RESUMEN
Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple (brdp) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b (Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.
Asunto(s)
Corteza Cerebral/anomalías , Genes Letales , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/patología , Mutación Missense , Tubulina (Proteína)/genética , Animales , Encéfalo/anomalías , Encéfalo/embriología , Encéfalo/metabolismo , Proliferación Celular , Supervivencia Celular , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Clonación Molecular , Evolución Molecular , Femenino , Humanos , Masculino , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Neuronas/fisiología , Fenotipo , Conformación Proteica , Alineación de Secuencia , Tubulina (Proteína)/químicaRESUMEN
BACKGROUND: Mental practice, the cognitive rehearsal of a task without physical movement, is known to enhance performance in sports and music. Investigation of this technique in surgery has been limited to basic operations. The purpose of this study was to develop mental practice scripts, and to assess their effect on advanced laparoscopic skills and surgeon stress levels in a crisis scenario. METHODS: Twenty senior surgical trainees were randomized to either conventional training or mental practice groups, the latter being trained by an expert performance psychologist. Participants' skills were assessed while performing a porcine laparoscopic jejunojejunostomy as part of a crisis scenario in a simulated operating room, using the Objective Structured Assessment of Technical Skill (OSATS) and bariatric OSATS (BOSATS) instruments. Objective and subjective stress parameters were measured, as well as non-technical skills using the Non-Technical Skills for Surgeons rating tool. RESULTS: An improvement in OSATS (P = 0.003) and BOSATS (P = 0.003) scores was seen in the mental practice group compared with the conventional training group. Seven of ten trainees improved their technical performance during the crisis scenario, whereas four of the ten conventionally trained participants deteriorated. Mental imagery ability improved significantly following mental practice training (P = 0.011), but not in the conventional group (P = 0.083). No differences in objective or subjective stress levels or non-technical skills were evident. CONCLUSION: Mental practice improves technical performance for advanced laparoscopic tasks in the simulated operating room, and allows trainees to maintain or improve their performance despite added stress.
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Competencia Clínica/normas , Educación de Postgrado en Medicina/métodos , Laparoscopía/normas , Práctica Psicológica , Especialidades Quirúrgicas/educación , Anastomosis en-Y de Roux/educación , Femenino , Lateralidad Funcional , Humanos , Yeyunostomía/educación , Laparoscopía/educación , MasculinoRESUMEN
The SGS1 gene of yeast encodes a DNA helicase with homology to the human WRN gene. Mutations in WRN result in Werner's syndrome, a disease with symptoms resembling premature aging. Mutation of SGS1 is shown to cause premature aging in yeast mother cells on the basis of a shortened life-span and the aging-induced phenotypes of sterility and redistribution of the Sir3 silencing protein from telomeres to the nucleolus. Further, in old sgs1 cells the nucleolus is enlarged and fragmented-changes that also occur in old wild-type cells. These findings suggest a conserved mechanism of cellular aging that may be related to nucleolar structure.
Asunto(s)
Nucléolo Celular/ultraestructura , Senescencia Celular , ADN Helicasas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , División Celular , Nucléolo Celular/química , Nucléolo Celular/metabolismo , ADN Helicasas/fisiología , Exodesoxirribonucleasas , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/análisis , Genes Fúngicos , Humanos , Mutación , Fenotipo , RecQ Helicasas , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae , Transactivadores/análisis , Síndrome de Werner/genética , Helicasa del Síndrome de WernerRESUMEN
The life cycle of many organisms involves a progressive decline in fitness and fecundity with age, and yeast is no exception. Many theories have been proposed to explain the mortality of yeast cells, including the increase in cell size and accumulation of bud scars on the cell surface. None of these has survived closed scrutiny. However, recent discoveries might have validated one aging model in which the triggering of a molecular aging clock results in the replication and accumulation of a senescence factor that eventually overwhelms old cells.
