Göran Akusjärvi

A novel adenovirus-2 E1A mRNA encoding a protein with transcription activation properties.

Two novel adenovirus‐2 early region 1A mRNAs, designated 10S and 11S, have been characterized. They differ from the previously described 9S, 12S and 13S mRNAs by having an additional intron removed during mRNA maturation. The 10S and 11S mRNAs encode proteins with mol. wts of 30 and 35 kd. These proteins are encoded in the same translational reading frame as the 12S and 13S mRNA products and differ by lacking 72 amino acids between position 27 and 98. A functional analysis showed that both the 10S and 11S mRNA products are non‐essential for lytic virus growth, and, furthermore, defective in cellular transformation. Interestingly the 11S mRNA product functioned as an efficient transcriptional activator in transient expression assays but was very ineffective as a gene activator during virus growth. Moreover, the virus expressing the 11S cDNA failed to block host cell gene expression although substantial amounts of late proteins were expressed. From the biological properties of the E1A cDNA mutants it was possible to localize two functional domains in the E1A proteins; one region required for transcriptional activation (amino acids 140‐185), and a second domain required for adenovirus transformation and the control of viral and cellular gene expression during a lytic infection (amino acids 27‐98).

Proteins with transcription regulatory properties encoded by human adenoviruses

Of the more than 30 genes encoded by the adenovirus genome, no less than six have been shown to encode proteins that have transcription regulatory properties. None of them is a sequence-specific DNA-binding protein. They act to modulate the activity of cellular transcription factors by causing their phosphorylation or dephosphorylation, by physically interacting with them, or by dissociating transcription factor inhibitory protein complexes.

An imporved nuclear extract preparation method

A rapid, efficient, and highly reproducible procedure for nuclear extract preparation is described. The method uses lysolecithin (lysophosphatidylcholine) to disrupt plasma membranes and requires no detergents or douncing. Soluble extracts prepared by this method are comparable to conventional nuclear extracts in all assays tested. Lysolecithin nuclear extracts are competent for RNA polymerase II and III transcription, DNA replication, pre-mRNA splicing, and sequence specific DNA-protein binding. Nuclear extracts can be prepared on a small scale (10(7) cells) as well as for preparative purposes by this method.

The carboxy-terminal exon of the adenovirus E1A protein is required for E4F-dependent transcription activation.

The adenovirus‐2 E1A 289R transcription activator protein contains a 49 amino acid sequence (designated CR3) that has been suggested to represent the minimal domain required for E1A‐induced activation of viral early transcription. We show here that the non‐conserved carboxy‐terminal E1A exon contains two interchangeable elements that are required for efficient CR3‐dependent transactivation of the adenovirus E4 promoter in HeLa cells. These two elements do not encode independent transactivation functions and have been designated auxiliary regions (ARs) 1 and 2. The effects of AR1 and AR2 are not additive, suggesting that they contribute a mechanistically analogous function in transcription. Previous studies have suggested that two cellular transcription factors, ATF‐2 and E4F, can function together with E1A to induce transcription of the E4 promoter. The importance of respective factors for E4 transcription has not been resolved. We find that E1A activation of E4F, but not ATF‐2 (or other ATF factors), is AR1‐ and AR2‐dependent. This result suggests that E1A induction of the E4 promoter in HeLa cells is primarily mediated by E4F.

Independent transformation activity by adenovirus-5 E1A-conserved regions 1 or 2 mutants.

Two conserved regions (CR1 and CR2) on the adenovirus E1A proteins have previously been shown to be required for cooperation with the ras oncogene in the transformation of primary rodent cells. Sequences within these regions are essential for the ability of E1A to associate with the 105K product of the retinoblastoma susceptibility gene, p105-RB, as well as with other cellular proteins, including a 107K (p107) and a 300K (p300) species. In this paper, we show that CR1 mutants deficient in p300 binding and CR2 mutants with lost or reduced binding of p105-RB and/or p107 have a low, but not abolished focus formation activity. In contrast, CR1/CR2 double mutants were deficient in focus formation, suggesting that the transformation activities displayed by the single CR1 or CR2 mutants were due to an independent transformation activity by both CR1 and CR2. No strict correlation between p105-RB binding and E1A-mediated transformation was observed. The E1A enhancer repression function was found to correlate with the binding of p300 but not with E1A-mediated transformation. Complex formation between E1A and p107, similar to the p105-RB binding, required sequences within both CR1 and CR2. The CR2 sequences required for binding of p107K or p105-RB were overlapping, but not identical. Finally, a larger segment of CR2 was required for stable complex formation between E1A and phosphorylated forms of p105-RB or p107 compared to corresponding unphosphorylated species.

Glucocorticoid hormones may partially substitute for adenovirus E1A in cooperation with ras

The effects of hormonal promotion of T24-ras oncogene-transfected rat embryo fibroblasts (REF) were compared to cotransformation of these cells with adenovirus E1A and ras. Cotransfection of E1A + ras resulted in the appearance of morphologically transformed cells which were very efficiently established into cell lines. Addition of glucocorticoid hormones to T24-ras-transfected REF cells resulted in cells with a transformed morphology and a capacity to form foci. These foci were, however, inefficiently established into stable cell lines. Removal of hormone from growing cells resulted in retarded growth, suggesting that the hormone acted as a growth factor on these cells. Both E1A-transformed cells and hormone-treated ras-transformed cells showed a reduction in synthesis of high molecular weight tropomyosin isoforms and a decreased expression of surface fibronectin. Control experiments demonstrated that the effects of hormone were mediated through the glucocorticoid receptor. Our findings suggest that glucocorticoid hormones may promote the in vitro growth of ras-initiated REF cells into stably transformed cell lines, but that this ability is limited compared to that of adenovirus E1A.

Progressive Factors in Oncogene Transfected Rodent Embryo Fibroblasts

Activated ras oncogenes are inefficient in the transformation of primary rodent embryo fibroblasts (REFs). However, in unison with viral or cellular genes such as the adenovirus E1A, polyoma large-T, mutant p53 or myc genes, transformation will occur. No common biochemical activity has been described for these cooperating oncogenes. We and others have demonstrated that treatment of T24-ras oncogene transfected REF cells with the glucocorticoid dexamethasone (DEX) facilitates transformation (Martens et al., 1988; Yamashita et al., 1988; Marshall et al., Exp. Cell. Res, in press;). During a critical period between 1–3 months after transfection, cellular growth was found to be dependent on glucocorticoid. Hormone independence invariably develops during subsequent culture. These observations raise the possibility of hormonal promotion and progression of rat embryo fibroblasts expressing activated ras genes, and may offer a convenient model for studies of promotion and progression in vitro.