Murine A type retroviruses promote high levels of gene expression in embryonal carcinoma cells

Abstract

The expression of Intracisternal A Particle (IAP) genes in the mouse embryonal carcinoma cell line PCC3 was investigated by cDNA cloning and transient gene expression assays. A group of 26 IAP cDNA clones, products of transcriptionally active IAP proviruses, were selected from a cDNA library made from undifferentiated PCC3 cell RNA. Several of these clones were characterized by restriction enzyme mapping and DNA sequence analysis. The DNA sequence in both the promoter and structural regions of two cDNAs closely resembles those of IAP genomic clones. Three new sequence elements were identified within the U3 region, an Sp1 transcription-factor-binding site, an adenovirus E1a enhancer sequence and a region of homology to a promoter element of adenovirus E4 gene. Hybrid constructs were made that place the U3/R region of the IAP cDNAs immediately 5' to the chloramphenicol acetyl transferase (CAT) gene. IAP-CAT constructs were transfected into PCC3 cells, and cell extracts prepared and analysed for CAT enzyme activity and CAT RNA levels. IAP-CAT transfected cells were shown to contain substantial levels of CAT enzyme activity and to accumulate much greater levels of CAT RNA than two standard promoters, pRSVcat and pSV2cat. The ability of these A type retroviral promoters to function in PCC3 cells is in direct contrast to the near total restriction of normal C type retroviral expression in EC cells.