Asunto(s)
Saccharomyces cerevisiae/citología , ADN de Hongos/metabolismo , ADN Ribosómico/metabolismo , Genes Fúngicos , Modelos Biológicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Polyglutamine (polyQ) diseases, such as Huntington's disease and Machado-Joseph disease (MJD), are caused by gain of toxic function of abnormally expanded polyQ tracts. Here, we show that expanded polyQ of ataxin-3 (Q79C), a gene that causes MJD, stimulates Ku70 acetylation, which in turn dissociates the proapoptotic protein Bax from Ku70, thereby promoting Bax activation and subsequent cell death. The Q79C-induced cell death was significantly blocked by Ku70 or Bax-inhibiting peptides (BIPs) designed from Ku70. Furthermore, expression of SIRT1 deacetylase and the addition of a SIRT1 agonist, resveratrol, reduced Q79C toxicity. In contrast, mimicking acetylation of Ku70 abolished the ability of Ku70 to suppress Q79C toxicity. These results indicate that Bax and Ku70 acetylation play important roles in Q79C-induced cell death, and that BIP may be useful in the development of therapeutics for polyQ diseases.
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Antígenos Nucleares/metabolismo , Citoprotección/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Péptidos/farmacología , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Fragmentación del ADN/efectos de los fármacos , Histona Acetiltransferasas/metabolismo , Humanos , Autoantígeno Ku , Proteínas Mutantes/metabolismo , Péptidos/toxicidad , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Resveratrol , Estilbenos/farmacología , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
STUDY QUESTION: Can maternal and offspring high-fat diet (HFD)-induced changes in mRNA expression levels in mice be ameliorated by interventions in female offspring? SUMMARY ANSWER: Our results indicate that exercise and nicotinamide mononucleotide (NMN) can ameliorate the negative effects of maternal and post-weaning HFD in female offspring. WHAT IS KNOWN ALREADY: Maternal and post-weaning HFD can perturb offspring developmental trajectories. As rates of maternal obesity are rising globally, there is a need for effective treatments in offspring to ameliorate the negative effects from a maternal obesogenic environment. Modulation of the nicotinamide adenine dinucleotide (NAD+) pathway by exercise and the NAD+ precursor NMN has previously been shown to reduce the effects of obesity. STUDY DESIGN SIZE DURATION: This study consisted of a multigenerational study using C57Bl6 mice. Mice were fed a control (chow) or HFD ad libitum throughout mating, pregnancy and lactation (n = 13-25). Female offspring (n = 72) were then also supplied either a chow or HFD post-weaning. At 9 weeks of age offspring from HFD dams were subjected to exercise on a treadmill for 9 weeks or at 16 weeks of age administered NMN (i.p.) for 2.5 weeks. At 18.5 weeks mice were euthanized and ovaries and cumulus-oocyte complexes (COC) were collected to examine the possibility of ameliorating the negative effects of maternal and post-weaning HFD. PARTICIPANTS/MATERIALS SETTING METHODS: Ovary and COC mRNA expression was analysed using RT-qPCR. An initial screen of candidate genes was developed to test which molecular pathways may be involved in generating adverse reproductive system effects. For histological analysis, ovarian tissue was fixed in paraformaldehyde and embedded in paraffin and stained with haematoxylin and eosin. The numbers of primordial, primary, secondary and antral follicles were counted. MAIN RESULTS AND THE ROLE OF CHANCE: In the offspring's COC, maternal obesity increased both growth differentiation factor 9 (Gdf9: 2-fold; P < 0.05, HFD versus chow) and bone morphogenetic protein 15 (Bmp15: 4-fold; P < 0.05, HFD versus chow) mRNA expression levels while exercise and NMN interventions did not regulate Gdf9 and Bmp15 in the same manner. In whole ovary, maternal diet programmed a 25-50% reduction in FSH receptor and sirtuin-3 mRNA expression levels in daughter ovaries (P < 0.05, HFD versus chow). There was a significant interaction between HFD and intervention on the proportion of large preantral and preovulatory follicles (P < 0.05). However, the increase in preovulatory follicles did not translate to increased oocyte yield. NMN administration resulted in reduced body weight in HFD-fed individuals. LIMITATIONS REASONS FOR CAUTION: It is unclear if the changes in oocyte mRNA expression levels reported here will impact oocyte quality and fertility in offspring. Offspring ovulation rate or fecundity could not be studied here and fertility trials are required to determine if the changes in gene expression do reduce fertility. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that maternal and offspring HFD perturbs key signalling pathways that are known to regulate fertility in mice, highlighting the importance of interventions in helping to prevent the declining rates of fertility in the context of the current obesity epidemic. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants and fellowships from the National Health and Medical Research Council to R.B.G. (APP1023210, APP1062762, APP1117538) and to M.J.M. and D.A.S. (APP1044295). DAS is a consultant to and inventor on patents licenced to Ovascience, Metrobiotech and GlaxoSmithKline. The other authors declare that there is no conflict of interest.
