High frequency trans-splicing in a cell line producing spliced and polyadenylated RNA polymerase I transcripts from an rDNA-myc chimeric gene.

The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3' end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5' exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells.

Modification of diamine oxidase activity in vitro by metabolites of asparagine and differences in asparagine decarboxylation in normal and virus-transformed baby hamster kidney cells

The oxidation of putrescine in vitro by pig kidney diamine oxidase (EC 1.4.3.6) was increased in the presence of 2-oxosuccinamic acid and malonamic acid. It was inhibited by 3-aminopropionamide, oxaloacetate and pyruvate. 2-Oxosuccinamate was derived from asparagine in virus-transformed baby hamster kidney (BHK) cells growing in tissue culture. Asparagine was decarboxylated more efficiently by transformed than by normal BHK cells. In BHK cells transformed by polyoma virus (Py BHK), 2-oxosuccinamate is the most likely immediate precursor of the 14 CO2 arising from [U-14C] asparagine, and there was some evidence for its formation in an asparagine-dependent clone of BHK cells before and after their transformation by hamster sarcoma virus (respectivey Asn- and HSV Asn-). The relationship between 2-oxosuccinamate and pyruvate and the possible roles of these two substances in controlling cellular diamine oxidase activity are discussed (AU)