RESUMEN
We report on the first measurement of the fission barrier height in a heavy shell-stabilized nucleus. The fission barrier height of 254No is measured to be Bf=6.0±0.5 MeV at spin 15â and, by extrapolation, Bf=6.6±0.9 MeV at spin 0â. This information is deduced from the measured distribution of entry points in the excitation energy versus spin plane. The same measurement is performed for 220Th and only a lower limit of the fission barrier height can be determined: Bf(I)>8 MeV. Comparisons with theoretical fission barriers test theories that predict properties of superheavy elements.
RESUMEN
In mammalian cells, extracellular signals can regulate the delivery of particular proteins to the plasma membrane. We have discovered a novel example of regulated protein sorting in the late secretory pathway of Saccharomyces cerevisiae. In yeast cells grown on either ammonia or urea medium, the general amino acid permease (Gap1p) is transported from the Golgi complex to the plasma membrane, whereas, in cells grown on glutamate medium, Gap1p is transported from the Golgi to the vacuole. We have also found that sorting of Gap1p in the Golgi is controlled by SEC13, a gene previously shown to encode a component of the COPII vesicle coat. In sec13 mutants grown on ammonia, Gap1p is transported from the Golgi to the vacuole, instead of to the plasma membrane. Deletion of PEP12, a gene required for vesicular transport from the Golgi to the prevacuolar compartment, counteracts the effect of the sec13 mutation and partially restores Gap1p transport to the plasma membrane. Together, these studies demonstrate that both a nitrogen-sensing mechanism and Sec13p control Gap1p transport from the Golgi to the plasma membrane.
Asunto(s)
Membrana Celular/fisiología , Proteínas de la Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Saccharomyces cerevisiae/fisiología , Sistemas de Transporte de Aminoácidos , Transporte Biológico , Proteínas Fúngicas/fisiología , Microscopía Fluorescente , Saccharomyces cerevisiae/ultraestructuraRESUMEN
The SEC13 gene was originally identified by temperature-sensitive mutations that block all protein transport from the ER to the Golgi. We have found that at a permissive temperature for growth, the sec13-1 mutation selectively blocks transport of the nitrogen-regulated amino acid permease, Gap1p, from the Golgi to the plasma membrane, but does not affect the activity of constitutive permeases such as Hip1p, Can1p, or Lyp1p. Different alleles of SEC13 exhibit different relative effects on protein transport from the ER to the Golgi, or on Gap1p activity, indicating distinct requirements for SEC13 function at two different steps in the secretory pathway. Three new genes, LST4, LST7, and LST8, were identified that are also required for amino acid permease transport from the Golgi to the cell surface. Mutations in LST4 and LST7 reduce the activity of the nitrogen-regulated permeases Gap1p and Put4p, whereas mutations in LST8 impair the activities of a broader set of amino acid permeases. The LST8 gene encodes a protein composed of WD-repeats and has a close human homologue. The LST7 gene encodes a novel protein. Together, these data indicate that SEC13, LST4, LST7, and LST8 function in the regulated delivery of Gap1p to the cell surface, perhaps as components of a post-Golgi secretory-vesicle coat.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Transporte Biológico , Liasas de Carbono-Oxígeno/genética , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Activación Enzimática , Proteínas Fúngicas/genética , Genes Fúngicos , Genes Letales , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis , Proteínas de Complejo Poro Nuclear , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/genéticaRESUMEN
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. Once released, it is removed from the extracellular space by cellular uptake catalyzed by GABA transporter proteins. Four GABA transporters (GAT1, GAT2, GAT3 and BGT1) have been identified. Inhibition of the GAT1 by the clinically available anti-epileptic drug tiagabine has been an effective strategy for the treatment of some patients with partial seizures. Recently, the investigational drug EF1502, which inhibits both GAT1 and BGT1, was found to exert an anti-convulsant action synergistic to that of tiagabine, supposedly due to inhibition of BGT1. The present study addresses the role of BGT1 in seizure control and the effect of EF1502 by developing and exploring a new mouse line lacking exons 3-5 of the BGT1 (slc6a12) gene. The deletion of this sequence abolishes the expression of BGT1 mRNA. However, homozygous BGT1-deficient mice have normal development and show seizure susceptibility indistinguishable from that in wild-type mice in a variety of seizure threshold models including: corneal kindling, the minimal clonic and minimal tonic extension seizure threshold tests, the 6Hz seizure threshold test, and the i.v. pentylenetetrazol threshold test. We confirm that BGT1 mRNA is present in the brain, but find that the levels are several hundred times lower than those of GAT1 mRNA; possibly explaining the apparent lack of phenotype. In conclusion, the present results do not support a role for BGT1 in the control of seizure susceptibility and cannot provide a mechanistic understanding of the synergism that has been previously reported with tiagabine and EF1502.