RESUMEN
Though widely recognized in higher eukaryotes, the regulation of Saccharomyces cerevisiae genes transcribed by RNA polymerase II by proteins that bind within the coding sequence remains largely speculative. We have shown for the LPD1 gene, encoding lipoamide dehydrogenase, that the coding sequence between +13 and +469 activated gene expression of an LPD1::lacZ fusion by up to sixfold in the presence of the upstream promoter. This downstream region, inserted upstream of a promoterless CYC1::lacZ fusion, activated gene expression in a carbon source-dependent manner by a factor of 15 to 111, independent of orientation. Deletion and mutational analysis identified two downstream activation sites (DAS1 and DAS2) and two downstream repressor sites (DRS1 and DRS2) that influence the rate of LPD1 transcription rather than mRNA degradation or translation. Activation from the DAS1 region (positions +137 to +191), encompassing a CDEI-like element, is twofold under derepressive conditions. Activation from DAS2 (+291 to +296), a CRE-like motif, is 12-fold for both repressed and derepressed states. DRS1, a pair of adjacent and opposing ABF1 sites (+288 to +313), is responsible for a 1.3- to 2-fold repression of transcription, depending on the carbon source. DRS1 requires the concerted action of DRS2 (a RAP1 motif at position +406) for repression of transcription only when the gene is induced. Gel mobility shift analysis and in vitro footprinting have shown that proteins bind in vitro to these downstream elements.
Asunto(s)
Dihidrolipoamida Deshidrogenasa/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Secuencia de Bases , Secuencia de Consenso , ADN de Hongos/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Reguladores , Operón Lac , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/metabolismo , Eliminación de Secuencia , Transcripción Genética , beta-Galactosidasa/genéticaRESUMEN
Tumors utilize aerobic glycolysis to support growth and invasion. However, the molecular mechanisms that link metabolism with invasion are not well understood. The nutrient sensor O-linked-ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) modifies intracellular proteins with N-acetylglucosamine. Cancers display elevated O-GlcNAcylation and suppression of O-GlcNAcylation inhibits cancer invasion and metastasis. Here, we show that the regulation of cancer invasion by OGT is dependent on the NAD+-dependent deacetylase SIRT1. Reducing O-GlcNAcylation elevates SIRT1 levels and activity in an AMPK (AMP-activated protein kinase α)-dependent manner. Reduced O-GlcNAcylation in cancer cells leads to SIRT1-mediated proteasomal degradation of oncogenic transcription factor FOXM1 in an MEK/ERK-dependent manner. SIRT1 is critical for OGT-mediated regulation of FOXM1 ubiquitination and reducing SIRT1 activity reverses OGT-mediated regulation of FOXM1. Moreover, we show that SIRT1 levels are required for OGT-mediated regulation of invasion and metastasis in breast cancer cells. Thus, O-GlcNAcylation is a central component linking metabolism to invasion and metastasis via an SIRT1/ERK/FOXM1 axis.
