RESUMO
The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.
Assuntos
Fosfatase Ácida/biossíntese , Repressão Enzimática , Regulação da Expressão Gênica , Saccharomyces cerevisiae/genética , Sulfato de Amônio/farmacologia , Divisão Celular/efeitos dos fármacos , Meios de Cultura , Repressão Enzimática/efeitos dos fármacos , Proteínas Fúngicas/biossíntese , Genes Reguladores , Concentração de Íons de Hidrogênio , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.
Assuntos
Fosfatase Ácida/genética , Saccharomyces cerevisiae/genética , Repressão Enzimática , Retroalimentação , Fosfatos/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Saccharomyces cerevisiae/enzimologia , Proteínas Virais/genéticaRESUMO
FK-506 is a novel and potent antagonist of T-cell activation and an inhibitor of fungal growth. Its immunosuppressive activity can be antagonized by the structurally related antibiotic rapamycin, and both compounds interact with cytoplasmic FK-506-binding proteins (FKBPs) in T cells and yeast cells. In this paper, we show that FK-506 and two analogs inhibit vegetative growth of Saccharomyces cerevisiae in a fashion that parallels the immunosuppressive activity of these compounds. Yeast mutants resistant to FK-506 were isolated, and at least three complementation groups (fkr1, fkr2, and fkr3) were defined. These fkr mutants show no alteration in their levels of FK-506-binding activity. Likewise, strains carrying null alleles of FKB1 (the yeast gene coding for the FKBP) remain FK-506 sensitive, indicating that depletion of yeast FKBP is not sufficient to confer an FK-506 resistance phenotype, although fkb1 null mutants are resistant to rapamycin. FKB1 does not map to the three fkr loci defined here. These results suggest that yeast FKBP mediates the inhibitory effect of rapamycin but that at least one other protein is directly involved in mediating the activity of FK-506. Interestingly, the ability of FK-506 to rescue a temperature-sensitive growth defect of the fkr3 mutant suggests that the FKR3 gene may define such a protein.
Assuntos
Antibacterianos/farmacologia , Antifúngicos , Genes Fúngicos/efeitos dos fármacos , Imunossupressores/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Antibacterianos/antagonistas & inibidores , Proteínas de Transporte/genética , Resistência Microbiana a Medicamentos/genética , Mutação , Polienos/farmacologia , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sirolimo , Tacrolimo , Proteínas de Ligação a Tacrolimo , TemperaturaRESUMO
We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.
Assuntos
Fosfatase Ácida/genética , Genes Reguladores , Genes , Saccharomyces cerevisiae/genética , Transcrição Gênica , Fosfatase Ácida/biossíntese , Cicloeximida/farmacologia , Repressão Enzimática , Genes Fúngicos , Genótipo , Cinética , Peso Molecular , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Saccharomyces cerevisiae/enzimologiaRESUMO
The nucleotide sequence of 5'-noncoding and N-terminal coding regions of two coordinately regulated, repressible acid phosphatase genes from Saccharomyces cerevisiae were determined. These unlinked genes encode different, but structurally related polypeptides of molecular weights 60,000 and 56,000. The DNA sequences of their 5'-flanking regions show stretches of extensive homology upstream of, and surrounding, a "TATA" sequence and in a region in which heterogeneous 5' ends of the p60 mRNA were mapped. The predicted amino acid sequences encoded by the N-terminal regions of both genes were confirmed by determination of the amino acid sequence of the native exocellular acid phosphatase and the partial sequence of the presecretory polypeptide synthesized in a cell-free protein synthesizing system. The N-terminal region of the p60 polypeptide was shown to be characterized by a hydrophobic 17-amino acid signal polypeptide which is absent in the native exocellular protein and thought to be necessary for acid phosphatase secretion.
Assuntos
Fosfatase Ácida/genética , Regulação da Expressão Gênica , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Fúngico/genética , Genes , Genes Reguladores , Peptídeos/fisiologia , Sinais Direcionadores de Proteínas , RNA Mensageiro/genéticaRESUMO
Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (sec1). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In sec18 the protoxin was stable after a chase; but in sec7 and sec1 the protoxin was unstable, and in sec1 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-l-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and sec1 cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-sec18 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kex1 but unstable in kex2. Protoxin stability in kex1 kex2 double mutants indicated the order kex1 --> kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEX1- and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion.
