1.Application Of Suppression Subtractive Hybridization (SSH) In Screening, Isolating And Cloning Relapse-related Genes Of Acute Leukemia 2.Development And Confirmation Of Polymerase Chain Transformation-block PCR: A Novel Method For Screening Differential

Object(1)Leukemia is a common hematological malignancy. Drug resistance and relapse are the main obstacles to clinical treatment. So far there is no report about relapse-related genes, moreover, the known mechanism of drug resistance can not explain all the leukemia cases. Suppression subtractive hybridization (SSH) is a new method of phenotypic cloning which had the characteristics of convenience, good sensitivity and reprodution. For this reason, it is an ideal tool for particular phenotypic cloning of target genes and has been applied more and more in cancer research. The objective of this study was to construct a subtractive relapse-related cDNA library from untreated and relapsed marrows of the same patient with acute lymphocytic leukemia, and screened, isolated and cloned the cDNA sequences which might be related to leukemia relapse(.2)Up to now, there are a variety of methods for screening of differentially expressed genes, including microarray, representational difference analysis (RDA), suppression subtractive hybridization (SSH) , mRNA differential display RT-PCR (DDRT-PCR) and serial analysis of gene express(SAGE).Although a lot of differentially expressed genes have been identified by these methods, all of them have some disadvantage and need to be improved,such as cost, labor intensive and short-length cDNA. Thus, development of new methods for screening of differentially expressed genes is still necessary. In this study, it is our goal to develop a novel method for screening of differentially expressed genes.Methods (1) Applying suppression subtractive hybridization (SSH) to construct a subtractive relapse-related cDNA library from untreated and relapsed marrows of the same patient with acute lymphocytic leukemia. Dot blot and DNA sequencing were used to confirm the ESTs(expressed sequence tags) of differentially expressed genes. One of unknown ESTs was chosed to clone full-length cDNA by in silicon cloning combination with RT-PCR, and the gene expression in normal individual and patients with acute leukemia was detected by RT-PCR.(2) Driver and tester templates prepared with long primer and short primer respectively were used to verify the selective amplification of tester templates by block PCR;Different distance segments of a same sequence were prepared to verify the transformation effect by polymerase chain transformation. Multidrug resistance gene (mdr-1) is expressed in KAR cells (K562 cells resistance to Doxorubicin) but not or low expressed in K562 cells. KAR cells were used as tester and K562 cells as driver. To demonstrate the selective amplification of tester templates, PCR was used to compare the abundance of common sequence (β-actin) and differentially expressed gene (mdr-1) before and after polymerase chain transformation-block PCR.Results (1) The subtractive relapse-related cDNA library from untreated and relapsed marrows of the same patient with acute lymphocytic leukemia was successfully constructed. (2) 34 clones were successfully sequenced. Homolog analysis confirmed 20 clones as unknown EST, and among them, 13 ESTs were confirmed as differentially expressed genes which might be relapse-related. 7 of them have been successfully submitted to GenBank. The GenBank accession numbers were CB412259- CB412265. (3) Two full-length sequences of eIF4E splicing variants were obtained, termed splicing variant1 and variant2 of eIF4E. The results of ORF finder and protein blast revealed that these two variants encode different proteins which are partially identical to eIF4E protein. (4)The results of RT-PCR showed that there was no different expression of eIF4E variant2 between normal individuals and acute lymphocytic patients, but the abundance of eIF4E variant2 in patients with acute mononuclear leukemia was significantly lower than normal individual and acute lymphocytic patients(P<0.05). (5) Block-PCR can selectively amplify the tester templates which were made with short primer, and simultaneously block driver template amplification. Polymerase chain transformation enables the short distant sequence to extent according to the long distant sequence. (6) The abundance ofβ-actin reduced about 10 cycles after polymerase chain transformation-block PCR, and mdr-1 show little change. The results indicated that the subtractive effect of polymerase chain transformation-block PCR was about 10 cycles.Conclusion1. The subtractive relapse-related cDNA library from untreated and relapsed marrows of the same patient with acute lymphocytic leukemia was successfully constructed.2. Two full-length sequences of eIF4E splicing variants were obtained. The protein encoded by these two variants are partially identical to eIF4E protein.3.Splicing variant2 of eIF4E was significant lower expressed in patients with acute mononuclear leukemia than normal individuals and acute lymphocytic patients(P<0.05).4. we have developed a novel method for screening differentially expressed genes termed polymerase chain transformation-block PCR, which has the characteristics of convenience, time-saving, and full-length cDNA being available directly.