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Neoplasias de la Mama/metabolismo , Proteína Forkhead Box M1/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Sirtuina 1/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Proteína Forkhead Box M1/genética , Glicosilación , Xenoinjertos , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Metástasis de la Neoplasia , Sirtuina 1/genéticaRESUMEN
The NAD(+)-dependent deacetylase SIRT1 plays key roles in numerous cellular processes including DNA repair, gene transcription, cell differentiation, and metabolism. Overexpression of SIRT1 protects against a number of age-related diseases including diabetes, cancer, and Alzheimer's disease. Moreover, overexpression of SIRT1 in the murine brain extends lifespan. A number of small-molecule sirtuin-activating compounds (STACs) that increase SIRT1 activity in vitro and in cells have been developed. While the mechanism for how these compounds act on SIRT1 was once controversial, it is becoming increasingly clear that they directly interact with SIRT1 and enhance its activity through an allosteric mechanism. Here, we present detailed chemical syntheses for four STACs, each from a distinct structural class. Also, we provide a general protocol for purifying active SIRT1 enzyme and outline two complementary enzymatic assays for characterizing the effects of STACs and similar compounds on SIRT1 activity.
Asunto(s)
Activadores de Enzimas/química , Activadores de Enzimas/farmacología , Pruebas de Enzimas/métodos , Sirtuina 1/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/síntesis química , Humanos , Bibliotecas de Moléculas Pequeñas/síntesis químicaRESUMEN
Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.
Asunto(s)
Drosophila melanogaster/genética , Variación Genética , Intrones , Familia de Multigenes , ARN de Transferencia de Serina/genética , Recombinación Genética , Animales , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , Cruzamientos Genéticos , Diploidia , Femenino , Masculino , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sondas de Oligonucleótidos , Cromosoma XRESUMEN
The present study consists of an investigation of P-induced male recombination in Drosophila melanogaster from a number of perspectives. In an initial set of experiments, male recombination induced by several different P strains was examined on both major autosomes. The ability of these P strains to evoke recombination is striking; in many cases it exceeded that of radiation treatment. Also of interest is the apparent nonrandom chromosomal distribution of P-exchange breakpoints. The data suggest that both recombinagenic capacity and distribution pattern of exchange breakpoints may be P-strain specific. In addition to these findings, we have confirmed previous indications that P-induced exchange is reasonably symmetrical and that it frequently occurs during premeiotic stages of spermatogenesis. Moreover, we have established that radiation and P background act additively with regard to the induction of male recombination. The second part of the work involved an analysis of heterochromatic vs. euchromatic recombination induced by several recombinagenically potent P strains. Results of these experiments have confirmed our earlier findings concerning the recombinagenic capacity of p strains. More importantly, it would appear that P-induced exchange in heterochromatin is rare. The induction of various kinds of mutations was also monitored in several of these experiments. The results indicate that the mutagenic potential of the P strains is substantial and of particular interest, that certain types of mutations are P-strain specific. For example, rare heterochromatic lesions were recovered exclusively in the experiment using the h12 strain, whereas a novel pleiotropic mutation occurred at a high frequency only in the T-007 experiment. Our findings are discussed within the context of a model of P-induced exchange.
