Cloning,Sequence Analysis,Expression Feature And Biological Function Prediction Of A Nove1 Leukemia-associated Gene LRP16

In order to find and clone relapse-related genes of acute leukemia (AL), we had ever compared the methylat ion difference of DNA CpG island between diagnostic and relapsed acute myeloid leukemia (AML) using restriction landmark genomic scanning technique. A 2948bp DNA fragment containing two Not I restriction enzyme sites had been cloned. Further research demonstrated that CpG within one Not I was significantly methylated in relapsed acute myeloid leukemia. In this research starting from this DNA fragment with the aid of electronic hybridization in nr and hEST (human Expression Sequence Tags) data bases, the complete coding cDNA sequence of a human gene which is associated with relapsed AML has been cloned employing 3'-and5'-RACE technique(Rapid Amplification of cDNA End ).After confirming it as a novel gene, our research group named it LRP16 and registered it in GenBank in 1999 December (Accession Number: AF202922) Subsequently, using its cDNA sequence and human genome sequencing information releasing by internet, LRP16 gene was exactly located on chromosome 11q12. 1. Within this subregion of chromosome 11q12. 1, one small gene cluster including LRP16, FLRT1, FLJ2O113 was observed. The gene LRP16 spans 8. 8kb on genomic DNA and contains 11 exons. The sequence of its promote region is CpG rich and lacks conserved TATA box. The full-length transcript of LRP16 is 1225bp and has high GC content (reaching to 67. 5%) Because two initiating codon ATG was observed within the same reading frame of LRP16 cDNA and the second context has nearly conserved Kozak feature, we postulated that LRP16 gene may translate two types of peptide chain, 5 the length type and the short . The length type has 325 amino acids and the short has 243aa, the only difference between these two is the Nerminal. The result of multi issue Northern blot hybridization displayed that LRP16 gene had different expression pattern in different tissues, highly in overy and colon mucosa, moderately in prostate and small intestine, lower in testis and lowest in spleen, thymus and peripheral blood. A fourthxoneleted splicing type was screened through the prokaryotic expression of LRP16 shortype coding chain using BL21 E.Coli bacterium, the expression amount of which is comparably lower in most tissues. To investigate the possible significance of LRP16 during tumor development and progression and during hematopoietic maturation, bioinformatic and experimental method were employed to detect the expression state of LRP16 in tumor development and different hematopoietic stages. Using CGAP/SAGEmap database provided by NCBI as experimental material, irtual Northern?program as tool, LRP16 expression pattern was compared among multiple tumor tissues, cell lines and corresponding normal tissues. Semiuantified RTCR was adopted to compare the expression amount among normal blood cells and leukemie cells. Results of SAGE showed that LRP16 gene was highly expressed in several human tumor cells than in their normal counterparts. LRP16 was not detected in cord blood cells. The expression amount of LRP16 gene in normal bone marrow and PBSC ( peripheral blood stem cells ) mobilized by GSF were significantly lower than in normal peripheral blood, priary leukemic cells and leukemic cell lines (P<0. 0001) suchasHL60, K562andU937. These results suggested that LRP16 is a gene associated with hematopoietic 6...