Asunto(s)
Proteínas Transportadoras de GABA en la Membrana Plasmática/deficiencia , Convulsiones/genética , Animales , Anticonvulsivantes/uso terapéutico , Convulsivantes/toxicidad , Cruzamientos Genéticos , Relación Dosis-Respuesta a Droga , Electrochoque/efectos adversos , Exones/genética , Femenino , Proteínas Transportadoras de GABA en la Membrana Plasmática/efectos de los fármacos , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/fisiología , Isoxazoles/uso terapéutico , Excitación Neurológica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácidos Nipecóticos/uso terapéutico , Pentilenotetrazol/toxicidad , ARN Mensajero/biosíntesis , Convulsiones/inducido químicamente , Convulsiones/etiología , Convulsiones/prevención & control , TiagabinaAsunto(s)
Células de la Médula Ósea , Trasplante de Médula Ósea , Leucemia Linfoide/terapia , Leucemia Mieloide Aguda/terapia , Adolescente , Adulto , Suero Antilinfocítico , Niño , Isótopos de Cobalto , Ciclofosfamida/uso terapéutico , Pruebas Inmunológicas de Citotoxicidad , Femenino , Reacción Injerto-Huésped/efectos de los fármacos , Prueba de Histocompatibilidad , Humanos , Prueba de Cultivo Mixto de Linfocitos , Masculino , Metotrexato/uso terapéutico , Persona de Mediana Edad , Radiación , Timo/inmunología , Trasplante HomólogoRESUMEN
The human coronary sinus is an important part of the cardiac venous system that serves as an anatomic landmark as well as a conduit for diagnostic and therapeutic procedures. We studied 50 human coronary sinuses of hearts of normal and increased cardiac weight in order to ascertain various functional anatomic features of the coronary sinus and differences between hearts of normal and increased weight. In the hearts of normal weight (195 to 300 gm) the Thebesian valve covered the ostium of the coronary sinus an average of 41%, with complete coverage in 20%. The valve of Vieussens covered the origin of the coronary sinus an average of 59%. The average volume of the coronary sinus was 1.26 +/- 0.45 cc. In hearts of increased weight (365 to 675 gm), the Thebesian valve covered the ostium an average of 26% and the valve of Vieussens covered the origin of the coronary sinus an average of 56.5%. The average volume of the coronary sinus was increased to 1.76 +/- 0.73 cc (p less than 0.005). Thus knowledge of these functional anatomic features and the differences in hearts of increased cardiac weight allows for better utilization of the human coronary sinus for diagnostic and therapeutic purposes.
Asunto(s)
Vasos Coronarios/anatomía & histología , Adulto , Cardiomegalia/patología , Femenino , Corazón/anatomía & histología , Humanos , Masculino , Tamaño de los ÓrganosRESUMEN
NMT1 is an essential Saccharomyces cerevisiae gene which encodes myristoyl-CoA:protein N-myristoyltransferase (Nmt1p). Nmt1p transfers myristate (C14:0), from myristoyl-CoA to the amino-terminal Gly residue of several essential cellular proteins. Little information is available about how myristoyl-CoA metabolism is regulated in eukaryotic cells. We have isolated and characterized three unlinked Fatty Acid Activation genes from S. cerevisiae, FAA1, FAA2, and FAA3. In vitro biochemical assays reveal that the myristoyl-CoA synthetase activity of purified Faa2p is approximately equal to that of Faa1p, and two orders of magnitude greater than that of Faa3p. Analysis of NMT1 strains containing faa1, faa2, and/or faa3 null alleles indicates that Faa1p, Faa2p, and Faa3p are not essential for vegetative growth when de novo acyl-CoA synthesis by fatty acid synthetase (Fas) is active. S. cerevisiae strains containing nmt1-181 exhibit temperature-sensitive growth arrest and myristic acid auxotrophy due to the reduced affinity of its mutant protein product (nmtGly451-->Asp) for myristoyl-CoA. Comparison of the growth characteristics of isogenic NMT1 and nmt1-181 strains with all possible combinations of faa1, faa2, and faa3 null alleles, in the presence or absence of an active Fas complex, indicates that (i) Faa1p is responsible for activation of imported fatty acids to their CoA derivatives; (ii) Faa2p and Faa3p are able to access endogenous but not imported fatty acid substrates; (iii) nmt181p requires myristoyl-CoA production from both Fas and Faas for cells to remain viable at nonpermissive temperatures; (iv) Faa2p is unique among the three Faas in its ability, when overproduced, to partially rescue growth of a nmt1-181 strain at nonpermissive temperatures on yeast/peptone/dextrose (YPD) media without C14:0 supplementation; (v) acyl-CoAs produced by Faa1p, Faa2p, or Faa3p are not specifically targeted for beta-oxidation; and (vi) the ability of NMT1, faa1 delta, faa2 delta, faa3 delta strains to remain viable in the absence of an active Fas complex on YPD plus C14:0, or on media that contains fatty acids as the sole carbon source, suggests that S. cerevisiae contains other acyl-CoA synthetases which can activate imported fatty acids.