Asunto(s)
Drosophila melanogaster/genética , Recombinación Genética , Animales , Mapeo Cromosómico , Cromosomas/efectos de la radiación , Femenino , Heterocromatina , Masculino , MutaciónRESUMEN
Saccharomyces cerevisiae can grow on glycine as sole nitrogen source and can convert glycine to serine via the reaction catalyzed by the glycine decarboxylase multienzyme complex (GDC). Yeast strains with mutations in the single gene for lipoamide dehydrogenase (lpd1) lack GDC activity, as well as the other three 2-oxoacid dehydrogenases dependent on this enzyme. The LPD1 gene product is also required for cells to utilize glycine as sole nitrogen source. The effect of mutations in LPD1 (L-subunit of GDC), SER1 (synthesis of serine from 3-phosphoglycerate), ADE3 (cytoplasmic synthesis of one-carbon units for the serine synthesis from glycine), and all combinations of each has been determined. The results were used to devise methods for isolating mutants affected either in the generation of one-carbon units from glycine (via GDC) or subsequent steps in serine biosynthesis. The mutants fell into six complementation groups (gsd1-6 for defects in conversion of glycine to serine). Representatives from three complementation groups were also unable to grow on glycine as sole nitrogen source (gsd1-3). Assays of the rate of glycine uptake and decarboxylation have provided insights into the nature of the mutations.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Dihidrolipoamida Deshidrogenasa/genética , Proteínas Fúngicas/genética , Glicina/metabolismo , Complejos Multienzimáticos/genética , Nitrógeno/metabolismo , Saccharomyces cerevisiae/genética , Serina/biosíntesis , Aminoácido Oxidorreductasas/metabolismo , Clonación Molecular , Dihidrolipoamida Deshidrogenasa/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Prueba de Complementación Genética , Glicina-Deshidrogenasa (Descarboxilante) , Complejos Multienzimáticos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiaeRESUMEN
A temperature-sensitive (ts) third chromosome Minute (M) mutation, designated Q-III, has been recovered and characterized. Q-III heterozygotes raised at 29 degrees exhibit all of the dominant traits of M mutants including small bristles, rough eyes, prolonged development, reduced viability and interactions with several unrelated mutations. Q-III homozygotes raised at 29 degrees are lethal; death occurs primarily during the first larval instar. When raised at 22 degrees, Q-III heterozygotes are phenotypically normal and Q-III homozygotes display moderate M traits. In addition, Q-III elicits ts sterility and maternal-effect lethality. As is true of M lesions, the dominant traits of Q-III are not expressed in triploid females raised at 29 degrees. Complementation tests suggest that Q-III is a ts allele of M(3)LS4, which is located in 3L near the centromere.--Reciprocal temperature-shift experiments revealed that the temperature-sensitive period (TSP) of Q-III lethality is polyphasic, extending from the first instar to the latter half of pupation. Heat-pulse experiments further resolved this into two post-embryonic TSPs: one occurring during the latter half of the second larval instar, and the other extending from the larval/pupal boundary to the second half of pupation. In addition, heat pulses elicited a large number of striking adult phenotypes in Q-III individuals. These included pattern alterations such as deficiencies and duplications and other morphological defects in structures produced by the eye-antennal, leg, wing and genital imaginal discs and the abdominal histoblasts. Each defect or pattern alteration is associated with a specific TSP during development.--We favor the interpretation that most of the major Q-III defects, particularly the structural duplications and deficiencies, result from temperature-induced cell death in mitotically active imaginal anlagen, while the small macrochaete phene probably results from the direct effects of Q-III on bristle synthesis. The hypothesis that the Q-III locus specifies a component required for protein synthesis is discussed, and it is concluded that this hypothesis can account for the pleiotropy of Q-III, and that perhaps it can be extended to M loci in general.
Asunto(s)
Drosophila melanogaster/genética , Genes Letales , Mutación , Temperatura , Animales , Cromosomas/ultraestructura , Cruzamientos Genéticos , Femenino , Heterocigoto , Masculino , FenotipoRESUMEN
Temperature-sensitive (ts) autonomous cell-lethal mutations have been used extensively to study important developmental phenomena, such as pattern formation, in Drosophila. Their utility would be enhanced considerably if it were possible to establish which cell type is primarily affected by each lesion. To facilitate such an approach we have isolated and characterized 21 EMS- induced X-linked adult-lethal (adl) mutants, 16 of which are ts. Most of these lesions also elicit ts lethal effects during preimaginal development. They represent 19 different loci distributed randomly along the X chromosome. The general properties of these mutations are described. In addition, results of an in-depth analysis (focus mapping and, in some cases, temperature shift and heat-pulse studies) of four strains, adl-1(ts) (1), sesE, adl-2(ts) ( 1) and rex are reported. Two major temperature-sensitive periods (TSPs) of adl-1 lethality were resolved: one during the second half of embryogenesis and the other coinciding with pupariation. Mosaic analysis revealed separate mesodermal foci for leg paralysis. Developmental analysis of adl-1 embryos suggest that the adl-1 product may be required for maintenance of muscle tissue. Two discrete TSPs of sesE lethality exist: one during the second instar and the other extending from late third instar to early pupation. Mosaic analysis of sesE lethality resolved a pair of neural foci, each of which appears to incorporate three separate foci for leg paralysis. Mosaic analysis of adl-2(ts) ( 1) revealed the existence of paired lethal foci that appear to map to the vicinity of the subesophageal ganglion. Analysis of rex mosaics resolved separate mesodermal foci for leg paralysis.