Assuntos
Micotoxinas/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores Matadores de Levedura , Proteínas de Membrana/metabolismo , Peso Molecular , Mutação , Micotoxinas/genética , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Taxa Secretória/efeitos dos fármacos , Tosilfenilalanil Clorometil Cetona/farmacologia , Tunicamicina/farmacologiaRESUMO
The plasmid determinants of killer phenotypes in type K1 and K2 killer yeast cells are the 1.9-kilobase (kb) M1 and 1.7-kb M2 double-stranded RNAs (dsRNAs), respectively. These are dependent for their maintenance and encapsidation, in Saccharomyces cerevisiae virus ScV-M1 or ScV-M2 virus-like particles, on the capsid provided by one of a group of moderately related 4.7-kb dsRNAs called LA. The L1A and L2A dsRNAs found in naturally isolated K1 and K2 killers encode 88-kilodalton VL1A-P1 and 86-kilodalton VL2A-P1 capsids, respectively. These are competent for encapsidating homologous LA dsRNAs as well as M dsRNAs. Most strains of S. cerevisiae, including killers, contain one of a second group of closely related 4.7-kb dsRNAs called LBC. These encode their own 82-kilodalton capsid protein, VLBC-P1, which, at least in strains containing only LBC, encapsidates homologous dsRNA in ScV-LBC virus-like particles. In a K1 killer strain containing both L1A and LBC, ScV-M1 particles contain only VL1A-P1. In such strains it is probable that each virus-like particle contains a single capsid type and that each L dsRNA is encapsidated by a homologous capsid.
Assuntos
Capsídeo/metabolismo , Plasmídeos , RNA de Cadeia Dupla/metabolismo , Saccharomyces cerevisiae/genética , Capsídeo/genética , Eletroforese em Gel de Poliacrilamida , Hibridização de Ácido Nucleico , Biossíntese de ProteínasRESUMO
We characterized the organization and expression of PHO5 and PHO3, the tightly linked repressible and constitutive acid phosphatase genes of Saccharomyces cerevisiae. The "constitutive" gene, PHO3, is expressed only when PHO5 is not. Altering PHO5 expression, either through promoter deletions or through mutations in trans-acting regulatory genes, showed that PHO5 expression is sufficient to block transcription of PHO3. An active genomic copy of PHO5 was able to block expression of PHO3 from a high-copy-number plasmid, showing that some trans-acting product of PHO5 is involved. This is probably a translation product, since the presence of a nontranslatable PHO5 RNA did not inhibit transcription of PHO3.
Assuntos
Fosfatase Ácida/genética , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica , Genes , Família Multigênica , Mutação , Fosfatos/fisiologia , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
We have isolated a 10.2-kb fragment of yeast DNA from a genomic library of recombinant centromeric YCp50 plasmids, which complements a mutation in the PHO4 gene of Saccharomyces cerevisiae. The identity of the PHO4 gene on this plasmid was established by integration of a subfragment into the PHO4 region of the yeast chromosome. Analysis of a series of plasmid subclones covering different regions of the original yeast DNA insert localized the PHO4 gene within a 2.25-kb sequence. Southern hybridization of total genomic DNA prepared from wild-type strains and from integrative transformants show that the PHO4 gene consists of unique yeast DNA sequences and is present at a single copy in the S. cerevisiae genome. RNA blot hybridization mapping of transcripts within this genomic region identify the PHO4 transcript as a 1.7-kb, low-abundancy, constitutively expressed and polyadenylated RNA.
Assuntos
Genes Fúngicos , Genes Reguladores , Genes , Saccharomyces cerevisiae/genética , Fosfatase Ácida/genética , Fosfatase Alcalina/genética , Enzimas de Restrição do DNA , Mutação , Hibridização de Ácido Nucleico , Plasmídeos , Saccharomyces cerevisiae/enzimologiaRESUMO
Homology between the coding regions of the chicken and yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes was directly demonstrated by the hybridization of a cDNA clone coding for GAPDH in the chicken with EcoRI-digested yeast DNA. A yeast EcoRI fragment library in bacteriophage lambda was screened using the chicken cDNA plasmid as probe, and two recombinant phages were isolated, each one containing a different GAPDH gene. The initiation and termination sites for the GAPDH mRNA were localized for the two different GAPDH genes and compared to those of other yeast genes. Measurements of the relative mRNA levels for the two genes show that both genes are transcribed at about the same level when yeasts are grown on glucose media.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/genética , Saccharomyces cerevisiae/genética , Animais , Sequência de Bases , Galinhas/genética , Mapeamento Cromossômico , DNA Fúngico/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Hibridização de Ácido Nucleico , RNA Fúngico/biossíntese , Especificidade da Espécie , Transcrição GênicaRESUMO
Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.
Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas Tipo A/metabolismo , Inibidores Enzimáticos/síntese química , Proteínas de Membrana , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ligação Competitiva , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Hidrólise , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Proteína 25 Associada a SinaptossomaRESUMO
A family of Schistosoma mansoni proteins (18-22 kDa, pI 5.3-5.8) are biosynthesized in juvenile worms and immunoprecipitated by antibodies uniquely present in protective Fischer rat antiserum. A cDNA clone, lambda gt11-40, expressing epitopes common to this protein family was used to obtain a genomic DNA clone, by hybridization with a lambda gt11-40 oligonucleotide probe. In the 1.37 kb of genomic DNA sequenced, an open reading frame of 182 amino acids was identified on the strand corresponding to lambda gt11-40 coding sequences, and those of identical independently isolated cDNA clones defining a 25-kDa surface membrane glycoprotein. The new S. mansoni gene is termed GP22. There are two candidate promoters, confirmed by primer extension studies with worm RNA. Promoter 1 (P1) is preceded by a G + C-rich region and potential CAAT sequences, and is to the 5'-side of P2. Transcription from P1 is initiated at 2 different sites, apparently producing mRNAs with different translation start sites (ATG). Decoding these mRNAs yields protein products of 182 (P1), 175 (P1), 140 (P2) and 136 (P2) amino acids. The polypeptides share the following features: a hydrophobic segment near the carboxy terminus sufficient to span a lipid bilayer, with a consensus sequence for thio-esterification by a fatty acid; an external domain containing 2 potential N-linked glycosylation sites; and a candidate leucine-zipper motif, suggesting the protein may exist as a dimer on the worm surface. While sharing these common features in their carboxy terminal regions, the three proteins differ in the length and properties of their amino termini. The 140-amino acid protein has a short hydrophobic amino terminus, while the 175- and 182-amino acid proteins have more extensive hydrophobic sequences, each preceded by a hydrophilic amino terminal sequence. The heterogeneity observed in 2-dimensional gels of the antigen may be explained in part by the size and charge differences among the proteins deduced from the sequence and transcription pattern of this gene. The possibility of stage-specific regulated expression of this candidate vaccine antigen family is an attractive concept, potentially accounting for the phenomenon of concomitant immunity observed in the rat and perhaps other schistosome hosts.
Assuntos
Antígenos de Helmintos/genética , Proteínas de Helminto , Glicoproteínas de Membrana/genética , Schistosoma mansoni/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA/genética , Genes , Dados de Sequência Molecular , Mapeamento por Restrição , Schistosoma mansoni/imunologia , Transcrição GênicaRESUMO
The toxic effects of microcystin-LR, a cyclic heptapeptide isolated from the cyanobacterium Microcystis aeruginosa, were studied in the fasted rat model and in subcellular fractions from fasted, toxin-treated and control rats. Hepatotoxic effects of a lethal dose (100 micrograms/kg) were examined 15-90 min post-injection. Elevations of serum enzymes, particularly sorbitol dehydrogenase, specific for liver mitochondria, correlated with hepatic damage. Electron micrographs showed progressive cellular disruption, including dilation of rough endoplasmic reticulum, incorporation of cellular components into cytolysosomes, hydropic mitochondria devoid of electron-opaque deposits, loss of desmosome-associated intermediate filaments, disruption of sinusoidal architecture and, ultimately, lysis of hepatocytes. The appearance of hydropic mitochondria correlated with loss of coupled electron transport. Changes in plasma membrane-associated cytoskeletal filaments correlated with loss of desmosome tonofilaments. In contrast to in vivo exposure to microcystin-LR, in vitro exposure to toxin had no effect on mitochondria or cytoskeletal filaments, suggesting that the toxic effects observed in vivo were indirect and may be dependent on bioactivation of the toxin or a cascade of events not supported in in vitro models.
