RESUMO
In Saccharomyces cerevisiae, three G1 cyclins (Clns) are important for Start, the event committing cells to division. Sic1, an inhibitor of C1b-Cdc28 kinases, became phosphorylated at Start, and this phosphorylation depended on the activity of Clns. Sic1 was subsequently lost, which depended on the activity of Clns and the ubiquitin-conjugating enzyme Cdc34. Inactivation of Sic1 was the only nonredundant essential function of Clns, because a sic1 deletion rescued the inviability of the cln1 cln2 cln3 triple mutant. In sic1 mutants, DNA replication became uncoupled from budding. Thus, Sic1 may be a substrate of Cln-Cdc28 complexes, and phosphorylation and proteolysis of Sic1 may regulate commitment to replication at Start.
Assuntos
Replicação do DNA , Inibidores Enzimáticos/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC28 de Saccharomyces cerevisiae/antagonistas & inibidores , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proteínas Inibidoras de Quinase Dependente de Ciclina , Ciclinas/metabolismo , DNA Fúngico/biossíntese , Ligases/metabolismo , Fosforilação , Saccharomyces cerevisiae/citologia , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína LigasesRESUMO
Since DNA polymerase requires a labile primer to initiate unidirectional 5'-3' synthesis, some bases at the 3' end of each template strand are not copied unless special mechanisms bypass this "end-replication" problem. Immortal eukaryotic cells, including transformed human cells, apparently use telomerase, an enzyme that elongates telomeres, to overcome incomplete end-replication. However, telomerase has not been detected in normal somatic cells, and these cells lose telomeres with age. Therefore, to better understand the consequences of incomplete replication, we modeled this process for a population of dividing cells. The analysis suggests four things. First, if single-stranded overhangs generated by incomplete replication are not degraded, then mean telomere length decreases by 0.25 of a deletion event per generation. If overhangs are degraded, the rate doubles. Data showing a decrease of about 50 base-pairs per generation in fibroblasts suggest that a full deletion event is 100 to 200 base-pairs. Second, if cells senesce after 80 doublings in vitro, mean telomere length decreases about 4000 base-pairs, but one or more telomeres in each cell will lose significantly more telomeric DNA. A checkpoint for regulation of cell growth may be signalled at that point. Third, variation in telomere length predicted by the model is consistent with the abrupt decline in dividing cells at senescence. Finally, variation in length of terminal restriction fragments is not fully explained by incomplete replication, suggesting significant interchromosomal variation in the length of telomeric or subtelomeric repeats. This analysis, together with assumptions allowing dominance of telomerase inactivation, suggests that telomere loss could explain cell cycle exit in human fibroblasts.
Assuntos
Cromossomos Humanos/fisiologia , Replicação do DNA , DNA/metabolismo , Telômero/fisiologia , Adulto , Sequência de Bases , Divisão Celular , Células Cultivadas , Deleção Cromossômica , DNA/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Cinética , Modelos Genéticos , Sondas de Oligonucleotídeos , Sequências Repetitivas de Ácido Nucleico , Fenômenos Fisiológicos da PeleRESUMO
The 2 micron circle is a small double stranded DNA plasmid that occurs at about 60 copies per cell in the nuclei of virtually all strains of Saccharomyces cerevisiae. The plasmid has no apparent phenotypic effect on host cells, and is the basis of many useful vectors for the transformation of yeast. Under certain circumstances, the plasmid is apparently able to replicate more than once per cell cycle; this over-replication allows the maintenance of the plasmid at high copy number. The plasmid has two inverted repeat sequences, and encodes a product that catalyses intra-molecular recombination between these two repeats. Models are proposed whereby recombination leads to copy number amplification. In particular, it is proposed that intra-molecular recombination during replication flips the orientation of one replication fork with respect to the other, so that both forks travel in the same direction around a circular monomer template, generating a large multimer from a monomer and a single initiation of replication.
