RESUMO
Proteins involved in the regulation of the cell cycle are highly conserved across all eukaryotes, and so a relatively simple eukaryote such as yeast can provide insight into a variety of cell cycle perturbations including those that occur in human cancer. To date, the budding yeast Saccharomyces cerevisiae has provided the largest amount of experimental and modeling data on the progression of the cell cycle, making it a logical choice for in-depth studies of this process. Moreover, the advent of methods for collection of high-throughput genome, transcriptome, and proteome data has provided a means to collect and precisely quantify simultaneous cell cycle gene transcript and protein levels, permitting modeling of the cell cycle on the systems level. With the appropriate mathematical framework and sufficient and accurate data on cell cycle components, it should be possible to create a model of the cell cycle that not only effectively describes its operation, but can also predict responses to perturbations such as variation in protein levels and responses to external stimuli including targeted inhibition by drugs. In this review, we summarize existing data on the yeast cell cycle, proteomics technologies for quantifying cell cycle proteins, and the mathematical frameworks that can integrate this data into representative and effective models. Systems level modeling of the cell cycle will require the integration of high-quality data with the appropriate mathematical framework, which can currently be attained through the combination of dynamic modeling based on proteomics data and using yeast as a model organism.
RESUMO
Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Fúngico/biossíntese , Proteínas Fúngicas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Fracionamento Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G1 , Complexo de Reconhecimento de Origem , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1). In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system. Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis. In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication.
Assuntos
Núcleo Celular/genética , Replicação do DNA/genética , Saccharomyces cerevisiae/genética , Centrifugação com Gradiente de Concentração , DNA Super-Helicoidal/genética , Nucleotídeos de Desoxiuracil , Eletroforese em Gel de Ágar , Imunofluorescência , Técnicas In Vitro , Plasmídeos , Fase SRESUMO
One of the principal environmental adaptations of certain fishes inhabiting polar and northern coastal waters is the synthesis of antifreeze proteins (AFPs). AFPs bind to and prevent the growth of nascent ice crystals, thus depressing the serum freezing point. The transgenic expression of AFP holds great promise for conferring freeze resistance to commercially important plant and animal species. Since fish at the greatest risk of freezing have multiple AFP gene copies in order to synthesize higher levels of this protein, we have evaluated this evolutionary strategy as a way to maximize AFP expression in a model transgenic host, the fruit fly Drosophila melanogaster. A construct in which AFP genes of the Atlantic wolffish are fused to the Drosophila yolk protein 1,2 promoter/enhancer region was transferred to flies through P-element mediated transformation. Several independent transgenic fly lines were used in genetic crosses to obtain multi-insert lines. Haemolymph freezing point depression (thermal hysteresis) was greater in homozygotes relative to heterozygotes for a given insert. Similarly, multi-insert lines consistently displayed greater haemolymph AFP activity than the single insert lines from which they were derived. The thermal hysteresis value obtained with a fly line harboring 8 AFP gene copies, 0.43 degree C, represents the highest such value to date recorded in a transgenic host, and is even higher than the levels found in some AFP-producing fish.
Assuntos
Drosophila melanogaster/genética , Dosagem de Genes , Glicoproteínas/genética , Animais , Animais Geneticamente Modificados , Proteínas Anticongelantes , Feminino , Hemolinfa/fisiologia , Regiões Promotoras GenéticasRESUMO
Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclina B/metabolismo , Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Animais , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Ciclina B/genética , Replicação do DNA/genética , DNA Fúngico/biossíntese , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fase G1 , Técnicas In Vitro , Mutação , Complexo de Reconhecimento de Origem , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fase S , Saccharomyces cerevisiae/genética , XenopusRESUMO
We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.
Assuntos
Adenosina Trifosfatases/fisiologia , Núcleo Celular/metabolismo , DNA Helicases , Replicação do DNA/genética , DNA Super-Helicoidal/metabolismo , Saccharomyces cerevisiae/enzimologia , Afidicolina/farmacologia , Ciclo Celular/fisiologia , Centrifugação com Gradiente de Concentração , Metilação de DNA , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , DNA Super-Helicoidal/ultraestrutura , Nucleotídeos de Desoxiadenina/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Eletroforese em Gel Bidimensional , Microscopia Eletrônica , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/genética , Plasmídeos/ultraestrutura , RNA/metabolismoRESUMO
Using Drosophila as a host, we have examined the effect that the presence of an intron has on the accumulation of a processed transgene mRNA. To provide a model system that was free from major position effects, two fish antifreeze protein (AFP) transgenes were arranged in divergent transcriptional orientation from a central Drosophila yolk protein promoter/enhancer region and introduced into the flies by P-element transformation and/or mobilization. In this way the organization of both the structural genes and the promoter elements mimicked their natural arrangements. When one member of the fish AFP transgene pair had its single 180 bp intron deleted, there was a 2- to 11-fold (average 5-fold) decrease in its mRNA level compared to that generated from the control gene containing the fish AFP intervening sequence. When the deleted intron was replaced by a 70 bp intervening sequence originating from the yolk protein 1 gene, mRNA accumulation was restored to its original level. Even for the streamlined genome of Drosophila, where the intron number and size are generally reduced compared to mammals, the presence of an intervening sequence appears to facilitate mRNA accumulation.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Íntrons/genética , Transgenes , Animais , Proteínas Anticongelantes , Sequência de Bases , Peixes , Glicoproteínas/genética , Dados de Sequência MolecularRESUMO
The circulating Type II antifreeze protein (AFP) in sea raven is 129 amino acids (aa) long (14 kDa) and is derived from an initial 163 aa translation product that is synthesised in the liver. Signal peptide cleavage algorithms, as well as transgenic expression studies in fall armyworm cells, predict the formation of a 146 aa (16 kDa) proprotein intermediate. A protein of this size that cross-reacted with anti-sea raven AFP antibody was detected in sea raven serum using phosphate/urea SDS-PAGE, and was purified by size-exclusion chromatography and reverse-phase HPLC. N-terminal sequencing and mass spectrometry identified the protein as the predicted proAFP, and immunoblotting suggested that it is the predominant form present in liver. These results are consistent with production and storage of a proAFP intermediate in the liver, and its subsequent processing to mature AFP during or soon after its release into the circulation.
