RESUMO
Entry into mitosis requires p34(cdc2), which activates downstream mitotic events through phosphorylation of key target proteins. In Aspergillus nidulans, the NIMA protein kinase has been identified as a potential downstream target and plays a role in regulating chromatin condensation at mitosis. nimA- mutants arrest in a state that physically resembles interphase even though p34(cdc2) is fully active. Despite evidence for the existence of NIMA-like activities in a variety of cell types, the only bona fide NIMA homologue that has been identified is the nim-1 gene of Neurospora crassa. We report here the isolation of a fission yeast NIMA homologue, and have designated this gene fin1 and the 83 kDa predicted protein p83(fin1). Overexpression of fin1 promotes premature chromatin condensation from any point in the cell cycle independently of p34(cdc2) function. Like NIMA, p83(fin1) levels fluctuate through the cell cycle, peaking in mitosis and levels are greatly elevated by removal of C-terminal PEST sequences. Deletion of fin1 results in viable but elongated cells, indicative of a cell cycle delay. Genetic analysis has placed this delay in G2 but, unlike in nimA mutants of Aspergillus, p34(cdc2) activation appears to be delayed. Interaction of fin1 mutants with other strains defective in chromatin organisation also support the hypothesis of p83(fin1) playing a role in this process at the onset of mitosis. These data indicate that NIMA-related kinases may be a general feature of the cell cycle and chromatin organisation at mitosis.
Assuntos
Proteínas de Ciclo Celular , Cromatina/metabolismo , Proteínas Fúngicas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Schizosaccharomyces/enzimologia , Schizosaccharomyces/fisiologia , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fase G2/genética , Meiose/genética , Dados de Sequência Molecular , Quinase 1 Relacionada a NIMA , Quinases Relacionadas a NIMA , Neurospora crassa , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Schizosaccharomyces/genéticaRESUMO
Combinatorial methods of chemical synthesis allow the creation of molecular libraries having immense diversity. The utility of such libraries is dependent upon identifying the structures of the molecules so prepared. We describe the construction of a peptide combinatorial library, having 117,649 different members, synthesized on beads and indexed with inert chemical tags. These tags are used as a binary code to record the reaction history of each bead. The code can be read directly from a single bead by electron capture capillary gas chromatography. We demonstrate the correct selection of members of the library on the basis of binding to a monoclonal antibody.
Assuntos
Peptídeos/química , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Cromatografia Gasosa , Epitopos , Dados de Sequência Molecular , Peptídeos/síntese química , Proteínas Proto-Oncogênicas c-myc/imunologiaRESUMO
We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100- to 700-fold enrichment of target sequences.
