Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae.

Entry into anaphase and proteolysis of B-type cyclins depend on a complex containing the tetratricopeptide repeat proteins Cdc16p, Cdc23p, and Cdc27p. This particle, called the anaphase-promoting complex (APC) or cyclosome, functions as a cell cycle-regulated ubiquitin-protein ligase. Two additional subunits of the budding yeast APC were identified: The largest subunit, encoded by the APC1 gene, is conserved between fungi and vertebrates and shows similarity to BIMEp from Aspergillus nidulans. A small heat-inducible subunit is encoded by the CDC26 gene. The yeast APC is a 36S particle that contains at least seven different proteins.

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins.

Entry into anaphase and exit from mitosis depend on a ubiquitin-protein ligase complex called the anaphase-promoting complex (APC) or cyclosome. At least 12 different subunits were detected in the purified particle from budding yeast, including the previously identified proteins Apc1p, Cdc16p, Cdc23p, Cdc26p, and Cdc27p. Five additional subunits purified in low nanogram amounts were identified by tandem mass spectrometric sequencing. Apc2p, Apc5p, and the RING-finger protein Apc11p are conserved from yeast to humans. Apc2p is similar to the cullin Cdc53p, which is a subunit of the ubiquitin-protein ligase complex SCFCdc4 required for the initiation of DNA replication.

A screen for genes required for meiosis and spore formation based on whole-genome expression.

BACKGROUND: Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS: To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS: Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Electrochemical behaviour of aluminium(III) and beryllium(II) perchlorates in dimethylformamide.

Well defined voltammetric peaks of A1(III) and Be(II) were obtained in DMF at the HMDE, and the nature of the electrochemical process in this medium was investigated. Adsorption and chemical reaction coupled with the charge transfer were observed in both cases. The optimum depolarizer concentration and scan-rate for analytical determination were established. Close similarities were found in the electrochemical behaviour of Al(III) and Be(II), and differences were in degree rather than of kind.

[Primary prevention of malignant tumors]. / Primární prevence zhoubných nádoru.

Primary prevention represents an effective instrument in the battle against tumour diseases. Prevention is aimed to lower the mortality for this disease by means of reduction of the cancer incidence. Our article reviews the main fields of primary prevention, namely the principles of proper nutrition and regimen. Effects of balance in energy and related nutrients, essential components of the diet, types of meals together with problems of food processing and storage are discussed from the view of the cancer prevention. Each chapter brings applicable preventive recommendations. Smoking is considered as a risks factor not only for tumours, but also for other serious diseases. Other carcinogenic factors and possible preventive measures are referred in the last chapter. Though we know that the primary cancer prevention requires active approach of the whole society, we are certain that medical professionals in every day contact with patients can highly improve the public informedness. The article submits substrate for such discussions.

APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5.

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)). Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7). We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref. 4). Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.

Characteristics of NADPH-dependent thymidylate synthetase purified from Streptomyces aureofaciens.

NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme.

Functional genomics identifies monopolin: a kinetochore protein required for segregation of homologs during meiosis i.

The orderly reduction in chromosome number that occurs during meiosis depends on two aspects of chromosome behavior specific to the first meiotic division. These are the retention of cohesion between sister centromeres and their attachment to microtubules that extend to the same pole (monopolar attachment). By deleting genes that are upregulated during meiosis, we identified in Saccharomyces cerevisiae a kinetochore associated protein, Mam1 (Monopolin), which is essential for monopolar attachment. We also show that the meiosis-specific cohesin, Rec8, is essential for maintaining cohesion between sister centromeres but not for monopolar attachment. We conclude that monopolar attachment during meiosis I requires at least one meiosis-specific protein and is independent of the process that protects sister centromere cohesion.

Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins.

Certain cell types give rise to progeny that adopt different patterns of gene expression in the absence of any differences in their environment. Cells of budding yeast give birth to mother and daughter cells that differ in that only mother cells express the HO endonuclease gene and thereby switch mating types. We describe the identification of five genes, called SHE1-SHE5, that encode cytoplasmic proteins required for mother-specific HO expression. She1p, which is identical to the minimyosin Myo4p, and She3p are not, however, mother-specific proteins. On the contrary, they accumulate in growing buds. She proteins might be required for the transport of factors that promote HO repression from the mother cell into its bud. In an accompanying paper, we show that SHE genes are needed for the accumulation in daughter nuclei of Ash1p, a repressor of HO.

Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication.

Sister chromatid cohesion is crucial for chromosome segregation during mitosis. Loss of cohesion very possibly triggers sister separation at the metaphase --> anaphase transition. This process depends on the destruction of anaphase inhibitory proteins like Pds1p (Cut2p), which is thought to liberate a sister-separating protein Esp1p (Cut1p). By looking for mutants that separate sister centromeres in the presence of Pds1p, this and a previous study have identified six proteins essential for establishing or maintaining sister chromatid cohesion. Four of these proteins, Scc1p, Scc3p, Smc1p, and Smc3p, are subunits of a 'Cohesin' complex that binds chromosomes from late G1 until the onset of anaphase. The fifth protein, Scc2p, is not a stoichiometric Cohesin subunit but it is required for Cohesin's association with chromosomes. The sixth protein, Eco1p(Ctf7p), is not a Cohesin subunit. It is necessary for the establishment of cohesion during DNA replication but not for its maintenance during G2 and M phases.

A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis.

A multisubunit complex, called cohesin, containing Smc1p, Smc3p, Scc1p, and Scc3p, is required for sister chromatid cohesion in mitotic cells. We show here that Smc3p and a meiotic version of Scc1p called Rec8p are required for cohesion between sister chromatids, for formation of axial elements, for reciprocal recombination, and for preventing hyperresection of double-strand breaks during meiosis. Both Rec8p and Smc3p colocalize with chromosome cores independently of synapsis during prophase I and largely disappear from chromosome arms after pachytene but persist in the neighborhood of centromeres until the onset of anaphase II. The eukaryotic cell's cohesion apparatus is required both for the repair of recombinogenic lesions and for chromosome segregation and therefore appears to lie at the heart of the meiotic process.

Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.