Assuntos
Peixes/metabolismo , Glicoproteínas/metabolismo , Fígado/metabolismo , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Animais , Proteínas Anticongelantes , Linhagem Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Congelamento , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Espectrometria de Massas , Biossíntese de Proteínas , Precursores de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
We have used Drosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting. Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 degree C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenic Drosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.
Assuntos
Proteínas Anticongelantes Tipo II , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Drosophila melanogaster/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Proteínas de Transporte/química , Cristalização , Feminino , Hemolinfa/química , Hemolinfa/metabolismo , Gelo , Masculino , Dados de Sequência Molecular , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genéticaRESUMO
Fish antifreeze proteins (AFPs) have been reported by some researchers to prolong the viability of tissues, organs, and embryos under hypothermic conditions, while others have observed no such effect or even AFP-mediated cryotoxicity. We examined the influence of Type III AFP from Atlantic wolffish on cold tolerance in a whole animal model system, transgenic Drosophila. The activity of the AFP, transgenically expressed under the transcriptional control of the female-specific yp1 and yp2 promoters and secreted into fly hemolymph, was confirmed through thermal hysteresis and differential scanning calorimetry measurements as well as through observations of ice crystal morphology. In cold exposure trials, at 0 degrees C and at -7 degrees C, transgenic adult flies of both sexes exhibited greater survival than nontransgenic controls even though the antifreeze was only produced in females. We attribute these observations to the expression of the xanthine dehydrogenase marker gene used to identify transgenics, rather than the production of AFP. Type III AFP therefore appears unable to enhance survival of adult Drosophila under hypothermic conditions.
Assuntos
Temperatura Baixa/efeitos adversos , Drosophila melanogaster/fisiologia , Glicoproteínas/fisiologia , Perciformes/genética , Animais , Animais Geneticamente Modificados , Proteínas Anticongelantes , Água Corporal/química , Varredura Diferencial de Calorimetria , Cristalização , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica , Glicoproteínas/genética , Hemolinfa/química , Masculino , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Caracteres Sexuais , Xantina Desidrogenase/genética , Xantina Desidrogenase/fisiologiaRESUMO
In winter flounder, the levels of type I antifreeze protein (AFP) and its mRNA vary seasonally by as much as 1000-fold. Elevated levels in the fall are prompted by the loss of long day-lengths, while higher spring temperatures correlate with AFP clearance. We have investigated the role of temperature on AFP accumulation using transgenic Drosophila melanogaster by expressing multiple AFP genes under control of the heat-inducible hsp70 promoter. AFP and AFP mRNA persisted far longer in flies reared at 10 degrees C compared to 22 degrees C. This difference appears to be mediated by cold-specific mRNA stability since no such temperature effect was observed with either an endogenous heat-inducible mRNA or a constitutively expressed mRNA.
Assuntos
Temperatura Baixa , Linguado/metabolismo , Glicoproteínas/metabolismo , RNA Mensageiro/biossíntese , Animais , Animais Geneticamente Modificados , Proteínas Anticongelantes , Drosophila/metabolismo , Regulação da Expressão Gênica , Glicoproteínas/genética , Água do MarRESUMO
Recombinant cystine-rich fish antifreeze protein (AFP) was produced by fall armyworm cells from a baculovirus expression vector containing sea raven AFP cDNA. The natural signal sequence encoded in the cDNA directed secretion of the antifreeze into the medium, from where it was recovered and purified to homogeneity. The M(r) of the exported protein (16k), as determined by SDS-PAGE, was larger than that (14k) of mature AFP isolated from sea raven serum. Sequencing of the recombinant AFP showed that it had a 17-amino-acid extension N-terminal to the 129-amino-acid mature AFP that began where signal peptide cleavage should occur according to current algorithms. Recombinant proAFP had antifreeze activity equivalent to that of the mature AFP, which indicates that the disulfide bonds were correctly formed and that the ice-binding site on the antifreeze is not sterically hindered by the 17-amino-acid N-terminal extension.