We are examining cellular mechanisms that regulate intracisternal A-particle (IAP) expression in normal and transformed mouse cells. As one aspect of the program, we are studying the interaction of cellular factors with cis-regulatory sequences in the IAP LTR. A novel enhancer-binding protein fraction has been isolated by DNA affinity chromatography from transformed human and mouse cells. The fraction, designated EBP-80, contains two protein components of 75 and 85 kDa. We have now shown that EBP-80 acts to stimulate transcription from IAP LTRs in a cell-free system. The EBP-80 binding domain in a number of recently expressed and/or transposed IAP elements contains a particular sequence variant which has higher affinity for the factor and confers greater promoter activity on the LTR. Methylation of a single CpG residue in the EBP-80 binding domain reduces both affinity for the factor and promoter activity of the LTR. EBP-80 has been isolated from cultured human cells in sufficient quantity to permit peptide separation, microsequencing and development of specific oligonucleotide probes for gene cloning. We are also studying whether particular elements of the multicopy IAP gene family are selectively activated in different cell types, and the means by which this might be accomplished. Isolation and sequencing of multiple cDNA clones from BALB/c thymocytes and stimulated splenic B-cells reveals that the two cell types express very limited and largely overlapping sets of IAP transcripts. Only a few highly related IAP elements seem to be responsible for most of the IAP expression in these cells. Efforts to clone these elements from genomic DNA are in progress. We plan to follow IAP expression during the stages of plasmacytoma development since enhanced expression may be a very early marker of B-cell transformation.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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Division of Cancer Biology and Diagnosis
United States
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