Assuntos
Jejum/fisiologia , Peptídeos Cíclicos/toxicidade , Difosfato de Adenosina/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Transporte de Elétrons , Eletroforese em Gel de Poliacrilamida , Técnicas In Vitro , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/ultraestrutura , Toxinas Marinhas , Microcistinas , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , RatosRESUMO
A model was developed in the rhesus monkey to determine if the marked wasting of body proteins associated with sepsis could be prevented by an intravenous supply of various nutritional substrates. All monkeys were given a basic infusion of 0.5 gm of amino acid nitrogen/kg body weight via an indwelling catheter in the jugular vein. Three groups were given diets with no added calories, 85 calories/kg from dextrose or 85 calories from lipid. In each group, six monkeys were inoculated with 3 x 10(8) Streptococcus pneumoniae and four with heatkilled organisms. In the monkeys infused with the amino acids alone, pneumococcal sepsis resulted in a fourfold increase in loss of body proteins compared with calorie-restricted controls. Addition of 85 calories/kg/day of either dextrose or lipid reduced body wasting associated with infectious disease. The calories from lipid were utilized bythe septic host as a source of energy, with a slightly reduced efficiency when compared with the isocaloric infusion of dextrose. The nitrogen sparing of the fat emulsion could not be accounted for by its glycerol content. Therefore, the septic monkey seemed to utilize fatty acids as an energy substrate. It appears that the carbohydrate calories tend to favor the synthesis of peripheral proteins (associated mainly with skeletal muscle), while lipid calories favor synthesis of visceral proteins such as plasma albumin and acute-phase proteins.
Assuntos
Proteínas Alimentares/administração & dosagem , Nutrição Parenteral , Infecções Pneumocócicas/terapia , Aminoácidos/administração & dosagem , Animais , Antibacterianos/uso terapêutico , Ingestão de Energia , Emulsões Gordurosas Intravenosas/uso terapêutico , Glucose/uso terapêutico , Haplorrinos , Hidroxibutiratos/urina , Insulina/sangue , Lipídeos/sangue , Macaca mulatta , Masculino , Modelos Biológicos , Nitrogênio/metabolismoAssuntos
Clofibrato/farmacologia , Cobre/metabolismo , Ferro/metabolismo , Zinco/metabolismo , Animais , Ceruloplasmina/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Músculos/efeitos dos fármacos , Músculos/metabolismo , Ratos , Transferrina/metabolismo , alfa-Macroglobulinas/metabolismoAssuntos
Espectrofotometria , Zinco/sangue , Adulto , Química Clínica , Humanos , Masculino , MétodosAssuntos
Ferro/sangue , Tularemia/sangue , Adulto , Aerossóis , Ritmo Circadiano , Ensaios Clínicos como Assunto , Colorimetria , Febre , Humanos , Contagem de Leucócitos , Masculino , Espectrofotometria , VacinaçãoRESUMO
Antimicrobial research is geared toward the discovery and development of novel chemical structures such as therapeutic antimicrobial agents. The continuing problem of development of resistance to existing antibacterial agents and the dearth of good antifungal agents motivates this effort toward innovation. Selection of possible new enzyme targets for antibiotic inhibition may be made on theoretical grounds, but it appears premature to select any single, previously unvalidated target for the intensive study required for rational drug design. Instead, a broad screen of chemical entities can be undertaken, dedicated to the discovery of novel antimicrobial inhibitors. A number of target areas are under investigation, including fungal mRNA splicing and bacterial DNA synthesis. A major part of the endeavor is in the historically productive area of natural product screening. To make the best use of natural product resources for the discovery of novel antibiotics, a balance is struct between screening for inhibitors of rationally chosen targets for which clinically useful inhibitors are not yet available, and screening more broadly to ensure that rare activities of unanticipated mode-of-action are not missed.
Assuntos
Antibacterianos/análise , Bactérias/genética , DNA Bacteriano/análise , Desenho de Fármacos , Fungos/genética , RNA Mensageiro/análise , HumanosRESUMO
We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Extracellular transmission of the killer viruses was judged by the following criteria and controls. Filter-sterilized virus inocula were shown to be free of viable yeast cells, and host cells treated in the absence of added virus did not yield killer progeny. Infected clones originating from spheroplasts or lithium acetate-treated cells were shown to possess the genotype of the host strain and the killer phenotype of the infecting virus. Infected clones derived from complementary mating pairs were found to be wild-type diploids, whose meiotic segregants exhibited 2:2 segregation for unlinked nutritional markers and 4:0 segregation for the killer phenotype. This technique is generally applicable to the study of interactions between yeast viruses and different hosts and suggests that extracellular transmission may be a natural route for the inheritance and dissemination of mycoviruses.