A t(3;5)(g25.1;g34) reciprocal chromosomal translocation has been identified in a subset of patients with acute nonlymphocytic leukemia (ANLL) or severe trilineage dysplasia characterized by increased bone marrow megakaryocytes. This proposal is based upon the hypothesis that the t (3;5) rearrangement leads to activation of critical gene products that contribute to leukemogenesis. The planned research has two specific aims: (i) to identify and clone the t(3;5) breakpoint regions on chromosomes 3 and 5, and (ii) to characterize the normal cellular genes and their encoded proteins involved in this translocation and the alteration in their structure or regulation that results from the rearrangement. Since known genes are not altered by the t(3;5), a """"""""reverse genetics"""""""" approach has been initiated to identify the critical genes bases on their proximity to the chromosomal breakpoints. Preliminary studies have localized the chromosome 5 breakpoint of the (3;5) translocation to within a cone megabase DNA segment bounded by two anonymous cosmid clones. The current studies are designed to identify the breakpoint region using yeast artificial chromosome (YAC) clone walking for longer distances and, conventional cosmid chromosome walking techniques for short distances together with fluorescence in situ hybridization analysis of metaphase chromosomes and interphase nuclei to aid in orienting the walk toward the breakpoint. Long rang restriction fragment mapping of the region using DNA from non translocated cells and from t (3;5) derived somatic cell hybrids and patient leukemic cells will help to guide this analysis. Genomic probes derived from the breakpoint region will be used to identify exons for the involved genes and to isolate CDNA clones for the normal and altered genes. Identification, characterization and retroviral expression of the proteins encoded by these genes will allow precise determination of their functional role in normal and malignant cells. Isolation of the t(3;5)-associated genes and their products should permit a more complete understanding of the molecular events that culminate in malignant transformation and may permit the development of more effective, targeted therapeutic strategies.
