RESUMO
The choice between meiosis and alternative developmental pathways in budding yeast depends on the expression and activity of transcriptional activator Ime1. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. IREu, a 32-bp element in the IME1 promoter, exhibits upstream activation sequence activity depending on Msn2 and -4 and the presence of acetate. We show that in the presence of glucose IREu functions as a negative element and that Sok2 mediates this repression activity. We show that Sok2 associates with Msn2. Sok2 functions as a general repressor whose availability and activity depend on glucose. The activity of Sok2 as a repressor depends on phosphorylation of T598 by protein kinase A (PKA). Relief of repression of Sok2 depends on both the N-terminal domain of Sok2 and Ime1. In the absence of glucose and the presence of Ime1 Sok2 is converted to a weak activator. Overexpression of Sok2 or mild expression of Sok2 with its N-terminal domain deleted leads to a decrease in sporulation. Previously it was reported that overexpression of Sok2 suppresses the growth defect resulting from a temperature-sensitive PKA; thus Sok2 has a positive role in mitosis. We show that Candida albicans Efg1, a homolog of Sok2, complements sok2 Delta in repressing IREu. Our results demonstrate that Sok2, a positive regulator of mitosis, and Efg1, a positive regulator of filamentation, function as negative regulators of meiosis. We suggest that cells use the same regulators with opposing effects to ensure that meiosis will be an alternative to mitosis.
Assuntos
Meiose , Proteínas Repressoras/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Western Blotting , Candida albicans/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Mitose , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Sporulation of Saccharomyces cerevisiae ordinarily requires the a1 function of the a mating type locus. SAD is a dominant mutation that allows strains lacking a1 (MAT alpha/MAT alpha and mata1/MAT alpha diploids) to sporulate. We provide functional and physical evidence that SAD is an extra cassette in the yeast genome, distinct from those at HML, MAT, and HMR. The properties of SAD strains indicate that the a cassette at SAD produces a limited amount of a1 product, sufficient for promoting sporulation but not for inhibiting mating and other processes. These conclusions come from the following observations. (i) SAD did not act by allowing expression of HMRa: mata1/MAT alpha diploids carrying SAD and only alpha cassettes at HML and HMR sporulated efficiently. (ii) SAD acted as an a cassette donor in HML alpha HMR alpha strains and could heal a mata1 mutation to MATa as a result of mating type interconversion. (iii) The genome of SAD strains contained a single new cassette locus, as determined by Southern hybridization. (iv) Expression of a functions from the SAD a cassette was limited by Sir: sir- SAD strains exhibited more extreme phenotypes than SIR SAD strains. This observation indicates that SAD contains not only cassette information coding for a1 (presumably from HMRa) but also sites for Sir action.
Assuntos
Saccharomyces cerevisiae/genética , Sequência de Bases , Regulação da Expressão Gênica , Mutação , Fenótipo , Reprodução , Saccharomyces cerevisiae/fisiologia , Esporos FúngicosRESUMO
The transcription of meiosis-specific genes, as well as the initiation of meiosis, in the budding yeast Saccharomyces cerevisiae depends on IME1. IME1 encodes a transcriptional activator which lacks known DNA binding motifs. In this study we have determined the mode by which Ime1 specifically activates the transcription of meiotic genes. We demonstrate that Ime1 is recruited to the promoters of meiotic genes by interacting with a DNA-binding protein, Ume6. This association between Ime1 and Ume6 depends on both starvation and the activity of a protein kinase, encoded by RIM11 In the absence of Ime1, Ume6 represses the transcription of meiotic genes. However, in the presence of Ime1, or when Ume6 is fused in frame to the Gal4 activation domain, Ume6 is converted from a repressor to an activator, resulting in the transcription of meiosis-specific genes and the formation of asci.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Proteínas Nucleares/metabolismo , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Regulação Fúngica da Expressão Gênica , Genótipo , Meiose , Modelos Genéticos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Supressão Genética , Transcrição GênicaRESUMO
