Han-chun Chen1, Qiao Meng. 1. Research Center for Molecular Biology, XiangYa School of Medicine, Central South University, Changsha 410078, China.
Abstract
OBJECTIVE: To identify hemin-induced gene expression in K562 cells. METHODS: Poly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively. RESULTS: Fifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins. CONCLUSIONS: Hemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.
OBJECTIVE: To identify hemin-induced gene expression in K562 cells. METHODS:Poly A(+) RNAs were isolated from hemin-induced (tester) and non-induced K562 cells (driver) respectively, and double-strand cDNAs were synthesized by reverse transcription. The forward subtracted cDNA library was constructed by using suppression subtractive hybridization (SSH) techniques. The recombinant plasmids were extracted and the positive clones were identified by EcoR I digestion after the amplification and screening of the library. The inserts were amplified by PCR. The upregulated cDNA transcripts were identified by reverse dot blot hybridization, DNA sequencing and homology analysis with GenBank database "blast" respectively. RESULTS: Fifteen upregulated clones were identified and most of them were homologous to the mRNA sequences of protein with known function, including globin epsilon1, glutathione S-transferase like glutathione transferase Omega (GSTTLp28), selenoprotein X1 (SEPX1), triosephosphate isomerase (TPI1), ribosomal protein L7 (RPL7), ribosomal protein S13 (RPS13), ferritin light polypeptide, globin A gamma, RAD 51 homolog C(RAD51C), ferritin heavy polypeptide, X-box binding protein (XBP1). A part of the hemin-induced cDNA clones exhibited sequence similarities to that of the GenBank registered mRNA with unknown function of their expressed proteins, including the cDNA clones of DKFZp434I116, hypothetical protein HSPC014 and NOL1R2 proteins. CONCLUSIONS:Hemin mainly induces the genes expression related to erythroid differentiation, protein synthesis and metabolism in K562 cell. There results provide comprehensive information useful for the differential gene expression in hemin-induced erythroid differentiation and for further function study of genes involved in hematopoiesis.