RESUMEN
Saccharomyces cerevisiae mutants deficient in folate synthesis have been constructed and employed to study the utilisation of exogenous folates in yeast. One mutant specifically lacked dihydropteroate synthase while the second lacked dihydrofolate synthase. Exogenous folinic acid restored optimal growth to both strains. Folic acid did not generally rescue growth but spontaneous isolates capable of utilising folic acid were selected. The folic acid synthesis pathway in the folate utilising isolates was restored via transformation with FOL1 or FOL3 expression plasmids and transformants were tested for resistance to sulfamethoxazole (SMX). The presence of elevated levels of folic acid led to greatly reduced SMX sensitivity regardless of whether strains were folate utilisers or not.
Asunto(s)
Antiinfecciosos/farmacología , Farmacorresistencia Fúngica , Ácido Fólico/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Sulfametoxazol/farmacología , Pruebas de Sensibilidad Microbiana , Mutación , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Sulfa drugs have been used as antimicrobials for decades but resistance is now a problem. For major eukaryotic pathogens, including Plasmodium and Pneumocystis, sulfa drug testing is difficult or impossible. We have shown that the eukaryote yeast can be used as a model for the study of sulfa drugs within certain parameters. Fifteen sulfa drugs inhibited yeast growth in a manner indicating competition with p-aminobenzoate (pABA). Such competition resulted from direct addition of pABA or through increased expression of the pABA synthase gene (ABZ1). The model system predicts that overexpression of the pABA synthase gene can lead to drug resistance.
Asunto(s)
Ácido 4-Aminobenzoico/farmacología , Antiinfecciosos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonas/farmacología , Clonación Molecular , Prueba de Complementación Genética , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death. Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities. Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important. Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects. In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated.
Asunto(s)
División Celular , Productos del Gen vpr/fisiología , VIH-1/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Muerte Celular , Clonación Molecular , ADN Viral/análisis , Escherichia coli , Productos del Gen vpr/química , Productos del Gen vpr/genética , Glicina/fisiología , VIH-1/patogenicidad , Histidina/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.
Asunto(s)
Membrana Celular/metabolismo , Productos del Gen nef/metabolismo , Ácidos Mirísticos/metabolismo , Membrana Celular/ultraestructura , Núcleo Celular , Centrifugación , Cromatografía en Gel , Productos del Gen nef/genética , Cuerpos de Inclusión/ultraestructura , Microscopía Inmunoelectrónica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , TransfecciónRESUMEN
The biological effects of the HIV-1 accessory protein, Vpr, have been studied in yeast expression systems. In our previous study [1], employing the pCUP1-vpr copper-inducible expression cassette, Vpr was shown to cause growth arrest and structural defects. In this study yeast constitutively expressing vpr, through elevated copy number and/or elevated transcription levels, displayed no growth arrest in fermentative growth conditions while Vpr was produced at much lower levels than in the inducible expression system. However, such cells were respiratory deficient and unable to utilise ethanol or glycerol as the sole carbon source. They exhibited gross mitochondrial dysfunction displayed in the loss of respiratory chain complex I, II, III, IV and citrate synthase activities. The effects on mitochondria required a C-terminal domain of Vpr that contains a conserved amino acid sequence motif HFRIGCRHSRIG. These results suggest that the widely observed phenomenon of 'Vpr-induced growth arrest' in human cells could be due to mitochondrial dysfunction.
Asunto(s)
Productos del Gen vpr/fisiología , VIH-1 , Mitocondrias/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Productos del Gen vpr/biosíntesis , Productos del Gen vpr/genética , Glutatión Transferasa/genética , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Saccharomyces cerevisiae/metabolismo , Transfección , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
We have produced human immunodeficiency virus type 1 (HIV-1) Nef (a myristylated 206-amino-acid protein) in Saccharomyces cerevisaie and shown that, while Nef is normally found as a predominantly intracellular protein, amounts up to 40 micrograms/ml of Nef are also released into the extracellular medium during stress. By electrophoretic (SDS-PAGE) analysis the extracellular Nef is indistinguishable from intracellular Nef. Conditions of stress that lead to the release of Nef include elevated levels of copper or magnesium ions or growth at elevated temperatures. This release appears to be dependent upon the N-terminal sequences of Nef, including the presence of a myristylation site. Our observations concerning Nef release in yeast suggest new ways in which the behaviour of Nef should be examined in order to gain further insights into the development of AIDS. If the release of Nef is important in the development of AIDS, our work reveals that Nef-associated symptoms may be reduced or delayed by reducing stresses, such as fevers.
