open access

https://doi.org/10.11501/3076826

https://hdl.handle.net/2115/50201

胚性腫瘍細胞分化の分子機構 : 制御転写調節エレメントとその結合タンパク質

英語

1994-03-25

Hokkaido University

78p.

BOX DNA was previously isolated from the DNA sequence inserted in the enhancer B domain of the mutant polyomavirus DNA （fPyF9）． We also reported that BOX DNA functioned negatively on DNA replication and transcription of another polyomavirus mutant （PyhrN2） in F9-28 cells, a subclone of mouse F9 embryonal carcinoma （EC） cells expressing the polyomavirus large T antigen． ln this study，1 demonstrate that BOX DNA enhances transcription from the thymidine kinase （TK） promoter in various EC cells． One or three copies of BOX DNA， linked to the bacterial chloramphenicol acetyltransferase （CAT） gene under the control of the herpes simplex virus （HSV） TK promoter， activated the promoter activity in F9， P19 and ECA2 cells． The band shift assays using BOX DNA as a probe revealed that specific binding protein(s) were present in all the EC cells examined： The patterns of BOX DNA-protein complexes were the same among them． A mutation introduced within BOX DNA abolished the enhancer activity as well as the formation of the specific DNA－protein complexes． ln non－EC cells including L and Balb3T3 cells， the enhancer activity of BOX DNA on the TK promoter was not observed， although binding proteins specific to the sequence exist． ln band shift assays， the patterns of the DNA－protein complexes of either L or Balb3T3 cells were different from those of EC cells． Furthermore， the enhancer activity of BOX DNA decreased upon differentiation induction in all the EC cells examined， of different origins and distinct differentiation ability． ln parallel with the loss of enhancer activity， the binding proteins specific for BOX DNA decreased in these cells. Moreover， 1 cloned a genomic DNA of F9， termed BOXF1， containing BOX DNA sequence approximately 400 bp upstream from the RNA start site of the gene． BOXF1， containing a TATA－like motif and the binding clements for Sp1 and Oct in addition to BOX DNA， possessed promoter activity deduced by a BOXF1-CAT construct． Deletion analyses of the construct revealed that the transcription of BOXF1 gene is regulated by BOX DNA，preferentially in undifferentiated EC cells than differentiated cells． Hence， BOX DNA isprobably a novel transcriptional element related to EC oe11 differentiation． Furthermore， I cloned a cDNA corresponding to BOXF1． The gene expressed highly in undifferentiated EC cells． lts expression decreased after differentiation induction． lt is thus thougt that the gene may be a target gene of BOX DNA binding proteins．Moreover， I purified BOX DNA binding proteins of P19， EC cells， and Raji， non－EC cells having no pluripotency． BOX DNA had no enhancer activity in Raji cells， where specific binding proteins were present． A 44kDa protein was purified from P19 cells. as the binding protein specific to BOX DNA 100/60 kDa proteins were mainly purified in Raji cells， although the 44kDa protein was slightly co-purified． Thus， it is speculated that BOX DNA enhancer activity is positively or repressively regulated by complex mechanisms under the influence of pluripotency and differentiated states of the cells.

1994-03-25

乙第4506号

博士(薬学)

博士論文

Version of Record