RESUMO
The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Dano ao DNA , Reparo do DNA , Proteínas Fúngicas/química , Genes Fúngicos , Dados de Sequência Molecular , Mutação , Fenótipo , Precursores de RNA/metabolismo , Splicing de RNA , Fatores de Processamento de RNA , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Spliceossomos , Esporos Bacterianos , Temperatura , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
A novel mutant isolate of Saccharomyces cerevisiae, sensitive to photoactivated mono- and bi-functional psoralens, to UV at 254 nm (UVC), and to nitrosoguanidine, was found to complement the photoactivated psoralen-sensitivity phenotype conferred by the pso1- pso7 mutations and was therefore named pso8-1. A constructed pso8-1 rad4-4 double mutant was super-sensitive to UVC, thus indicating a synergistic interaction of the two mutant alleles. Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6. While a pso8-1 mutant had low mutagen-induced mutability, homoallelic diploids showed nearly wild-type sporulation. Sequence analysis of the mutant allele showed pso8-1 to contain a novel, hitherto undescribed T-->C transition in nucleotide position 191, leading to a substitution by leucine of a highly conserved proline at position 64, Rad6-[P64L], which may have severe consequences for the tertiary structure (and hence binding to Rad18p) of the mutant protein.
Assuntos
Furocumarinas/farmacologia , Genes Fúngicos , Mutação de Sentido Incorreto , Fármacos Fotossensibilizantes/farmacologia , Saccharomyces cerevisiae/genética , Raios Ultravioleta , Alelos , Sequência de Aminoácidos , Sequência Conservada , Metilnitronitrosoguanidina/farmacologia , Dados de Sequência Molecular , Mutagênicos/farmacologia , Fenótipo , Fotoquímica , Filogenia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
OBJECTIVE: To determine whether initial effects on metabolic control and quality of life associated with a behavioral intervention combined with intensive diabetes management (IDM) can be sustained over 1 year in youth implementing intensive therapy regimens. STUDY DESIGN: Seventy-seven patients (43 females, 95% white) 12 to 20 years (mean = 14.2 +/- 1.9; duration, 8.7 +/- 3.9) electing to initiate IDM were randomly assigned to one of two groups: with or without coping skills training (CST), which consists of 6 small group sessions and monthly follow-up to help youth cope with their lives in the context of diabetes management; skills included social problem solving, cognitive behavior modification, and conflict resolution. Data were collected before the intervention and at 3, 6, and 12 months after the intervention by using the Self-Efficacy for Diabetes Scale, Children's Depression Inventory, Issues in Coping with IDDM, and the Diabetes Quality of Life: Youth scales. Clinical data (glycosylated hemoglobin level, height, weight, adverse effects) were collected monthly. RESULTS: The CST and IDM groups were comparable at baseline. CST subjects had lower glycosylated hemoglobin (P =.001) and better diabetes (P =.002) and medical (P =. 04) self-efficacy, and less impact of diabetes on their quality of life (P =.005) than youth receiving IDM alone after 1 year. In males, CST did not affect adverse outcomes of IDM hypoglycemia, diabetic ketoacidosis, and weight gain, but CST decreased the incidence of weight gain (P =.05) and hypoglycemia in females (P =.03). CONCLUSIONS: The addition of behavioral intervention to IDM in adolescence results in improved metabolic control and quality of life over 1 year.
Assuntos
Adaptação Psicológica , Terapia Comportamental , Diabetes Mellitus Tipo 1/terapia , Qualidade de Vida , Adolescente , Adulto , Criança , Diabetes Mellitus Tipo 1/sangue , Feminino , Hemoglobinas Glicadas/análise , Humanos , MasculinoRESUMO
Sequencing of the yeast gene that complemented the sensitivity to the photoactivated monofunctional 3-carbethoxypsoralen of the pso6-1 mutant strain revealed that the ERG3 locus, encoding sterol C-5 desaturase involved in biosynthesis of ergosterol, is allelic to PSO6. Disruption of the ERG3 gene yielded an erg3Delta mutant viable in ergosterol-containing YEPD media with the same pleiotropic mutant phenotype known for pso6-1 and erg3 mutants, including sensitivity to hydrogen peroxide and paraquat. Thus, the erg3/pso6 yeast mutant seems to be more sensitive than the WT to 3-CPs+UVA because of the oxidative damage contributed by this treatment and not because of an impaired repair of the furocoumarin-thymine monoadducts formed in the DNA. We found a significant increase of petites amongst erg3Delta and pso6-1 yeast mutant strains grown in conditions where respiration was mandatory. Mutant pso6-1, with its lowered content of ergosterol, exhibited enhanced synthesis of chitin that was maldistributed and not confined to the bud scars. Chitin overproduction in pso6/erg3 mutants resulted in hypersensitivity to Calcofluor White.
Assuntos
Alelos , Furocumarinas/farmacologia , Genes Fúngicos , Oxirredutases/genética , Saccharomyces cerevisiae/genética , Quitina/análise , Quitina/biossíntese , Clonagem Molecular , Mutação , Estresse Oxidativo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use.
Assuntos
Formaldeído , Engenharia Genética , Plasmídeos , Saccharomyces cerevisiae/genética , Leveduras/genética , Resistência Microbiana a Medicamentos , Vetores GenéticosRESUMO
The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H2O2 and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.
Assuntos
Dano ao DNA , Reparo do DNA/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alelos , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Fúngico/efeitos da radiação , Teste de Complementação Genética , Ligação Genética , Oxirredução , Tolerância a Radiação/genética , Saccharomyces cerevisiae/efeitos da radiação , Raios UltravioletaRESUMO
Ethylene diamine tetra-acetic acid-induced hypocalcemia was used as provocative test of parathyroid reserve in eight normocalcemic patients with thalassemia major (age 8 to 26 years) and five young adult control subjects (age 22 to 35). In response to an intravenous infusion of disodium EDTA (50 mg/kg), serum immunoreactive parathyroid hormone rose by 1.97 +/- 1.93 (SD) microliterEq/ml in the patients, controls showing a rise of 10.6 +/- 3.6 microliterEq/ml (t = 5.46, P less than 0.001). There was no relationship between parathyroid response and total iron burden as measured by serum ferritin- or desferrioxamine-induced urinary iron excretion. Impairment of parathyroid reserve is common in transfused patients with thalassemia major and may serve as a marker of significant iron overload.