IME1 encodes a transcriptional activator required for the transcription of meiosis-specific genes and initiation of meiosis in Saccharomyces cerevisiae. The transcription of IME1 is repressed in the presence of glucose, and a low basal level of IME1 RNA is observed in vegetative cultures with acetate as the sole carbon source. Upon nitrogen depletion a transient induction in the transcription of IME1 is observed in MATa/MATalpha diploids but not in MAT-insufficient strains. In this study we demonstrate that the transcription of IME1 is controlled by an extremely unusual large 5' region, over 2,100 bp long. This area is divided into four different upstream controlling sequences (UCS). UCS2 promotes the transcription of IME1 in the presence of a nonfermentable carbon source. UCS2 is flanked by three negative regions: UCS1, which exhibits URS activity in the presence of nitrogen, and UCS3 and UCS4, which repress the activity of UCS2 in MAT-insufficient cells. UCS2 consists of alternate positive and negative elements: three distinct constitutive URS elements that prevent the function of any upstream activating sequence (UAS) under all growth conditions, a constitutive UAS element that promotes expression under all growth conditions, a UAS element that is active only in vegetative media, and two discrete elements that function as UASs in the presence of acetate. Sequence analysis of IME1 revealed the presence of two almost identical 30- to 32-bp repeats. Surprisingly, one repeat, IREd, exhibits constitutive URS activity, whereas the other repeat, IREu, serves as a carbon-source-regulated UAS element. The RAS-cyclic AMP-dependent protein kinase cAPK pathway prevents the UAS activity of IREu in the presence of glucose as the sole carbon source, while the transcriptional activators Msn2p and Msn4p promote the UAS activity of this repeat in the presence of acetate. We suggest that the use of multiple negative and positive elements is essential to restrict transcription to the appropriate conditions and that the combinatorial effect of the entire region leads to the regulated transcription of IME1.
Assuntos
Proteínas Fúngicas/genética , Meiose/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transcrição Gênica , Ativação Transcricional/genética , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rm- enables the diploids a*/alpha, a/a*, and alpha/alpha to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.
Assuntos
Replicação do DNA , Meiose , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Alelos , Diploide , Genótipo , Haploidia , Mutação , Fenótipo , Poliploidia , Esporos Fúngicos/metabolismoRESUMO
Mutations leading to expression of the silent HMRa information in Saccharomyces cerevisiae result in sporulation proficiency in mata1/MAT alpha diploids. An example of such a mutation is sir5-2, a recessive mutation in the gene SIR5. As expected, haploids carrying the sir5-2 mutation are nonmaters due to the simultaneous expression of HMRa and HML alpha, resulting in the nonmating phenotype of an a/alpha diploid. However, sir5-2/sir5-2 mata1/MAT alpha diploids mate as alpha yet are capable of sporulation. The sir5-2 mutation is unlinked to sir1-1, yet the two mutations do not complement each other: mata1/MAT alpha sir5-2/SIR5 SIR1/sir1-1 diploids are capable of sporulation. In this case, recessive mutations in two unlinked genes form a mutant phenotype, in spite of the presence of the normal wild-type alleles. The PAS1-1 mutation, Provider of a Sporulation function, is a dominant mutation tightly linked to HMRa. PAS1-1 does not affect the mating ability of a strain, yet it allows diploids lacking a functional MATa locus to sporulate. It is proposed that PAS1-1 leads to partial expression of the otherwise cryptic a1 information at HMRa.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Animais , Regulação da Expressão Gênica , Fator de Acasalamento , Mutação , Peptídeos/genética , Feromônios/genética , Esporos FúngicosRESUMO
Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.
