In vivo detection of the BCR/ABL1 protein [thesis]

Ying Chen
The BCR/ABL1 fusion protein is found in virtually all cases chronic myeloid leukemia (CML) and a large proportion of acute lymphoblastic leukemia (ALL). The fact that the BCR/ABL1 fusion protein is crucial for the development of leukemia makes this fusion protein an attractive target for therapy development. We have developed a strategy for the in vivo detection of the BCR/ABL1 fusion protein, in which the presence of the BCR/ABL1 fusion protein is detected intracellularly and if the fusion
more » ... ein is present an arbitrary action is initiated in the cell (e.g. mark the cells or selectively kill the cells). Our BCR/ABL1 detection strategy is based on protein-protein interactions. Two detection proteins are expressed in the cells: 1) protein A, a GAL4-DNA binding domain/BCR interacting protein fusion protein (GAL4DBD-BAP-1) and 2) protein B, a GAL4-activation domain/ABL interacting protein fusion protein (GAL4AD-CRKL). Only when BCR/ABL1 is present in the cell, do protein A, protein B, and BCR/ABL1 form a trimeric complex which activates the transcription of reporter genes under the control of GAL4-upstream activating sequence (UAS). A proof of principle for the strategy was implemented in the yeast system. We did not use full length BAP-1 or CRKL but only those portions of the proteins that directly interacted with BCR or ABL, respectively. We showed in the yeast two hybrid system, that the C-terminus of BAP-1(amino acids 617-879) binds to full length BCR. The site of interaction of CRKL and ABL was confirmed to be the N-terminal SH3 domain (SH3n) of CRKL as described in the literature. Yeast cells (strain CG1945) transformed with a protein A expressing plasmid (pGBT9-BAP), a protein B expressing plasmid (pGAD424-CRKLSH3n), and a BCR/ABL expressing plasmid (pES1/BCR-ABL) showed expression of the reporter genes HIS3 and LACZ. The expression of the HIS3 reporter gene was assayed by growth of the yeast cells on medium lacking histidine. The expression of the LACZ gene was verified by a beta-galactosidase filter assay. [...]
doi:10.5282/edoc.5319 fatcat:tiwdr2u5k5a5dnffvlgmayezvy