Characterisation of the Fit-1 Common Integration Locus

Genetics, Veterinary science

Barr, Nighean I

University:University of Glasgow

关键词: Genetics, Veterinary science;   

Others  :  http://theses.gla.ac.uk/75389/1/13815572.pdf

来源: University of Glasgow

【 摘 要 】

The aim of this study was to identify the gene which was affected by proviral integration within the Fit-1 common integration locus. The Fit-1 locus was identified in the cat as a common site of insertion for feline leukaemia virus (FeLV) in thymic lymphomas induced by FeLV-myc recombinant viruses. Thus precedent suggested that it harbours a gene which cooperates with c-myc in T-cell leukaemogenesis. Using a zooblot positive probe from the major insertion cluster, the mouse homologue was mapped to chromosome 10 and the human homologue to chromosome 6 (N. Barr et al, in press and Appendix 2). A cosmid clone containing homologous human sequences was isolated and used to generate a PAC clone contig around the human Fit-l locus, revealing that it is only 100kb upstream of the c-myb gene. A number of retroviral insertion sites have been mapped close to c-myb on mouse chromosome 10, Mml1, Ahi-l and Mis-2. Thus, the close proximity of c-myh and Fit-1 in the human genome and the lack of obvious transcribed sequences at these loci might suggest that retroviral insertions within this cluster of loci activates the c-myb gene by long-range activation. However, analysis of c-myb expression in tumours rearranged at Ahi-l, Mis-2 and Mml1 failed to show evidence of long- range activation. In contrast, careful analysis of the FT1 cell line which carries an insertion at Fit-1 and c-myc revealed evidence of approximately 2-fold over-expression of c-myb which may, at least in part, be due to a decreased turnover of c-myb RNA. This subtle change in expression may have been missed in previous analyses of primary tumour RNA from tumours rearranged at Fit-1, Ahi-1 or Mis-2. These results raised the unexpected prospect that Myc and Myb can collaborate in T-lymphoma development. In addition to over-expression of the c-myb gene in the FT1 cell line, there was also evidence of an altered c-myb transcript structure in this cell line. Further analysis revealed that this was due to alternative splicing of the c-myb gene involving the alternative exon 9A, a form which was approximately 5-fold over-expressed in FT1 cells. Although this form has been associated with both normal and activated forms of c-myb, levels seen in the FT1 cell line were higher than in previous studies. Thus, it appears that c-myb may be the true target of insertions at the Fit-1/Mml1/Ahi-1/Mis-2 cluster and that, at least for Fit-1 retroviral integration may promote the alternative splicing of c-myb, possibly favouring a more oncogenic isoform.