To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization （SSH） was performed to detect the differences in gene expression between porcine longissimus dorsi of Meishan X Large Wh...To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization （SSH） was performed to detect the differences in gene expression between porcine longissimus dorsi of Meishan X Large White （MS × LW） F1 hybrids and their parents Meishan pigs. An expression sequence tag （EST） differentially expressed was found, designated as ML556, which was homologous to a hypothetical protein HSPC117, from human hematopoietic stem/progenitor cells （HSPCs）, and the full-length cDNA of porcine HSPC117 was cloned using rapid amplification of cDNA ends （RACE） method. Translation of the mRNA transcript revealed an open reading frame （ORF） of 505 amino acid residues encoding a peroxisomal targeting signal （PTS） with theoretical molecular weight of 55 kDa. Alignment analysis revealed that the deduced protein sequence exhibit 98, 98, 98, 97, and 97% identity with that of cattle, human, dog, rat, and mouse, respectively. The tissue expression analysis indicated that the porcine HSPC117 gene is highly expressed in muscle, spleen, lung, kidney, uterus, ovary and testis, moderately expressed in fat, heart, and liver, and not expressed in stomach and small intestine. The possible role of porcine HSPC117 and its relationship with porcine heterosis were discussed.展开更多
The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of ...The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the c/s-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of - 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.展开更多
基金financially supported by National Basic Research Program of China(973 Program,2006CB102102)National Natural Science Foundation of China(30400313).
文摘To investigate the molecular basis of porcine heterosis, suppression subtractive hybridization （SSH） was performed to detect the differences in gene expression between porcine longissimus dorsi of Meishan X Large White （MS × LW） F1 hybrids and their parents Meishan pigs. An expression sequence tag （EST） differentially expressed was found, designated as ML556, which was homologous to a hypothetical protein HSPC117, from human hematopoietic stem/progenitor cells （HSPCs）, and the full-length cDNA of porcine HSPC117 was cloned using rapid amplification of cDNA ends （RACE） method. Translation of the mRNA transcript revealed an open reading frame （ORF） of 505 amino acid residues encoding a peroxisomal targeting signal （PTS） with theoretical molecular weight of 55 kDa. Alignment analysis revealed that the deduced protein sequence exhibit 98, 98, 98, 97, and 97% identity with that of cattle, human, dog, rat, and mouse, respectively. The tissue expression analysis indicated that the porcine HSPC117 gene is highly expressed in muscle, spleen, lung, kidney, uterus, ovary and testis, moderately expressed in fat, heart, and liver, and not expressed in stomach and small intestine. The possible role of porcine HSPC117 and its relationship with porcine heterosis were discussed.
基金This work was supported by the National Natural Science Foundation of China (Grant Nos. 39770006 & 39925001)
文摘The thermoacidophilic archaeon Sulfolobus shibatae synthesizes a large amount of the 7-ku DMA binding proteins known as Ssh7. Our hybridization experiments showed that two Ssh7-encoding genes existed in the genome of S. shibatae. These two genes, designated ssh7a and ssh7b, have been cloned, sequenced and expressed in Escherichia coli. The two Ssh7 proteins differ only at three amino acid positions. In addition, the c/s-regulatory sequences of the ssh7a and ssh7b genes are highly conserved. These results suggest the presence of a selective pressure to maintain not only the sequence but also the expression of the two genes. We have also found that there are two genes encoding the 7-ku protein in Sulfolobus solfataricus. Based on this and other studies, we suggest that the gene encoding the 7-ku protein underwent duplication before the separation of Sulfolobus species. Binding of native Ssh7 and recombinant (r)Ssh7 to short duplex DNA fragments was analyzed by electrophoretic mobility shift assays. Both native and recombinant forms of the protein behaved in a similar fashion in the assays, suggesting that the interaction of Ssh7 with DNA is not affected either by specific lysine methylation found in the native Ssh7 proteins or by the difference between the two Ssh7 isomers in amino acid sequence. Our data show that Ssh7 binds duplex DNA fragments with a binding size of - 6.6 base pairs and an apparent dissociation constant of (0.7-1.0)×10-7 mol/L under the assay conditions employed in the present study. In addition, Ssh7 binds more tightly to negatively supercoiled DNA than to linear or relaxed DNA.
