A cDNA derived from the human PML/RARA (promyelocytic leukemia/retinoic acid receptor, alpha) fusion gene resulting from the T(15;17) chromosomal translocation associated with acute promyelocytic leukemia was modified to introduce into the PML protein an SV40 nuclear localization signal and a K160R point mutation that removes a sumoylation site. The modified cDNA has replaced the protein-coding region of the human S100A8 (also called MRP8) gene in the pUCMRP8d vector. The resultant construct contains the promoter, exon 1, intron 1 and the untranslated portion of exon 2 from S100A8 followed by the PML/RARA cDNA and the untranslated part of S100A8 exon 3. S100A8 is known to be expressed in early stages of the myeloid cell lineage and in peripheral neutrophils and monocytes, but not in tissue macrophages. The translocation, between breakpoint cluster region 1 (bcr1) in intron 6 of PML and a site in intron 2 of RARA, joins most of the 5' region of the PML gene to most of the 3' portion of the RARA gene. The resultant chimeric mRNA, which includes the alternatively spliced PML exon 5, contains a single open reading frame encoding a protein comprising the SV40 nls, 530 N-terminal amino acids (aa) of PML including its endogenous nls and two of the three sumoylation sites - the lysine at aa position 160 having been replaced by arginine (K160R) - and 402 C-terminal aa from RARA including the DNA- and retinol-binding domains, but not the N-terminal A region. All transgenic lines selected for study were shown by both western blot and immunofluorescence analysis to express the chimeric protein. (J:95945)
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