Preliminary Screening GAGA-like Element Binding Protein Cloning And Gene Regulatory Role Of Hsp90

To explore GAGA-like element binding protein in human Jurkat cells. Yeast one-hybrid system was used to screen the GAGA-like element binding proteins in HTLV-1 transformed Jurkat cell Matchmaker cDNA fusion library. 9 positive clones were obtained, and 6 out of the 9 clones positively hybridize with Jurkat cell the cDNA probe. 2 positive clones designated as GLBP1 and GLBP2 respectively, which could bind specifically with GAGA-like element, were confirmed by electrophoretic mobility shift assay (EMSA). Sequence analysis indicated that complete open reading frame of GLBP1 was obtained and encodes 478 amino acids, which is highly homologous to BPOZ protein.To study the effect of GLBP1 protein on the hsp90 gene expression, a competitive RT-PCR-based approach was established to quantify promoter activity of stress gene. With experimental conditions optimized, the relatively transcriptional levels can be detected without using any sophisticated instruments. CAT reporter genes driven by either the 5' upstream sequences of human hsp90α or the regulatory fragment of hsp90β was co-transfected with the sense or antisense GLBP1 cDNA expression plasmid into Jurkat cells followed by competitive RT-PCR-based CAT assay. The results indicated that GLBP1 protein had almost no effect on the expression of hsp90β gene. On the other hand, the protein could promote the heat shock inducible expression of hsp90α, and only work on chromatin level. The result suggests the importance of the research of gene regulation on chromatin level.To reconstitute the chromatin in vitro, the insect expression system was employed to express and purify the chromatin assembly factors ACF and NAP-1, and core histone was purified from calf thymus, which is the preliminary works for the further research of gene regulation on chromatin template.