Assuntos
Amplificação de Genes , Plasmídeos , Saccharomyces cerevisiae/genética , Replicação do DNA , DNA Circular/genética , DNA Fúngico/genética , Modelos Genéticos , Recombinação Genética , Sequências Repetitivas de Ácido NucleicoRESUMO
Two families of cyclin-like proteins have been found in S. cerevisiae. The clb proteins are the mitotic cyclins. The cln proteins provide an essential function, are required for the G1/S transition, and appear to be rate-limiting for START, but have no obvious role elsewhere in the cycle. The cln proteins are unstable; they form complexes with cdc28; the complexes have protein kinase activity; and at least one of the clns oscillates in abundance through the cell cycle. The action of the cln cyclins at START suggests that they may be 'G1 cyclins'.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/fisiologia , Ciclinas/fisiologia , Saccharomyces cerevisiae/fisiologia , Ciclo Celular/fisiologia , Fase G1/fisiologia , Mitose/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologiaRESUMO
A quick and convenient assay for depurination and AP endonuclease activities has been developed. (The term 'AP endonuclease' refers to a nuclease that acts on apurinic and probably apyrimidinic sites on DNA.) It is based on the observation that different topological forms of DNA, such as open circular DNA and covalently closed circular DNA, bind different amounts of the fluorescent intercalator ethidium bromide, and can therefore be distinguished by their fluorescence. This assay has been used to measure AP endonuclease activity in 22 repair-deficient mutants of Saccharomyces cerevisiae. All 22 had normal or nearly normal AP endonuclease activity. The AP endonuclease activity was partially characterized.
Assuntos
DNA/metabolismo , Desoxirribonucleases/metabolismo , Endonucleases/metabolismo , Saccharomyces cerevisiae/enzimologia , Ácido Apurínico/metabolismo , Mutação , Saccharomyces cerevisiae/genéticaRESUMO
The 2 microns circle plasmid is maintained at high frequencies in populations of yeast cells. To find out how the plasmid is maintained, three forces were measured: the selective advantage or disadvantage conferred by 2 microns circles, the rate of generation of [Cir0] cells, and the rate of illegitimate transfer of 2 microns circles from cell to cell. It was found that under the conditions used, 2 microns circles confer a selective disadvantage of about 1%, that [Cir0] cells are generated at the rate of 7.6 x 10(-5) per [Cir+] cell per generation, and that illegitimate transfer of 2 microns circles occurs at a rate less than 10(-7) per recipient cell per generation. The most likely explanation of 2 microns circle maintenance is that the plasmid is sexually transmitted at such a rate that it spreads through populations despite selection against it.
Assuntos
Plasmídeos , Saccharomyces cerevisiae/genética , Conjugação Genética , Saccharomyces cerevisiae/fisiologia , Transformação GenéticaRESUMO
The copy number and stability of artificial 2-micron circle-based plasmids have been accurately measured in [Cir+] and [Cir0] strains of Saccharomyces cerevisiae. We conclude that (i) instability and copy number vary greatly from plasmid to plasmid; (ii) instability and copy number are negatively correlated--that is, high copy number is associated with low instability; (iii) it is difficult to reconcile this variability with a strict and direct system of copy number control; (iv) instabilities are much higher than expected from random partition and the observed copy numbers: this may imply partition which is less efficient than random. Even so, (v) the partitioning of 2-micron circle-like plasmids is more efficient than that of ARS-based plasmids, which hints at the existence of a system for the (inefficient) distribution of 2-micron circles.
Assuntos
Plasmídeos , Saccharomyces cerevisiae/genética , Quimera , DNA Fúngico/genética , Variação Genética , Cinética , Transformação GenéticaRESUMO
The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.
Assuntos
Envelhecimento , Cromossomos/ultraestrutura , Sobrevivência Celular , Células Cultivadas , Fibroblastos , HumanosRESUMO
Two plasmids containing Saccharomyces cerevisiae 2 µm DNA sequences and the S. cerevisiae LEU2 gene have been found to display incompatibility with 2 µm DNA; in the presence of the LEU2 plasmids, 2 µm DNA can be lost. The LEU2 plasmids can be lost spontaneously after (and before) 2 µm DNA loss has occurred, so that strains completely lacking 2 µm DNA sequences can be obtained routinely.