RESUMEN
The histone genes of wild-type Drosophila melanogaster are reiterated 100-150 times per haploid genome and are located in the segment of chromosome 2 that corresponds to polytene bands 39D2-3 to E1-2. The influence of altered histone gene multiplicity on chromatin structure has been assayed by measuring modification of the gene inactivation associated with position effect variegation in genotypes bearing deletions of the 39D-E segment. The proportion of cells in which a variegating gene is active is increased in genotypes that are heterozygous for a deficiency that removes the histone gene complex. Deletions that remove segments adjacent to the histone gene complex have no effect on the expression of variegating genes. Suppression of position effect variegation associated with reduction of histone gene multiplicity applies to both X-linked and autosomal variegating genes. Position effects exerted by both autosomal and sex-chromosome heterochromatin were suppressible by deletions of the histone gene complex. The suppression was independent of the presence of the Y chromosome. A deficiency that deletes only the distal portion of the histone gene complex also has the ability to suppress position effect variegation. Duplication of the histone gene complex did not enhance position effect variegation. Deletion or duplication of the histone gene complex in the maternal genome had no effect on the extent of variegation in progeny whose histone gene multiplicity was normal. These results are discussed with respect to current knowledge of the organization of the histone gene complex and control of its expression.
RESUMEN
Position effect variegation (PEV) results from the juxtaposition of a euchromatic gene to heterochromatin. In its new position the gene is inactivated in some cells and not in others. This mosaic expression is consistent with variability in the spread of heterochromatin from cell to cell. As many components of heterochromatin are likely to be produced in limited amounts, the spread of heterochromatin into a normally euchromatic region should be accompanied by a concomitant loss or redistribution of the protein components from other heterochromatic regions. We have shown that this is the case by simultaneously monitoring variegation of a euchromatic and a heterochromatic gene associated with a single chromosome rearrangement. Secondly, if several heterochromatic regions of the genome share limited components of heterochromatin, then some variegating rearrangements should compete for these components. We have examined this hypothesis by testing files with combinations of two or more different variegating rearrangements. Of the nine combinations of pairs of variegating rearrangements we studied, seven showed nonreciprocal interactions. These results imply that many components of heterochromatin are both shared and present in limited amounts and that they can transfer between chromosomal sites. Consequently, even nonvariegation portions of the genome will be disrupted by re-allocation of heterochromatic proteins associated with PEV. These results have implications for models of PEV.
Asunto(s)
Drosophila melanogaster/genética , Regulación de la Expresión Génica , Reordenamiento Génico , Heterocromatina/genética , Animales , Cromosomas/genética , Cromosomas/ultraestructura , Cruzamientos Genéticos , Femenino , Masculino , Modelos GenéticosRESUMEN
Genomic imprinting is a phenomenon in which the expression of a gene or chromosomal region depends on the sex of the individual transmitting it. The term imprinting was first coined to describe parent-specific chromosome behavior in the dipteran insect Sciara and has since been described in many organisms, including other insects, plants, fish, and mammals. In this article we describe a mini-X chromosome in Drosophila melanogaster that shows genomic imprinting of at least three closely linked genes. The imprinting of these genes is observed as mosaic silencing when the genes are transmitted by the male parent, in contrast to essentially wild-type expression when the same genes are maternally transmitted. We show that the imprint is due to the sex of the parent rather than to a conventional maternal effect, differential mitotic instability of the mini-X chromosome, or an allele-specific effect. Finally, we have examined the effects of classical modifiers of position-effect variegation on the maintenance and the establishment of the imprint. Factors that modify position-effect variegation alter the somatic expression but not the establishment of the imprint. This suggests that chromatin structure is important in maintenance of the imprint, but a separate mechanism may be responsible for its initiation.
