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1.
Cell ; 107(6): 715-26, 2001 Dec 14.
Article in English | MEDLINE | ID: mdl-11747808

ABSTRACT

Separation of sister chromatids in anaphase is mediated by separase, an endopeptidase that cleaves the chromosomal cohesin SCC1. Separase is inhibited by securin, which is degraded at the metaphase-anaphase transition. Using Xenopus egg extracts, we demonstrate that high CDC2 activity inhibits anaphase but not securin degradation. We show that separase is kept inactive under these conditions by a mechanism independent of binding to securin. Mutation of a single phosphorylation site on separase relieves the inhibition and rescues chromatid separation in extracts with high CDC2 activity. Using quantitative mass spectrometry, we show that, in intact cells, there is complete phosphorylation of this site in metaphase and significant dephosphorylation in anaphase. We propose that separase activation at the metaphase-anaphase transition requires the removal of both securin and an inhibitory phosphate.


Subject(s)
CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Chromatids/metabolism , Endopeptidases , Metaphase/physiology , Anaphase/physiology , Animals , CDC2 Protein Kinase/genetics , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone , Cyclin B/genetics , Cyclin B/metabolism , Cyclin B1 , HeLa Cells , Humans , Mass Spectrometry , Nuclear Proteins , Oocytes/physiology , Peptide Mapping , Phosphoproteins , Phosphorylation , Saccharomyces cerevisiae Proteins , Separase , Xenopus laevis
2.
Genes Dev ; 13(9): 1140-55, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10323865

ABSTRACT

We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins-Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1-as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein-DNA conformation that evokes the active centromere.


Subject(s)
Cell Cycle Proteins , Fungal Proteins/metabolism , Kinetochores/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Base Sequence , Centromere/genetics , Centromere/metabolism , DNA Primers/genetics , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Fungal/metabolism , Fungal Proteins/chemistry , Fungal Proteins/genetics , Macromolecular Substances , Models, Biological , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
3.
Proc Natl Acad Sci U S A ; 96(7): 3757-62, 1999 Mar 30.
Article in English | MEDLINE | ID: mdl-10097110

ABSTRACT

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.


Subject(s)
Centromere/ultrastructure , DNA, Fungal/metabolism , DNA, Fungal/ultrastructure , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/ultrastructure , Fungal Proteins/metabolism , Fungal Proteins/ultrastructure , Nuclear Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Chromosome Mapping , Chromosomes, Fungal/ultrastructure , DNA, Fungal/chemistry , DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , Kinetochores , Microscopy, Atomic Force
4.
EMBO J ; 15(14): 3611-20, 1996 Jul 15.
Article in English | MEDLINE | ID: mdl-8670864

ABSTRACT

We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.


Subject(s)
DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Kinetochores/metabolism , Nuclear Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Base Sequence , Centromere/metabolism , Cyclin-Dependent Kinases/isolation & purification , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , DNA, Fungal , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Fungal Proteins/genetics , Fungal Proteins/isolation & purification , Molecular Sequence Data , Rabbits , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
5.
J Bacteriol ; 178(6): 1614-22, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8626289

ABSTRACT

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.


Subject(s)
Bacterial Proteins/metabolism , Cyclic AMP Receptor Protein/metabolism , Escherichia coli/genetics , Promoter Regions, Genetic , Repressor Proteins/metabolism , Uridine Phosphorylase/genetics , Base Sequence , Carrier Proteins , DNA Footprinting , Enzyme Repression , Escherichia coli Proteins , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Mutagenesis , Protein Binding , Transcription, Genetic , Uridine Diphosphate/analysis
6.
Z Exp Angew Psychol ; 38(2): 264-78, 1991.
Article in German | MEDLINE | ID: mdl-1858433

ABSTRACT

This experiment provides a test for the common sense knowledge that moderate physical exercise leads to mood improvement. Furthermore, it was tested whether light exercise intensifies negative feeling states or alleviates them. 30 female and 30 male students of psychology served as subjects (mean age 25.3 years, SD = 4.8). After being exposed to a mood induction procedure designed to elicit either a positive or negative feeling state, the subjects had to pedal a bicycle ergometer with 0, 50 or 75 Watt load. Cardiovascular variables and self-reports of mood states were assessed during a baseline period, after the mood induction, following the ergometer exercise, and after a follow-up period. The mood induction procedures were successful, but only for a short duration. Physiological activation was observed according to the ergometer loadings. Moderate physical exercise led to an increase of positive feeling states (Concentration) and a decrease of negative feeling states (Tiredness) in the follow-up period. Corresponding changes in tension related states could not be observed, probably due to the weakness of the mood induction procedure employed and the low level of energetic activation reached.


Subject(s)
Affect , Arousal , Exercise , Adult , Anxiety/psychology , Exercise Test/psychology , Female , Humans , Male
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