth temperature for Saccharomyces cerevisiae.
RESUMO
Specific host-parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.
Assuntos
Bactérias Gram-Positivas Formadoras de Endosporo/fisiologia , Parasitos/fisiologia , Tylenchoidea/anatomia & histologia , Tylenchoidea/parasitologia , Animais , Aderência Bacteriana , Feminino , Interações Hospedeiro-Parasita , Masculino , Parasitologia/métodos , Partenogênese , Raízes de Plantas/parasitologia , Reprodução/fisiologia , Especificidade da Espécie , Esporos Bacterianos/fisiologia , Tylenchoidea/imunologiaRESUMO
The mutation rate to antimycin A resistance was determined for strains of Sacchromyces cerevisiae lacking a functional copy of the structural gene for alcohol dehydrogenase I (ADH1). One type of mutation that can cause antimycin A resistance in these strains is insertion of the transposable element Ty 5' to ADH2, the structural gene for the glucose-repressed isozyme of alcohol dehydrogenase, resulting in expression of this gene during growth on glucose. Here we show that after growth at 15 or 20 degrees C on glucose, 30% of the antimycin A resistance mutations are Ty insertions at ADH2 and another 65% of the mutations are Ty insertions at ADH4, a new locus identified and cloned as described in this paper. At 30 degrees C only 6% of the mutations are Ty insertions at either of these two loci. In addition, we show that the transposition rate is lower in mating-incompetent (a/alpha) cells than in either haploid or diploid mating-competent cells. Our results suggest that under certain conditions Ty transposition may be a major cause of spontaneous mutations in S. cerevisiae.
Assuntos
Álcool Desidrogenase/genética , Antimicina A/farmacologia , Elementos de DNA Transponíveis , Genes Fúngicos , Genes , Mutação , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Cruzamentos Genéticos , Resistência a Medicamentos , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Seven cis-dominant, constitutively expressed mutations of the normally glucose-repressible isozyme of alcohol dehydrogenase (ADHII) from the yeast Saccharomyces cerevisiae are caused by insertion of transposable elements from the Ty1 family in front of the ADHII structural gene (ADR2) (V. M. Williamson, E. T. Young, and M. Ciriacy, Cell 23:605-614, 1981). We cloned ADR2 with its associated Ty1 element from five S. cerevisiae strains carrying these mutations. Comparison of the Ty1 elements by heteroduplex studies and restriction enzyme analyses indicated that four were very similar; the fifth, although the same size as the others (about 5.6 kilobases), differed by the presence of two large substitutions of approximately 1 and 2 kilobases. The DNA sequences of the terminal direct repeats (deltas) were very homologous but not identical and were similar to previously reported Ty1 element direct repeats. We determined the 5'-flanking sequences of the ADR2 gene isolated from a wild-type strain and from five Ty1-associated mutations. The 5-base pair target sequence at the site of Ty1 insertion was present at both ends of each Ty1 element. The sites of insertion of the elements were all different and occurred from 125 to 210 base pairs in front of the coding region of ADR2. The 5' end of the major transcript as determined by S1 mapping was the same in wild-type cells and in Ty1-associated constitutive mutants and was approximately 54 base pairs upstream from the coding region. ADR2 transcripts were not detected when a solo delta sequence was present in the 5'-flanking region of this gene.
Assuntos
Oxirredutases do Álcool/genética , Elementos de DNA Transponíveis , Isoenzimas/genética , Saccharomyces cerevisiae/genética , Álcool Desidrogenase , Sequência de Bases , Clonagem Molecular , Genes , Mutação , Plasmídeos , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Root-knot and cyst nematodes cause severe damage to crops throughout the world. Genes conferring resistance against nematodes have been identified in many plant species and several of these have been, or soon will be, cloned. Nematode biotypes that can infect resistant plants have been identified. Investigation of cloned resistance genes and of virulent nematodes is likely to lead to improved host resistance.
Assuntos
Genes de Plantas , Nematoides/fisiologia , Plantas/parasitologia , AnimaisRESUMO
The gene Mi, which confers resistance to several species of root-knot nematode, is present in many modern tomato cultivars. Recent cloning of this gene revealed that it encodes a member of the plant resistance protein family characterized by the presence of a putative nucleotide binding site and a leucine-rich repeat. Analysis of transgenic plants revealed the unexpected result that Mi also confers resistance to potato aphids. Although highly effective in many conditions, Mi fails to confer resistance at high soil temperature, and Mi-virulent nematode isolates have been identified in many areas of the world. These findings have stimulated efforts to identify new sources of root-knot nematode resistance. Resistance genes that differ from Mi in properties and genetic position have been identified in Lycopersicon peruvianum. These genes, as well as the cloned Mi gene, provide a resource for broadening the base of root-knot nematode resistance in tomato and other crops.
RESUMO
Five isolates of M. hapla originating from the Netherlands and California were inbred by sequential transfer of single egg masses to produce six strains. Cytological examination showed that oocytes of these strains underwent meiosis and had n = 16 chromosomes. Strains were tested for ability to infect and to develop on several hosts by in vitro assays. The two strains from California infected tomato roots at a higher rate than those from the Netherlands, but no difference among strains was seen for ability to develop on tomato with or without the resistance gene Mi-1. All strains developed on the common bean cultivar Kentucky Wonder, but strains differed in ability to develop on the nematode-resistant cultivar NemaSnap. Strain-specific differences were also seen in ability to infect and to develop on Solanum bulbocastanum clone SB-22. Strain VW13, derived from nematodes treated with the mutagen EMS, was defective in ability to infect tomato and potato roots in vitro. Comparison of DNA using AFLP markers showed an average of 4% of the bands were polymorphic across the six strains, but no correlation was observed between the geographical origin or virulence and DNA polymorphism pattern.
