Abstract: The PML gene is involved in the 15;17 chromosomal translocation of acute promyelocytic leukemias (APL). It encodes a nuclear phosphoprotein which is associated, in vitro, with growth suppressor activity. We are currently studying the biochemical and biological properties of the coiled-coil region of the PML protein. It consists of four clusters of hydrophobic aminoacids and is thought to mediate homodimerization and heterodimerization with the PML/RARa mutant protein. The integrity of this region is required for the biological activity of PML and PML/RARa mutant protein. In this report we show the production of PML-heptads as a fusion protein with p-GEX vector. The fused protein was first solubilized, then purified with affinity chromatography. The heptads were obtained after column proteolitic cleavage, then were further analized with FPLC. The FPLC chromatograms, carried out in non denaturing conditions, show the ability of the heptads to elute as a high molecular weight aggregate confirming the evidence that this region is responsible for protein complex formation in "in vivo" experiments. Finally the purified peptide was structurally characterized by means of circular dichroism (CD) measurements. The CD spectra indicate the presence of an a-helix structure. A molecular model of heptad dimer, based on the sequence comparison with other homologue sequences, is also presented.

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Abstract: We describe here the rationale, implementation and results of a bioinformatic strategy for large-scale identification and annotation of chromosomal translocations in tumours, based on sequence and annotation comparison between human transcriptome and EST partial cDNA sequences derived from tissues or cell lines. We also illustrate how the sequencing and subsequent careful bioinformatic analysis of a number of identified candidate translocation cDNAs revealed the complexity of distinguishing recombination from true translocation events. Finally, we suggest some EST filtering and cleaning strategy for pursuing EST-based “in silico” translocation identification projects.