The long term objective of this research is to develop an understanding of the role of Myc in the control of cell proliferation and differentiation. One discovery that would help clarify this role is the identification and characterization of cellular proteins that interact with Myc in vivo. Thus, the specific goal of this research application is to develop the technology needed to isolate these cellular proteins. This will be accomplished by expressing in human cells a fusion protein which contains he Myc oligomerization domains fused to the bacterial lac repressor protein, followed by purification of the fusion protein complex using DNA affinity chromatography. HeLa cell lines which express high levels of the lac repressor-Myc fusion protein will be obtained by infecting cells with retroviruses which contain a lac repressor-Myc fusion gene. This research will establish that the fusion protein is capable of binding to lac operator DNA, and that it interacts specifically with cellular proteins via the Myc oligomerization domains. The fusion protein complex will be purified using a lac operator DNA affinity column, and proteins that interact with the fusion protein via the Myc oligomerization domains will be identified by SDS-polyacrylamide gel electrophoresis. The procedure will be scaled-up in order to obtain sufficient amounts of each protein for a partial amino acid sequence determination. Antibodies to each protein will be raised in rabbits, and used to screen a HeLa cell-lambda bacteriophage CDNA library to obtain cDNAs will subjected to DNA sequencing and the amino acid sequence of each protein deduced. The amino acid sequences will be compared to all sequences contained in the Protein Data Bank to identify domains that are homologous to known proteins. Northern and Western blot analysis will be used to test the hypothesis that the expression of these proteins is regulated in a growth-specific, differentiation-specific and developmental fashion. The knowledge gained from this research will lead to a better understanding of the molecular basis of cancer, and could be used to design new ways to reverse the cancer process.
| Freytag, S O; Paielli, D L; Gilbert, J D (1994) Ectopic expression of the CCAAT/enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells. Genes Dev 8:1654-63 |
