RESUMO
Completion of human genome sequencing has greatly accelerated functional genomic research. Full-length cDNA clones are essential experimental tools for functional analysis of human genes. In one of the projects of the New Energy and Industrial Technology Development Organization (NEDO) in Japan, the full-length human cDNA sequencing project (FLJ project), nucleotide sequences of approximately 30 000 human cDNA clones have been analyzed. The Gateway system is a versatile framework to construct a variety of expression clones for various experiments. We have constructed 33 275 human Gateway entry clones from full-length cDNAs, representing to our knowledge the largest collection in the world. Utilizing these clones with a highly efficient cell-free protein synthesis system based on wheat germ extract, we have systematically and comprehensively produced and analyzed human proteins in vitro. Sequence information for both amino acids and nucleotides of open reading frames of cDNAs cloned into Gateway entry clones and in vitro expression data using those clones can be retrieved from the Human Gene and Protein Database (HGPD, http://www.HGPD.jp). HGPD is a unique database that stores the information of a set of human Gateway entry clones and protein expression data and helps the user to search the Gateway entry clones.
Assuntos
Bases de Dados de Proteínas , Proteínas/genética , Proteínas/metabolismo , Proteômica , Clonagem Molecular , DNA Complementar/química , Eletroforese em Gel de Poliacrilamida , Genes , Humanos , Internet , Biossíntese de Proteínas , Proteínas/química , Interface Usuário-ComputadorRESUMO
Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.
Assuntos
Clonagem Molecular/métodos , Genoma Humano/genética , Engenharia de Proteínas/métodos , Proteoma/genética , Proteoma/metabolismo , Proteínas Recombinantes/metabolismo , Sistema Livre de Células , HumanosRESUMO
A new member of the piericidin family, JBIR-02, was isolated from mycelium of Streptomyces sp. ML55 together with two known piericidin derivatives, piericidin A(1) and IT-143-B. The structure was determined on the basis of spectroscopic data. JBIR-02 inhibited nuclear export of beta-arrestin in HeLa cells at the concentration of 20 microM.
Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Piridinas/química , Piridinas/farmacologia , Piridonas/farmacologia , Streptomyces , Células HeLa/efeitos dos fármacos , Humanos , Análise EspectralRESUMO
The centromere is a chromatin structure essential for correct segregation of sister chromatids, and defects in this region often lead to aneuploidy and cancer. We have previously reported purification of the interphase centromere complex (ICEN) from HeLa cells, and have demonstrated the presence of 40 proteins (ICEN1-40), along with CENP-A, -B, -C, -H and hMis6, by proteomic analysis. Here we report analysis of seven ICEN components with unknown function. Centromere localization of EGFP-tagged ICEN22, 24, 32, 33, 36, 37 and 39 was observed in transformant cells. Depletion of each of these proteins by short RNA interference produced abnormal metaphase cells carrying misaligned chromosomes and also produced cells containing aneuploid chromosomes, implying that these ICEN proteins take part in kinetochore functions. Interestingly, in the ICEN22, 32, 33, 37 or 39 siRNA-transfected cells, CENP-H and hMis6 signals disappeared from all the centromeres in abnormal mitotic cells containing misaligned chromosomes. These results suggest that the seven components of the ICEN complex are predominantly localized at the centromeres and are required for kinetochore function perhaps through or not through loading of CENP-H and hMis6 onto the centromere.
Assuntos
Autoantígenos/metabolismo , Centrômero/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Complexos Multiproteicos/metabolismo , Aneuploidia , Autoantígenos/genética , Proteínas de Ciclo Celular , Centrômero/genética , Proteína Centromérica A , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Interfase , Cinetocoros/metabolismo , Metáfase/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The sequence of the human genome has been determined. The next task is to determine the function of the genes. Classifying cellular forms of proteins encoded by human cDNA clones is a primary step toward understanding the biological role of proteins and their coding genes. We report here our ongoing work on an automatic system to facilitate this classification. Our system handles the transfection, incubation, acquisition of microscopic images of the cells, and the classification of forms there appearing in the images. Our system correctly classified proteins by their forms at a rate of 90% in feasibility studies.