RESUMO
[2 µm(+) and [2µm°] yeast were transformed to stable leucine prototrophy with the hybrid yeast - E. coli plasmid, pJDB219. This plasmid contains the entire sequence of the endogenous 2 µm yeast DNA plasmid in addition to the yeast nuclear LEU2 (+) gene and the Co1E1 derivative, pMB9. In the [2 µm(+)] transformants, a new wholly yeast LEU2 (+) plasmid, pYX, was generated, probably by a recombination event between pJDB219 and 2 µm DNA. The plamid, pYX, in the absence of 2 µm DNA, was found to exist in equimolar amounts of two forms, A and B, which probably arise by intramolecular recombination across the inverted repeat sequences of the 2 µm DNA portion of the plasmid. pJDB219 was found to require the presence of 2 µm DNA to undergo this intramolecular recombination. The results suggest that 2, µm DNA and pYX code for a gene product required in this recombination event which pJDB219 cannot produce.
RESUMO
WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.
Assuntos
Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular/efeitos dos fármacos , Divisão Celular , Clonagem Molecular , DNA Fúngico/genética , Genes Dominantes , Fator de Acasalamento , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Peptídeos/farmacologia , Fenótipo , Antígeno Nuclear de Célula em Proliferação , Mapeamento por Restrição , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+ phenotype. Finally, selection for Ura- cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.
Assuntos
Epitopos , Proteínas Fúngicas/análise , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Proteínas Fúngicas/imunologia , Vetores Genéticos , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Transformação GenéticaRESUMO
Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae. We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S. cerevisiae without previously cloning them. In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker. An indefinite number of disruptions can therefore be made in the same strain.
Assuntos
Mutagênese Insercional/métodos , Saccharomyces cerevisiae/genética , Sequência de Bases , Marcadores Genéticos , Dados de Sequência Molecular , Orotidina-5'-Fosfato Descarboxilase/genética , Reação em Cadeia da Polimerase , Seleção Genética , Transformação GenéticaRESUMO
cdc28-1N is a conditional allele that has normal G1 (Start) function but confers a mitotic defect. We have isolated seven genes that in high dosage suppress the growth defect of cdc28-1N cells but not of Start-defective cdc28-4 cells. Three of these (CLB1, CLB2, and CLB4) encode proteins strongly homologous to G2-specific B-type cyclins. Another gene, CLB3, was cloned using PCR, CLB1 and CLB2 encode a pair of closely related proteins; CLB3 and CLB4 encode a second pair. Neither CLB1 nor CLB2 is essential; however, disruption of both is lethal and causes a mitotic defect. Furthermore, the double mutant cdc28-1N clb2::LEU2 is nonviable, whereas cdc28-4 clb2::LEU2 is viable, suggesting that the cdc28-1N protein may be defective in its interaction with B-type cyclins. Our results are consistent with CDC28 function being required in both G1 and mitosis. Its mitotic role, we believe, involves interaction with a family of at least four G2-specific cyclins.
Assuntos
Ciclinas/fisiologia , Mitose/fisiologia , Proteínas Quinases/fisiologia , Saccharomyces cerevisiae/fisiologia , Alelos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Ciclinas/genética , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Genes Supressores , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Protamina Quinase/metabolismo , Proteínas Quinases/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido NucleicoRESUMO
When human fibroblasts from different donors are grown in vitro, only a small fraction of the variation in their finite replicative capacity is explained by the chronological age of the donor. Because we had previously shown that telomeres, the terminal guanine-rich sequences of chromosomes, shorten throughout the life-span of cultured cells, we wished to determine whether variation in initial telomere length would account for the unexplained variation in replicative capacity. Analysis of cells from 31 donors (aged 0-93 yr) indicated relatively weak correlations between proliferative ability and donor age (m = -0.2 doubling per yr; r = -0.42; P = 0.02) and between telomeric DNA and donor age (m = -15 base pairs per yr; r = -0.43; P = 0.02). However, there was a striking correlation, valid over the entire age range of the donors, between replicative capacity and initial telomere length (m = 10 doublings per kilobase pair; r = 0.76; P = 0.004), indicating that cell strains with shorter telomeres underwent significantly fewer doublings than those with longer telomeres. These observations suggest that telomere length is a biomarker of somatic cell aging in humans and are consistent with a causal role for telomere loss in this process. We also found that fibroblasts from Hutchinson-Gilford progeria donors had short telomeres, consistent with their reduced division potential in vitro. In contrast, telomeres from sperm DNA did not decrease with age of the donor, suggesting that a mechanism for maintaining telomere length, such as telomerase expression, may be active in germ-line tissue.