Asunto(s)
Aciltransferasas/genética , Coenzima A Ligasas/genética , Proteínas Fúngicas/genética , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Coenzima A Ligasas/metabolismo , ADN de Hongos , Ácidos Grasos/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Ligamiento Genético , Datos de Secuencia Molecular , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de AminoácidoRESUMEN
Dimorphic growth of the budding yeast Saccharomyces cerevisiae is regulated by the quality of the nitrogen supply. On a preferred nitrogen source diploid cells grow as ellipsoidal cells by using a bipolar pattern of budding, whereas on a poor nitrogen source a unipolar pattern of budding is adopted, resulting in extended pseudohyphal chains of filamentous cells. Here we report that the quality of the nitrogen source is signaled by the glutamine tRNA isoform with a 5'-CUG anticodon (tRNACUG). Mutations that alter this tRNA impair assessment of the nitrogen supply without measurably affecting protein synthesis, so that mutant cells display pseudohyphal growth even on a preferred nitrogen source. The nitrogen status for other nitrogen-responsive processes such as catabolic gene expression and sporulation also is signaled by this tRNA: mutant cells inappropriately induce the nitrogen-repressed gene CAR1 and undergo precocious sporulation in nitrogen-rich media. Therefore, in addition to its role in mRNA translation, this tRNA also transduces nitrogen signals that regulate development.
Asunto(s)
Nitrógeno/metabolismo , ARN de Transferencia de Glutamina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Esporas Fúngicas , Secuencia de Bases , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Codón , Proteínas Fúngicas/metabolismo , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN de Transferencia de Glutamina/química , ARN de Transferencia de Glutamina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37 degrees C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation.
Asunto(s)
Genes Fúngicos/genética , Proteínas de Choque Térmico/genética , Proteínas de la Membrana/genética , Mitocondrias/metabolismo , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia Conservada , ADN de Hongos/análisis , ADN Mitocondrial/análisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Calor , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mitocondrias/química , Datos de Secuencia Molecular , Desnaturalización Proteica , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Alineación de Secuencia , Análisis de Secuencia de ADN , Esporas FúngicasRESUMEN
The regulatory step Start in the cell cycle of the budding yeast Saccharomyces cerevisiae is inhibited by nalidixic acid (Nal). To study this inhibition, mutations were identified that alter the sensitivity of yeast cells to Nal. Nal-sensitive mutations were sought because the inhibitory effects of Nal on wild-type cells are only transient, and wild-type cells naturally become refractory to Nal. Three complementation groups of Nal-sensitive mutations were found. Mutations in the first complementation group were shown to reside in the ARO7 gene, encoding chorismate mutase; tyrosine and phenylalanine synthesis was inhibited by Nal in these aro7 mutants, whereas wild-type chorismate mutase was unaffected, These aro7 alleles demonstrate 'recruitment', by mutation, of an innately indifferent protein to an inhibitor-sensitive form. The Nal-sensitive aro7 mutant cells were used to show that the resumption of Nal-inhibited nuclear activity and cell proliferation takes place while cytoplasmic Nal persists at concentrations inhibitory for the mutant chorismate mutase. Mutations in the second complementation group, nss2 (Nal-supersensitive), increased intracellular Nal concentrations, and may simply alter the permeability of cells to Nal. The third complementation group was found to be the ERG6 gene, previously suggested to encode the ergosterol biosynthetic enzyme sterol methyltransferase. Mutation or deletion of the ERG6 gene had little effect on the inhibition of Start by Nal, but prevented recovery from this inhibition. Mutation of ERG3, encoding another ergosterol biosynthetic enzyme, also caused Nal sensitivity, suggesting that plasma membrane sterol composition, and plasma membrane function, mediates recovery from Nal-mediated inhibition of Start.
Asunto(s)
Ácido Nalidíxico/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Corismato Mutasa/metabolismo , Metiltransferasas/genética , Mutación , Saccharomyces cerevisiae/genéticaRESUMEN
Large-scale systematic sequencing has generally depended on the availability of an ordered library of large-insert bacterial or viral genomic clones for the organism under study. The generation of these large insert libraries, and the location of each clone on a genome map, is a laborious and time-consuming process. In an effort to overcome these problems, several groups have successfully demonstrated the viability of the whole-genome random 'shotgun' method in large-scale sequencing of both viruses and prokaryotes. Here we report the sequence of Saccharomyces cerevisiae chromosome IX, determined in part by a whole-chromosome 'shotgun', and describe the particular difficulties encountered in the random 'shotgun' sequencing of an entire eukaryotic chromosome. Analysis of this sequence shows that chromosome IX contains 221 open reading frames (ORFs), of which approximately 30% have been sequenced previously. This chromosome shows features typical of a small Saccharomyces cerevisiae chromosome.