Assuntos
Ciclo Celular , DNA/biossíntese , Mutação , Recombinação Genética , Saccharomyces cerevisiae/genética , Hidroxiureia/farmacologia , Meiose , Mutagênicos , FenótipoRESUMO
In the budding yeast Saccharomyces cerevisiae initiation and progression through the mitotic cell cycle are determined by the sequential activity of the cyclin-dependent kinase Cdc28. The role of this kinase in entry and progression through the meiotic cycle is unclear, since all cdc28 temperature-sensitive alleles are leaky for meiosis. We used a "heat-inducible Degron system" to construct a diploid strain homozygous for a temperature-degradable cdc28-deg allele. We show that this allele is nonleaky, giving no asci at the nonpermissive temperature. We also show, using this allele, that Cdc28 is not required for premeiotic DNA replication and commitment to meiotic recombination. IME2 encodes a meiosis-specific hCDK2 homolog that is required for the correct timing of premeiotic DNA replication, nuclear divisions, and asci formation. Moreover, in ime2Delta diploids additional rounds of DNA replication and nuclear divisions are observed. We show that the delayed premeiotic DNA replication observed in ime2Delta diploids depends on a functional Cdc28. Ime2Delta cdc28-4 diploids arrest prior to initiation of premeiotic DNA replication and meiotic recombination. Ectopic overexpression of Clb1 at early meiotic times advances premeiotic DNA replication, meiotic recombination, and nuclear division, but the coupling between these events is lost. The role of Ime2 and Cdc28 in initiating the meiotic pathway is discussed.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular , DNA/biossíntese , Proteínas Fúngicas/fisiologia , Meiose , Proteínas Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Animais , Western Blotting , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Separação Celular , Diploide , Citometria de Fluxo , Proteínas Fúngicas/genética , Genótipo , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Mitose , Modelos Genéticos , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Recombinação Genética , Temperatura , Fatores de TempoRESUMO
Normally, meiosis and sporulation in Saccharomyces cerevisiae occur only in diploid strains and only when the cells are exposed to starvation conditions. Diploidy is determined by the mating-type system (the genes MAT, RME1, IME1), whereas the starvation signal is transmitted through the adenylate cyclase - protein kinase pathway (the genes CDC25, RAS2, CDC35 (CYR1), BCY1, TPK1, TPK2, TPK3). The two regulatory pathways converge at the gene IME1, which is a positive regulator of meiosis and whose early expression in sporulating cells correlates with the initiation of meiosis. Sites upstream (5') of IME1 appear to mediate in the repression of the gene by repressors originating from both the mating-type and the cyclase--kinase pathways.
Assuntos
Saccharomyces cerevisiae/genética , Adenilil Ciclases/fisiologia , Diferenciação Celular/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Genes Fúngicos/fisiologia , Genes Fúngicos Tipo Acasalamento , Meiose/genética , Proteínas Quinases/fisiologia , Saccharomyces cerevisiae/citologia , Esporos Fúngicos/genéticaRESUMO
In meiotic cells of Saccharomyces cerevisiae, reduction in molecular weights of DNA in alkaline sucrose gradients is observed concomittantly with premeiotic DNA replication and with commitment to recombination. Following the completion of the latter processes, higher molecular weights are obtained. These single-stranded breaks are found in both old and newly synthesised strands. Similar scissions in DNA are also found in a temperature-sensitive mutant (cdc40/cdc40), which does not undergo commitment to recombination at the restrictive temperature, and in vegetative wild type cells that were previously exposed to sporulation medium. The suggestion that these scissions are the physical manifestation of commitment to recombination is therefore rejected.
RESUMO
IME1 (Inducer of MEiosis) was cloned due to its high copy number effect: it enabled MAT insufficient strains to undergo meiosis. Disruption of IME1 results in a recessive Spo- phenotype. Diploids homozygous for the two mutations ime1-0, rme1-1 are also meiosis deficient. We conclude that IME1 is a positive regulator of meiosis that normally is repressed by RME1. RME1 is repressed by a complex of MATa1 and MAT alpha 2 gene products. IME1 is also regulated by the environment: no transcripts could be detected in glucose growing cells, in contrast to acetate growing cells. Starvation for nitrogen further induced (6- to 8-fold) transcription of IME1, but, as expected, the induction was found only in MATa/MAT alpha or rme1-1/rme1-1 diploids. Furthermore, the IME1 multicopy plasmids promoted sporulation in rich media.
Assuntos
Genes Fúngicos , Genes Reguladores , Saccharomyces cerevisiae/genética , Transcrição Gênica , Clonagem Molecular , Marcadores Genéticos , Genótipo , Meiose , Plasmídeos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Previous studies have shown that the IME1 gene is required for sporulation and the expression of meiosis specific genes in Saccharomyces cerevisiae. However, sequence analysis has not revealed the precise functional role of the Ime1 protein. By engineering constructs which express various portions of the Ime1p fused to either the DNA binding or transcriptional activation domains of GAL4, we have conclusively demonstrated that IME1 is a transcription factor, apparently required for sporulation to activate the transcription of meiosis specific genes. The full Ime1p, when fused to the GAL4 DNA binding domain, can both activate GAL1-lacZ expression, and complement ime1-0 (a null allele) for the ability to sporulate, and transcriptionally activate IME2, a meiosis specific gene. As successively larger portions of the encoded Ime1p N-terminus are deleted from the GAL4(bd)-IME1 construct, the encoded fusion proteins retain the ability to complement an ime1 null allele, despite a decreasing ability to activate GAL1-lacZ transcription. However, a fusion construct which retains only the last 45 C-terminal amino acids of IME1 provides neither transcriptional activation of GAL1-lacZ nor complementation of ime1-0. Fusion of a GAL4 activation domain to this portion of IME1, results in a construct with a restored ability to complement an ime1-0 allele. This restored ability is dependent upon galactose induction. We conclude, therefore, that IME1 functions in meiosis as a transcriptional activator.
