RESUMO
Anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase in cell division, which catalyses ubiquitination of cell-cycle regulators. Studying this complex could reveal important information regarding its application in cancer research and therapy. In this study, 4 synthesized small interfering RNAs (siRNAs) were transfected into HEK293T cells to suppress messenger RNA (mRNA) of Apc11; 2 of these reduced the amount of Apc11 mRNA by over 50%. Further experiments showed that rather than causing apoptosis, siRNA transfection led to cell-cycle distributions characterized by less time spent in G2/M phase and more time spent in G1 phase. This phenomenon was specifically induced by Apc11 silencing, as co-transfection of siRNA and an Apc11 plasmid could reverse this distribution bias. Our results suggested that siRNA targeted against Apc11 could hamper entry into G2/M phase. Current efforts are focused on elucidating the function and utility of the APC complex for clinical applications.
Assuntos
Divisão Celular , Fase G2 , Técnicas de Silenciamento de Genes , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase , Células HEK293 , Humanos , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
We have identified LASS2, a previously unknown human homologue of the yeast longevity assurance gene LAG1. The LASS2 transcript is highly expressed in liver and kidney, which is very different from the expression of the previously identified human LAG1 homologue LAG1Hs-1. Radiation hybrid mapping studies indicated that LASS2 is located on chromosome 1q11. Yeast two-hybrid screening and glutathione S-transferase pull-down assays showed that the LASS2 protein interacts with several membrane-associated receptors or transporters. Furthermore, LASS2 protein was able to inhibit the colony formation of human hepatoma cells in vitro, which suggests that this gene may be involved in the regulation of cell growth.
Assuntos
Genes/genética , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Mapeamento Cromossômico , Cromossomos Humanos Par 1/genética , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Feminino , Proteínas Fúngicas/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Íntrons , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento de Híbridos Radioativos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esfingosina N-Aciltransferase , Distribuição Tecidual , Transfecção , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Proteínas Supressoras de Tumor , Técnicas do Sistema de Duplo-HíbridoRESUMO
Amplification of 3q25-q26 was one of the most frequent chromosomal alterations in human ovarian carcinoma. A chromosome microdissection-hybrid selection method was applied to isolate transcribed sequences from a primary ovarian cancer containing high-copy-number amplification of 3q26 using 3q26 band-specific DNAs generated by chromosome microdissection. Using this method, we have isolated a novel candidate oncogene eIF-5A2 (eukaryotic initiation factor 5A2). eIF-5A2 shares 82% identity of amino acid sequence with eIF-5A including the minimum domain needed for eIF-5A maturation by hypusine modification at lysine-50 residue. Amplification and overexpression of eIF-5A2 was frequently detected in primary ovarian cancers and ovarian cancer cell lines. The proliferation-related function of eIF-5A supports that eIF-5A2 is a candidate oncogene related to the development of ovarian cancer.
Assuntos
Neoplasias Ovarianas/genética , Fatores de Iniciação de Peptídeos/genética , Sequência de Aminoácidos , Animais , Galinhas , Aberrações Cromossômicas , Mapeamento Cromossômico , Cromossomos Humanos Par 3 , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Feminino , Amplificação de Genes , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
According to the synonymous codons used in 28 open reading frames from Pichia pastoris, the codon usage in this species was calculated and 19 codons have been inferred to be its optimal codons. The results show that pattern of the codon usage in P. pastoris is similar to that in S. cerevisiae and in K. lactis except for the synonymous codon of glutamic acid, which may be the special bias of P. pastoris.
Assuntos
Códon , Pichia/genéticaRESUMO
Two genes of Kluyveromyces lactis, KlPDI1 and KlMPD1, were studied. They code for a protein disulphide isomerase and its structural and functional homologue, respectively. The KlPDI1 product was 52.6% identical to Pdi1p and the KlMPD1 product 47% identical to Mpd1p of S. cerevisiae. Both genes contained the thioredoxin-active site-related signature. Their C-termini showed a new variant of the endoplasmic reticulum-retention signal, QDEL. A single copy of KlPDI1 was able to complement the growth defect of a pdi1 mutation. KlMPD1 on a multicopy vector partially suppressed the klpdi1 and pdi1 mutations. The Klpdi1 null mutation was lethal. The klmpd1 disruptant was viable, but showed an increased sensitivity to high temperature. Several stress response motifs were present in the upstream sequence of KlMPD1, but not of KlPDI1, whilst the opposite is known for the S. cerevisiae homologues. The viability of the klmpd1 mutant under starvation for nitrogen or carbon source was not different from that of the wild-type. The syntenic relationship is discussed for the KlPDI1 gene regions with respect to the duplicated segments PDI1/EUG1 in S. cerevisiae.
Assuntos
Kluyveromyces/enzimologia , Kluyveromyces/genética , Isomerases de Dissulfetos de Proteínas/genética , Sequência de Aminoácidos , Sítios de Ligação , Carbono/metabolismo , Deleção de Genes , Dosagem de Genes , Genes Essenciais , Genes Fúngicos , Teste de Complementação Genética , Kluyveromyces/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Nitrogênio/metabolismo , Plasmídeos/genética , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Yeast high stable plasmid vector pHC11 was constructed by introducing pEM-BL Yi27 cleaved with SmaI into the SnaBI site of intact 2 microns plasmid. The result of plasmid stability assay revealed that 82% of the host cells still harbored the vector after 50-generations growth in non-selective medium, which confirmed the existence of a non-functional region in 2 microns plasmid. The human interferon alpha A (IFN alpha A) gene expression-secretion cassette was inserted into pHC11, and the yeast transformant was cultured in complex medium. The data showed that the expressed product was 36.8% of the total protein amount in the culture supernatant and the IFN alpha A biological activity was 2.6 x 10(10) units per liter, demonstrating that high-level expression and secretion of IFN alpha A were achieved in yeast by using the stable vector pHC11.