RESUMO
The first epidermal growth factor-like (EGF1) domain of human factor VII (FVII) is essential for binding to tissue factor (TF). We hypothesized that the previously observed increased coagulant activity of rabbit plasma (i.e. FVII) with human TF might be explained by the five non-conserved amino acids in the rabbit vs. the human FVII EGF1 domain. Accordingly, we 'rabbitized' the human FVII EGF1 domain either by exchanging the entire EGF1 domain creating human FVII(rabEGF1) or by the single amino acid substitutions S53N, K62E, P74A, A75D and T83K. After transient expression in HEK293 cells, the recombinant FVII (rFVII) mutant proteins were analyzed for biological activity and binding affinity to human TF by competitive enzyme-linked immunosorbent assay (ELISA). Biological activity of the unpurified rFVII mutant proteins was either depressed or statistically unchanged vs. rFVII(WT). However, three of six rFVII mutant proteins had increased affinity for human TF in the rank order rFVII(rabEGF1) (3.3-fold) > rFVII(K62E) (2.9-fold) > rFVII(A75D) (1.7-fold). The mutant protein rFVII(K62E) was then permanently expressed and purified. Fully activated, purified rFVIIa(K62E) had a twofold greater clotting activity and 2.8-fold greater direct FVIIa amidolytic activity when compared with rFVIIa(WT). Quantitation of the affinity of TF binding by surface plasmon resonance indicated that the KD of purified rFVII(K62E) for human soluble TF (sTF) was 1.5 nM compared with 7.5 nM for rFVII(WT), i.e. fivefold greater affinity. We conclude that substitution of selected amino acid residues of the FVII EGF1 domain facilitated the creation of human rFVII chimeric proteins with both enhanced biological activity and increased affinity for TF.
Assuntos
Fator VII/genética , Proteínas Recombinantes de Fusão/farmacologia , Substituição de Aminoácidos , Animais , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular , Fator de Crescimento Epidérmico/química , Fator VII/metabolismo , Fator VII/farmacologia , Humanos , Análise Serial de Proteínas , Engenharia de Proteínas , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Tromboplastina/metabolismo , TransfecçãoRESUMO
A spontaneous antimycin A-resistant mutant carrying approximately four extra copies of ADH2 on chromosome XII was isolated from yeast strain 315-1D which lacks a functional copy of ADH1 and thus is antimycin A-sensitive. The additional copies of the normally glucose-repressed ADH2 are expressed during growth on glucose accounting for the antimycin A resistance. These extra copies are inserted into nonadjacent ribosomal DNA sequences (rDNA) near the recombination stimulating sequence HOT1. Each extra copy of the ADH2 gene (1548 bp) replaces most of the 37S transcript (approximately 7400 bp) in one of the approximately 200 copies of the rDNA present in the yeast genome. All four extra copies of ADH2 are lost at a rate of approximately 1 x 10(-5) deletions per cell per generation. One of the joints between the rDNA and ADH2 DNA is located 7 nucleotides downstream from 20 adenine residues in the normal copy of ADH2. This joint occurs at the end of a stretch of 16-29 thymidines in the rDNA which has been expanded to 57-59 thymidines. The other novel joint is located in a short region of sequence similarity between ADH2 and the rDNA. These observations suggest that amplification of ADH2 was a two step process: first the ADH2 gene was inserted into the rDNA, then multiple copies were generated by unequal crossing over or gene conversion within the rDNA.
Assuntos
Saccharomyces cerevisiae/genética , Álcool Desidrogenase/genética , Antimicina A/farmacologia , Sequência de Bases , Cromossomos Fúngicos , Clonagem Molecular , DNA Fúngico/genética , DNA Ribossômico/genética , Resistência Microbiana a Medicamentos/genética , Amplificação de Genes , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Mapeamento por Restrição , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologiaRESUMO
We describe the cloning of a cDNA encoding tomato alcohol dehydrogenase 2 (Adh2) by screening plasmid cDNA clones in phage plaques. A cDNA library constructed in a plasmid vector containing a unique SstI site at the 5' end of the cDNA insert was transferred into the SstI site of the lacZ gene of phage lambda Charon16, and screened by anti-Adh2 antibody to identify reactive plaques. Plasmid cDNA clones were recovered by SstI digestion, ligation, and transformation from phage minipreps for subsequent characterization. This system preserves the original plasmid library for subsequent screening with nucleic acid probes to identify full-length, multiple independent, or related cDNA clones not subject to the selection pressure of phage growth or lysogeny, or negative antibody reactivity. Thirty-two cDNA clones were identified with polyclonal antiserum to Adh2. Three of these reacted with monoclonal anti-Adh2 and only those three hybridized to maize adh1 sequence. One of these cDNAs, Adh31, was further characterized as encoding Adh2 by hybrid-selected translation and high sequence homology with the maize adh1 gene.