RESUMO
Deficiency of human glycerate kinase leads to D-glycerate acidemia/D-glyceric aciduria. Through PCR cloning assisted by in silico approach, we isolated the human glycerate kinase genes--Glycerate Kinase 1 (GLYCTK1) and its alternatively splicing variant--Glycerate Kinase 2 (GLYCTK2), which might be associated with D-glycerate acidemia/D-glyceric aciduria. The locus of GLYCTK gene is mapped to 3p21. PCR amplification in seventeen human tissue cDNAs revealed that both GLYCTK1 and GLYCTK2 are expressed widely almost in all these tissues. The expression of mouse Glyctk in various tissues was demonstrated by in situ hybridization. Both GLYCTK1 and GLYCTK2 proteins are localized in cytosol, and GLYCTK2 proteins are specifically localized in mitochondria. Present results revealed the characteristic expression pattern of murine Glyctk in neural system, skeleton muscle, supporting that glycerate kinase is implicated in D-glycerate acidemia/D-glyceric aciduria.
Assuntos
Ácidos Glicéricos/sangue , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Isoformas de Proteínas/genética , Processamento Alternativo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , DNA Complementar/isolamento & purificação , Variação Genética , Humanos , Hibridização In Situ , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Proteínas Recombinantes de Fusão/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Distribuição Tecidual/genéticaRESUMO
The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Expressão Gênica , Músculo Esquelético/química , Filogenia , Sequência de Bases , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Primers do DNA , DNA Complementar/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Funções Verossimilhança , Modelos Genéticos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Present work reported the cloning and characterization of a human novel RNA binding gene Partner of NOB1 (PNO1), with a length of 1637bp and a putative open reading frame of 759 bp, isolated from human kidney. It is composed of seven exons and is localized on chromosome 2p14. Western blot showed that the molecular weight of PNO1 is about 35kDa. RT-PCR results in 16 human tissues indicated that PNO1 is expressed mainly in liver, lung, spleen and kidney, slightly in thymus, testis, ovary, respectively, but not in heart, brain, skeletal muscle, placenta, pancreas, prostate, small intestine, colon and peripheral blood leukocytes. GFP fusion expression in mammalian cells exhibited its localization in the nucleus, especially in nucleoli. Subcellular localization of thirteen GFP fusion PNO1 deletion proteins showed that the region of 92-230 aa is solely responsible for its nucleolar retention, and KH domain alone is not sufficient for nucleolar retention. The PNO1 family shows significant conservation in both eukaryotes and prokaryotes.