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Drosophila melanogaster/genética , Impresión Genómica , Alelos , Animales , Cromosomas/genética , Cruzamientos Genéticos , Drosophila melanogaster/anatomía & histología , Femenino , Expresión Génica , Genes de Insecto , Masculino , Fenotipo , Caracteres Sexuales , TemperaturaRESUMEN
Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.
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Drosophila melanogaster/genética , Regulación de la Expresión Génica/genética , Genes Homeobox/genética , Alelos , Animales , Mapeo Cromosómico , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/embriología , Mutación , FenotipoRESUMEN
The Polycomb (Pc) group genes of Drosophila are negative regulators of homeotic genes, but individual loci have pleiotropic phenotypes. It has been suggested that Pc group genes might form a regulatory hierarchy, or might be members of a multimeric complex that obeys the law of mass action. Recently, it was shown that polyhomeotic (ph) immunoprecipitates in a multimeric complex that includes Pc. Here, we show that duplications of ph suppress homeotic transformations of Pc and Pcl, supporting a mass-action model for Pc group function. We crossed ph alleles to all members of the Polycomb group, and to E(Pc) and Su(z)2 to look for synergistic effects. We observed extragenic noncomplementation between ph503 and Pc, Psc1 and Su(z)2(1) in females, and between ph409 and Sce1, ScmD1 and E(z)1 mutations in males, suggesting that these gene products might interact directly with ph. Males hemizygous for a temperature-sensitive allele, ph2, are lethal when heterozygous with mutants in Asx, Pc, Pcl, Psc, Sce and Scm, and with E(Pc) and Su(z)2. Mutations in trithorax group genes were not able to suppress the lethality of ph2/Y; Psc1/+ males. ph2 was not lethal with extra sex combs, E(z), super sex combs (sxc) or l(4)102EFc heterozygotes, but did cause earlier lethality in embryos homozygous for E(z), sxc and l(4)102EFc. However, ph503 did not enhance homeotic phenotypes of esc heterozygotes derived from homozygous esc- mothers. We examined the embryonic phenotypes of ph2 embryos that were lethal when heterozygous or homozygous for other mutations. Based on this phenotypic analysis, we suggest that ph may perform different functions in conjunction with differing subsets of Pc group genes.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes Homeobox , Genes de Insecto , Familia de Multigenes , Nucleoproteínas/fisiología , Proteínas/fisiología , Alelos , Animales , Proteínas de Unión al ADN/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/ultraestructura , Embrión no Mamífero/ultraestructura , Desarrollo Embrionario , Femenino , Dosificación de Gen , Genes Letales , N-Metiltransferasa de Histona-Lisina , Sustancias Macromoleculares , Masculino , Modelos Genéticos , Nucleoproteínas/genética , Fenotipo , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas/genéticaRESUMEN
Cytogenetic region 31 of the second chromosome of Drosophila melanogaster was screened for recessive lethal mutations. One hundred and thirty nine new recessive lethal alleles were isolated that fail to complement Df(2L)J2 (31A-32A). These new alleles, combined with preexisting mutations in the region, define 52 complementation groups, 35 of which have not previously been described. Among the new mutations were alleles of the cdc2 and mfs(2)31 genes. Six new deficiencies were also isolated and characterized identifying 16 deficiency subintervals within region 31. The new deficiencies were used to further localize three loci believed to encode non-histone chromosomal proteins. Suvar(2)1/Su(var)214, a dominant suppressor of position-effect variegation (PEV), maps to 31A-B, while the recessive suppressors of PEV mfs(2)31 and wdl were localized to regions 31E and 31F-32A, respectively. In addition, the cytological position of several mutations that interact with heterochromatin were more precisely defined.