RESUMO
A unique structural variant of the cardiac L-type voltage-dependent calcium channel alpha 1 subunit cDNA was isolated from libraries derived from normal human heart mRNA. The deduced amino acid sequence shows significant homology to other calcium channel alpha 1 subunits. However, differences from the rabbit heart alpha 1 include a shortened N-terminus, a unique C-terminal insertion, and both forms of an alternatively spliced motif IV S3 region. The shortened N-terminus provides optimal access to consensus sequences thought to facilitate translation. Northern blot analysis revealed a single hybridizing mRNA species of 9.4 kb. The gene for the human heart alpha 1 subunit was localized specifically to the distal region of chromosome 12p13. The cloned alpha 1 subunit was expressed in Xenopus oocytes and single-channel analyses revealed native-like pharmacology and channel properties.
Assuntos
Canais de Cálcio , Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Clonagem Molecular , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Cricetinae , DNA/química , DNA/isolamento & purificação , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Coelhos , RatosRESUMO
The temperature-sensitive cell cycle mutation bimE7 of Aspergillus nidulans causes cells to become blocked in mitosis at a restrictive temperature. Previous work has shown that this mitotic block is induced even when cells are arrested in the S or G2 phase. The mitotic block is also observed in cells carrying a null mutation in bimE, obtained by molecular disruption of the gene (Osmani, S.A., Engle, D.B., Doonan, J.H., and Morris, N.R. (1988) Cell 52, 241-251), indicating that a lack of bimE function is responsible for the phenotype. We have cloned the bimE gene by complementation of the mutant phenotype and have isolated and sequenced its corresponding cDNA. The gene product is encoded by a 6.5-7-kilobase mRNA. The deduced amino acid sequence suggests a protein with three transmembrane domains. The sequence contains numerous potential N-glycosylation sites and several putative cAMP-dependent phosphorylation sites. No homologous protein sequences were found in the common data bases. The bimE gene product is a novel component in the regulation of mitosis.
Assuntos
Aspergillus/genética , Proteínas de Ciclo Celular , Genes Fúngicos , Mutação , Sequência de Aminoácidos , Aspergillus/citologia , Aspergillus/crescimento & desenvolvimento , Sequência de Bases , Northern Blotting , Southern Blotting , Ciclo Celular , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/genética , Teste de Complementação Genética , Mitose , Dados de Sequência Molecular , Fenótipo , Plasmídeos , Poli A/genética , RNA/genética , RNA Mensageiro , Mapeamento por Restrição , TemperaturaRESUMO
Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L-type VDCC. The alpha 1 and alpha 2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the alpha 1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle alpha 1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxy-nucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue-specific isoforms of the L-type VDCC.
Assuntos
Canais de Cálcio/metabolismo , Miocárdio/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Clonagem Molecular , Dados de Sequência Molecular , Oócitos/metabolismo , RNA Mensageiro/genética , Coelhos , Mapeamento por Restrição , XenopusRESUMO
We describe here recent work on the molecular genetics of mitosis in the filamentous fungus Aspergillus nidulans. Aspergillus is one of three simple eukaryotes with powerful genetic systems that have been used to analyze mitosis. The modern molecular biological techniques available with this organism have made it possible to use mutations to identify genes and proteins that play an important role in mitosis. Three Aspergillus genes that affect mitosis are described. One gene, nimA, is specifically expressed late in the cell cycle and codes for a putative protein kinase that induces mitosis, even in cells blocked in S-phase. The second gene, bimG, codes for a putative phosphatase that interacts functionally with the nimA kinase. The third gene, bimE, codes for a protein that suppresses mitosis during interphase, apparently by keeping nimA turned off. None of these genes appear to be similar to any of the genes affecting mitosis that have been characterized in other eukaryotes, but rather appear to be elements of a system that prevents mitosis from occurring during interphase.
Assuntos
Aspergillus nidulans/genética , Genes Fúngicos , Genes Reguladores , Sequência de Aminoácidos , Aspergillus nidulans/citologia , Humanos , Mitose , Dados de Sequência Molecular , Mutação , Proteínas Quinases/genética , Homologia de Sequência do Ácido NucleicoRESUMO
In Aspergillus nidulans the temperature-sensitive cell cycle mutation bimE7 causes chromosome condensation and pre-anaphase spindle formation to occur at restrictive temperature. By constructing double mutants between bimE7 and S phase or G2 phase mutants and blocking DNA replication with hydroxyurea, we demonstrate that bimE7 can cause chromatin condensation and spindle formation in cells held in S or G2. Thus bimE7 overrides normal control systems that prevent mitosis from prematurely occurring during S or G2. We show that bimE7 is a loss of function mutation and propose that bimE normally functions to negatively control a positive mitotic inducing factor, possibly the cell cycle gene nimA.
Assuntos
Aspergillus nidulans/genética , Ciclo Celular , Cromatina/ultraestrutura , Genes Fúngicos , Interfase , Fuso Acromático/ultraestrutura , Aspergillus nidulans/ultraestrutura , DNA/genética , Replicação do DNA/efeitos dos fármacos , DNA Recombinante , Imunofluorescência , Hidroxiureia/farmacologia , Microtúbulos/ultraestrutura , Índice Mitótico , Mutação , Plasmídeos , Temperatura , Transformação Genética , Tubulina (Proteína)/análiseRESUMO
MPM-2 is a monoclonal antibody that interacts with mitosis-specific phosphorylated proteins in many different organisms. Immunocytochemistry of tissue culture cells has shown that MPM-2 stains centrosomes, chromosomes, kinetochores, and spindles. In this paper, we demonstrate that MPM-2 staining colocalizes with the spindle pole body (SPB) of Aspergillus nidulans and that SPB staining varies during the mitotic cycle. In an unsynchronized population, about one-fourth to one-third of the cells stain with MPM-2 at the spindle plaques or SPBs. Nuclei in mitosis have two SPBs localized at the ends of the spindle, both of which stain with MPM-2. To determine when MPM-2 staining appears, we have examined the effects of temperature-sensitive cell-cycle mutations that block nuclear division in S or G2. Only a very small fraction of cells blocked in S-phase stain with MPM-2. In contrast, a large fraction of cells blocked in G2 stain brightly at the SPB. These data suggest that MPM-2 reactivity of SPBs appears in G2. Moreover, the fact that cells blocked in G2 showed MPM-2 staining but no spindles suggests that reactivity of SPBs occurs prior to mitosis but is not sufficient to trigger spindle formation. When G2-blocked cells were downshifted to permissive temperature, they generated a mitotic spindle with an SPB at each end. Both SPBs stained with MPM-2 in all of the mitotic cells.