Assuntos
Proteínas de Ciclo Celular , Proteínas Fúngicas/genética , Meiose/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Sequência de Bases , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/fisiologia , Esporos Fúngicos/genética , Fatores de Transcrição/metabolismo , beta-Galactosidase/metabolismoRESUMO
Hydroxyurea (HU) inhibits the premeiotic DNA replication and the meiotic events that followed, namely readiness, recombination commitment, haploidisation, sporulation commitment and ascus formation. Short incubations with HU (2-4 hrs) during the premeiotic replication (i.e. starting between 3 and 6.5 hrs in sporulation medium) allow the resumption of the replication at a normal rate following the removal of the drug. The other meiotic events are similarly delayed by the approximate length of the treatment. In these experiments, intragenic recombination in ade2 reached a higher level than in the controls (x 1.3-2.0 in one pair of heteroalleles and x 3.0-4.0 in another pair). The recombination response to short HU treatments was not observed for a pair of heteroalleles in ade2 that normally shows a high level of meiotic recombination (750 per 10(6) cells), nor was the response observed in a pair of heteroalleles in lys2. HU treatments have almost no effect on sporulating cells from 8 hrs onwards. At 7-7.5 hrs the meiotic cells are very sensitive to the drug and even short treatments cause cell death and massive DNA degradation.
Assuntos
Replicação do DNA/efeitos dos fármacos , Hidroxiureia/farmacologia , Meiose , Recombinação Genética , Saccharomyces cerevisiae/metabolismo , Diploide , Frequência do Gene , Esporos Fúngicos , Fatores de TempoRESUMO
The cdc40 mutation has been previously shown to be a heat-sensitive cell-division-cycle mutation. At the restrictive temperature, cdc40 cells arrest at the end of DNA replication, but retain sensitivity to hydroxyurea (Kassir and Simchen 1978). The mutation has also been shown to affect commitment to meiotic recombination and its realization. Here we show that mutant cells are extremely sensitive to Methyl-Methane Sulfonate (MMS) when the treatment is carried out at restrictive temperature. Incubation at 37 degrees C prior to, or after MMS treatment at 23 degrees C, does not result in lower survival. It is concluded that the CDC40 gene product has a role in DNA repair, possibly holding together or protecting the DNA during the early stages of repair. The CDC40 gene was cloned on a 2.65 kb DNA fragment. A 2 mu plasmid carrying the gene was integrated and mapped to chromosome IV, between trp4 and ade8, by the method of marker loss. Conventional tetrad analysis has shown cdc40 to map 1.7 cM from trp4.
Assuntos
Mapeamento Cromossômico , Reparo do DNA , Genes Fúngicos , Saccharomyces cerevisiae/genética , Clonagem Molecular , Escherichia coli/genéticaRESUMO
A diploid strain of yeast, homozygous for the mutation cdc5-1, undergoes a normal meiosis at 25 degrees C. At the nonpermissive temperature of 34 degrees C, meiosis is arrested at the first meiotic division, after premeiotic DNA replication and recombination commitment have taken place. Haploidisation commitment does not occur at 34 degrees C. Electron microscopy reveals that synaptons (synaptonemal complexes) are formed and the stage of arrest is characterised by a prevalence of "modified synaptons", which consist of paired lateral elements lacking the central elements. Prolonged incubation at this stage of arrest results in unusually high recombination levels, perhaps related to the synaptonal structures observed. Temperature shift-up experiments (transfers of cell from 25 degrees C to 34 degrees C at various times during meiosis) reveal that the CDC5 function is required for both the first and the second divisions of meiosis.
