Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA).

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

Purification and characterization of the repressor of the shiga toxin-encoding bacteriophage 933W: DNA binding, gene regulation, and autocleavage.

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (OR). Also typical, when bound at OR, 933W repressor functions as an activator at the PRM promoter and a repressor at the PR promoter. In contrast to other lambdoid bacteriophages, 933W left operator (OL) contains only two repressor binding sites, but the OL-bound repressor still efficiently represses PL transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.