Asunto(s)
Productos del Gen nef/metabolismo , VIH-1/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cobre/farmacología , Productos del Gen nef/química , Calor , Magnesio/farmacología , Datos de Secuencia Molecular , Ácido Mirístico , Ácidos Mirísticos/metabolismo , Procesamiento Proteico-Postraduccional , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Vpr is a virion-associated protein of human immunodeficiency type 1 (HIV-1) whose function in acquired immunodeficiency syndrome (AIDS) has been uncertain. Employing the yeast Saccharomyces cerevisiae as a model to examine the effects of HIV-1 auxiliary proteins on basic cellular functions, we found that the vpr gene caused cell growth arrest and structural defects indicated by osmotic sensitivity and gross cell enlargement. Production of various domains by gene expression showed that this effect arose from within the carboxyl-terminal third of the Vpr protein and implicated the sequence HFRIGCRHSRIG, containing two H(S/F)RIG motifs. Electroporation with a series of peptides containing these motifs caused structural defects in yeast that resulted in osmotic sensitivity. A protein with functions relating to the yeast cytoskeleton, Sac1p [Cleves, A. E., Novick, P.J. & Bankaitis, V.A. (1989) J. Cell Biol. 109, 2939-2950], shows sequence similarity to Vpr, and Vpr's effect in yeast may be to disrupt normal Sac1p functions. The Sac1p equivalent has not yet been described in mammalian cells, but in rhabdomyosarcoma and osteosarcoma cell lines Vpr also caused gross cell enlargement and replication arrest [Levy, D.N., Fernandes, L.S., Williams, W.V. & Weiner, D.B. (1993) Cell 72, 541-550]. We note that there is a correlation between the region containing the H(S/F)RIG motifs and the pathogenicity of primate lentiviruses and we suggest that the function of Vpr may be to bring about cell growth arrest and/or cytoskeletal changes as an early step in HIV-1 infection.
Asunto(s)
Productos del Gen vpr/química , Productos del Gen vpr/farmacología , VIH-1/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Síndrome de Inmunodeficiencia Adquirida/virología , Secuencia de Aminoácidos , Secuencia de Bases , División Celular/efectos de los fármacos , Clonación Molecular , Citometría de Flujo , Productos del Gen vpr/biosíntesis , Genes prv , VIH-1/metabolismo , VIH-1/patogenicidad , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/citología , Homología de Secuencia de Aminoácido , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
Influenza NS2 protein was expressed in Saccharomyces cerevisiae using a copper-inducible promoter. The protein produced had a molecular weight of 13 kDa, was reactive with anti-NS2 antiserum and was localised to the yeast cell nucleus. Two-hybrid analysis identified a direct protein-protein interaction between NS2 and the M2 protein of the virus, involving the C-terminal 163 residues of M1. A filter-binding assay localised the M1 binding region to the C-terminal 70 amino acids of NS2.
Asunto(s)
Virus de la Influenza A/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/química , Virus de la Influenza A/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes , Saccharomyces cerevisiae , Proteínas no Estructurales Virales/genéticaRESUMEN
We have examined heterologous protein secretion from Candida glabrata with the aid of a stable C. glabrata vector and a secretion reporter cassette comprising the Saccharomyces cerevisiae PGK-gene promoter and a Kluveromyces Iactis secretion signal to drive secretion of Escherichia coli beta-lactamase. Abundant secretion of beta-lactamase from C. glabrata indicates that the S. cerevisiae PGK promoter functions in C. glabrata. Furthermore, we show that C. glabrata processes the secreted beta-lactamase in a manner similar to, but not identical with, S. cerevisiae and K. lactis. C. glabrata may be a suitable new host for the expression of foreign genes.
Asunto(s)
Candida/genética , Proteínas Recombinantes/genética , Secuencia de Aminoácidos , Secuencia de Bases , Candida/metabolismo , Clonación Molecular , ADN Recombinante , Datos de Secuencia Molecular , Fosfoglicerato Quinasa/genética , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , beta-Lactamasas/metabolismoRESUMEN
We have sought to obtain a convenient system for the high-level production of secreted proteins in yeast. With the aid of a secretion reporter cassette we examined the secretion of beta-lactamase (Bla) as a model protein and found the highest expression in Saccharomyces cerevisiae using a high-copy-number plasmid. We further developed the high-copy-number plasmid introducing a secretion cassette that has a convenient cloning site coinciding with the sequence encoding the KEX2 cleavage site. Large quantities of correctly-processed product can therefore be obtained. We show that 0.3 g/l of correctly processed beta-lactamase can be obtained in fed-batch cultures without the need for selective media or significant loss of the plasmid.
Asunto(s)
Vectores Genéticos , Plásmidos , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/enzimología , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Medios de Cultivo , ADN de Hongos , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , beta-Lactamasas/genéticaRESUMEN
We describe six new yeast episomal vectors which encode glutathione S-transferase (GST) affinity tags. These allow for the production of GST-fusion proteins in Saccharomyces cerevisiae under the control of the CUP1 promoter. Affinity chromatography with glutathione-Sepharose permits convenient purification of the fusion protein from a yeast lysate. The presence of a protease cleavage site facilitates subsequent removal of the GST tag. The expression and single-step purification of both GST and a functional GST-metallothionein fusion from yeast are shown as an example of the application of these vectors.