RESUMO
Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.
Assuntos
Desenvolvimento Embrionário/fisiologia , Manose/metabolismo , Processamento de Proteína Pós-Traducional , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Clonagem Molecular , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Glicosilação , Manosiltransferases/genética , Manosiltransferases/metabolismo , Dados de Sequência Molecular , Filogenia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genéticaRESUMO
A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.
Assuntos
Ciona intestinalis/embriologia , Ciona intestinalis/genética , Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Perfilação da Expressão Gênica , Larva/genética , Regulação para Cima , ZigotoRESUMO
Although membrane-associated proteins are related to many diseases and are important targets for drug discovery, their expression is often difficult in bacterial hosts such as Escherichia coli. To overcome this limitation, here, we focused on a novel host-vector system in zebrafish for the expression of human protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 (hPOMGnT1) which is related to muscle-eye-brain disease. For the expression of hPOMGnT1, the vector pZex-EGFP-pXI-hPOMGnT1 was constructed and injected into fertilized eggs. Using this system, we demonstrated that recombinant hPOMGnT1 was successfully expressed in the whole bodies of zebrafish embryos.
Assuntos
N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Embrião não Mamífero/metabolismo , Vetores Genéticos/genética , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferases/biossíntese , Peixe-Zebra/embriologiaRESUMO
The genome of the basal choradate Ciona intestinalis contains a basic set of genes with less redundancy compared to the vertebrate genome. Extensive EST analyses, cDNA sequencing, and clustering yielded "Ciona intestinalis Gene Collection Release 1," which contains cDNA clones for 13,464 genes, covering nearly 85% of the Ciona mRNA species. This release is ready for use in cDNA cloning, micro/macroarray analysis, and other comprehensive genome-wide analyses for further molecular studies of basal chordates.