Search

Adriamycin (Adr) is one of the most powerful antitumor drugs. Its therapeutic effect may be due to its cyclic reduction-oxidation and, thus, generation of oxygen radicals. Using the spin-trap 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) and EPR we have demonstrated that in an enzymatic system consisting of NADPH, NADPH-cytochrome P-450 reductase, and Fe(EDTA)2 Adr stimulates formation of .OH radicals in the presence of DNA or RNA with equal efficiency. Incubation of nucleic acids in the Adr-dependent reaction generating .OH radicals resulted in extensive degradation of double- and single-stranded DNA, but did not effect RNA. In contrast, both DNA and RNA were effectively destroyed in a footprinting system, ascorbate-Fe(EDTA)2-H2O2, which generates .OH radicals in massive quantities. Fluorescence assays indicated that Adr forms stable complexes with ds- and ss-DNA but reacts only slightly with RNA. We conclude that the formation of Adr-nucleic acid complex is necessary for .OH radical-mediated cleavage of the latter, and thus, Adr may be regarded as a chemical nuclease acting in situ.

The complete nucleotide sequence of human abl RNA containing exon Ia was determined. It spans 5598 nucleotides and codes for a protein of 1130 amino acids. The 3' untranslated region contains two short open reading frames and multiple ATTT(A) motifs characteristic of short lived mRNAs. Computer analysis of the abl protein predicts four domains distinct with regard to surface probability and chain flexibility. Nucleotide analysis of the abl segment within a bcr-abl cDNA cloned from the K562 cell line indicated no further alterations within the coding region. A bcr-abl construct containing this segment transformed, together with c-myc, RAT-1 cells and produced a highly active tyrosine kinase.

The unintegrated circular DNA form of Moloney murine sarcoma virus (MSV) has been cloned in bacteriophage lambda. Discrete deletions in the viral genome were shown to occur during propagation of recombinant phage in Escherichia coli. Heteroduplex and restriction enzyme analyses indicated the deletion of tandemly repeated sequences within certain of the cloned MSV DNA inserts. Cloned MSV DNA was used to prepare a probe composed of its acquired cellular (src) sequences, shown previously to be necessary for MSV transformation. Analysis of EcoRI digests of normal mouse cellular DNA revealed the presence of a single 14-kilobase-pair fragment containing these sequences which lacked contiguity with endogenous type C helper viral information of the same cells. Thus, the sarcoma virus-specific sequences of MSV are represented within the normal mouse genome in a manner analogous to that of a cellular gene.

Several natural RNAs were compared with respect to their template activities for the DNA polymerase of Rous Sarcoma Virus during a 2-hr incubation period. 60-70S viral RNA was found to be a 5- to 10-fold better template than heat-dissociated Rous viral RNA, influenza virus RNA, tobacco mosaic virus RNA, or ribosomal RNA. Denatured salmon DNA is a little better, and poly(dAT) is 2-4 times better as a template for the enzyme than is 60-70S Rous viral RNA. The 60-70S RNAs of different strains of avian tumor viruses have very similar template activities for a given avian tumor virus DNA polymerase. Oligo(dT) or oligo(dC) were found to enhance the template activity of heat-dissociated Rous viral RNA 20- to 30-fold, and that of other natural RNAs tested one- to several-fold. DNA syntheses of 1-24% were obtained during a 2-hour incubation of the enzyme with the above RNA templates. The results suggest that the enzyme prefers partially doublestranded or hybrid regions of RNAs for optimal DNA synthesis, but certain regions of single-stranded RNA can also serve as templates.Poly(dAT) competes with viral RNA for purified DNA polymerase during DNA synthesis, as would be expected if RNA- and DNA-dependent DNA synthesis was performed by at least one common active site of the same enzyme.

The human ALL-1 gene is involved in acute leukemia through gene fusions, partial tandem duplications or a specific deletion. Several sequence motifs within the ALL-1 protein, such as the SET domain, PHD fingers and the region with homology to DNA methyl transferase are shared with other proteins involved in transcription regulation through chromatin alterations. However, the function of these motifs is still not clear. Studying ALL-1 presents an additional challenge because the gene is the human homologue of Drosophila trithorax. The latter is a member of the trithorax-Polycomb gene family which acts to determine the body pattern of Drosophila by maintaining expression or repression of the Antennapedia-bithorax homeotic gene complex. Here we apply yeast two hybrid methodology, in vivo immunoprecipitation and in vitro `pull down' techniques to show self association of the SET motifs of ALL-1, TRITHORAX and ASH1 proteins (Drosophila ASH1 is encoded by a trithorax-group gene). Point mutations in evolutionary conserved residues of TRITHORAX SET, abolish the interaction. SET–SET interactions might act in integrating the activity of ALL-1 (TRX and ASH1) protein molecules, simultaneously positioned at different maintenance elements and directing expression of the same or different target genes. Oncogene (2000) 19, 351–357. [ABSTRACT FROM AUTHOR]