Assuntos
Meiose , Mutação , Recombinação Genética , Temperatura Alta , Cinética , Fenótipo , Saccharomyces cerevisiae/citologiaRESUMO
The IME1 gene of Saccharomyces cerevisiae is required for initiation of meiosis. Transcription of IME1 is detected under conditions which are known to induce initiation of meiosis, namely starvation for nitrogen and glucose, and the presence of MATa1 and MAT alpha 2 gene products. In this paper we show that IME1 is also subject to translational regulation. Translation of IME1 mRNA is achieved either upon nitrogen starvation, or upon G1 arrest. In the presence of nutrients, constitutively elevated transcription of IME1 is also sufficient for the translation of IME1 RNA. Four different conditions were found to cause expression of Ime1 protein in vegetative cultures: elevated transcription levels due to the presence of IME1 on a multicopy plasmid; elevated transcription provided by a Gal-IME1 construct; G1 arrest due to alpha-factor treatment; G1 arrest following mild heat-shock treatment of cdc28 diploids. Using these conditions, we obtained evidence that starvation is required not only for transcription and efficient translation of IME1, but also for either the activation of Ime1 protein or for the induction/activation of another factor that, either alone or in combination with Ime1, induces meiosis.
Assuntos
Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Meiose/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA Fúngico , Genes Fúngicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , Ribonucleases , Transcrição GênicaRESUMO
Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.
Assuntos
Adenilil Ciclases/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/genética , Eletroforese em Gel Bidimensional , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/isolamento & purificação , Genótipo , Temperatura Alta , Meiose , Plasmídeos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esporos Fúngicos/fisiologia , Transcrição GênicaRESUMO
The FER locus of the mouse encodes two mRNA species: one is constitutively transcribed, giving rise to a 94 kDa tyrosine kinase (p94ferT); the second is a meiosis-specific RNA that gives rise to a 51 kDa tyrosine kinase (p51ferT). The p51ferT RNA and protein accumulate in primary spermatocytes that are in prophase of the first meiotic division. By using polyclonal antibodies directed against synthetic peptides derived from the unique amino-terminus of the mouse p51ferT, a 51 kDa phosphotyrosyl protein --p51y-- was identified in Saccharomyces cerevisiae. The p51y protein is constitutively expressed in yeast, but in meiotic cells, concomitantly with commitment to meiotic recombination, its level of phosphorylation on tyrosine residues is increased. A different pattern of phosphorylation is observed on serine residues: at early meiotic times the level is decreased, while in later meiotic time the level increases, reaching the vegetative level. When p51ferT is ectopically expressed in yeast, it is active, leading to preferential phosphorylation of an approx. 65 kDa protein. A similar pattern of phosphorylation by p51ferT is seen in mammalian cells.
Assuntos
Proteínas Fúngicas/biossíntese , Proteínas Nucleares/biossíntese , Proteínas Tirosina Quinases/biossíntese , Saccharomyces cerevisiae/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Immunoblotting , Masculino , Meiose , Camundongos , Peso Molecular , Proteínas Nucleares/genética , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/isolamento & purificação , RNA Mensageiro/biossíntese , Saccharomyces cerevisiae/genética , Espermatócitos/enzimologia , Tirosina/metabolismoRESUMO
In this report we study the regulation of premeiotic DNA synthesis in Saccharomyces cerevisiae. DNA replication was monitored by fluorescence-activated cell sorting analysis and by analyzing the pattern of expression of the DNA polymerase alpha-primase complex. Wild-type cells and cells lacking one of the two principal regulators of meiosis, Ime1 and Ime2, were compared. We show that premeiotic DNA synthesis does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. At late meiotic times, ime2 delta diploids exhibit an additional round of DNA synthesis. Furthermore, we show that in wild-type cells the B-subunit of DNA polymerase alpha is phosphorylated during premeiotic DNA synthesis, a phenomenon that has previously been reported for the mitotic cell cycle. Moreover, the catalytic subunit and the B-subunit of DNA polymerase alpha are specifically degraded during spore formation. Phosphorylation of the B-subunit does not occur in ime1 delta diploids, but does occur in ime2 delta diploids with an 8-9 h delay. In addition, we show that Ime2 is not absolutely required for commitment to meiotic recombination, spindle formation and nuclear division, although it is required for spore formation.