CDNA Genes Formed after Infection with Retroviral Vector Particles Lack the Hallmarks of Natural Processed Pseudogenes

Abstract

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.