To identify mRNAs with altered expression in Rous sarcoma virus (RSV)-transformed cells, we screened a chicken embryo fibroblast (CEF) cDNA library by differential hybridization. One clone, designated R1H, showed markedly elevated mRNA expression in RSV-transformed cells. Nucleotide sequence analysis indicated that R1H mRNA encodes 78-kilodalton glucose-regulated protein (GRP78). Chicken GRP78 was found to be very highly conserved in comparison with rat GRP78 (96% identity between chicken and rat amino acid sequences). In contrast to previous observations, we found that GRP78 was induced in RSV-transformed cells in the absence of glucose deprivation. When cells were grown in glucose-supplemented medium, the level of GRP78 mRNA was approximately fivefold higher in RSV-transformed CEF than in transformation-defective virus-infected or uninfected CEF. Similar changes in GRP78 protein content were also found. Using a temperature-sensitive mutant of RSV and supplemental glucose, we found a gradual increase in the level of GRP78 mRNA beginning at 4 h after shiftdown to permissive temperature. Uridine supplementation did not block the induction seen in CEF infected with a temperature-sensitive mutant. These results indicate that GRP78 is induced by p60`VS in the absence ot glucose deprivation.One approach to understanding how oncogenes induce neoplasia is to identify genes with altered expression in transformed cells. Changes in the expression of cellular genes may be relevant to phenotypic features of the transformed cell, such as uncontrolled growth, invasiveness, resistance to immune rejection, and metastatic spread. In addition, studying the signals that control the expression of such genes can provide insight into the mechanisms of action of oncogenes.We have been investigating the changes in cellular gene expression that occur in chicken embryo fibroblasts (CEF) after transformation by Rous sarcoma virus (RSV). In the initial stage in this project, we have isolated clones from CEF cDNA or RSV-transformed CEF cDNA libraries that are expressed at different levels in RSV-transformed cells compared with uninfected cells (38). It is likely that many of the mRNAs identified by this approach are regulated in normal cell growth. Altered expression Qf such mRNAs in RSV-transformed CEF may contribute to or result from altered growth processes after transformation. One such gene, designated 9E3, was found to encode a protein similar to human connective tissue-activating peptide (1, 38). In this paper we describe the isolation and characterization of another cDNA and report on the expression of this gene in RSV-transformed and normal cells. MATERIALS AND METHODSCell cultures and viruses. Secondary CEF were maintained in Ham F10 medium supplemented with 5% calf serum, 1% chicken serum, 10% tryptose pentose broth, 100 U of penicillin per ml, 50 ,ug of streptomycin per ml, and 2 pLg of * Corresponding author. amphotericin B per ml. The medium was changed every 24 h unless otherwise noted. In some experiments, glucose was added ...

We assayed phosphatidylinositol (PI) kinase (EC 2.7.1.67) activity in detergent extracts of nontransformed or virus-transformed cells. Nontransformed chicken embryo fibroblasts (CEF) contain PI kinase activity with an apparent specific activity of 20 pmol/min per mg of protein. This activity sedimented as a single peak with a molecular weight of approximately 60,000 in a glycerol gradient, although immunoprecipitation with anti-p60src sera showed that the PI kinase activity is distinct from p60c-src. Extracts from CEF transformed by Rous sarcoma virus, Fujinami sarcoma virus, or avian sarcoma virus UR2 showed no elevation of PI kinase activity over nontransformed CEF. Removal of the oncogene products from extracts by immunoprecipitation did not change the level of PI kinase activity in extracts, suggesting that putative virus-coded PI kinases do not make a significant contribution to overall levels of PI kinase activity in transformed cells. Additionally, P140Wagf-Is was separated from cellular PI kinase by phosphocellulose chromatography. This partially purified fraction contained low PI kinase activity distinct from Pl40W-fPs; indicating that p140Wzg.-fiS has no detectable PI kinase activity.There has been a great deal of interest in an intracellular signaling system that utilizes inositol lipids and might be involved in regulating cell growth (1,26,29,32). Various stimuli induce the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C to diacylglycerol and inositol trisphosphate which act as second messengers. Diacylglycerol activates protein kinase C, and inositol trisphosphate mobilizes intracellular calcium. This hydrolysis is followed by the phosphorylation of phosphatidylinositol (PI) and phosphatidylinositol-4-monophosphate (PIP). PIP2 is formed from PI through stepwise phosphorylation by PI kinase (EC 2.7.1.67) and PIP kinase (EC 2.7.1.68). These kinases are known to exist in a variety of tissues (7,8,15,17,(20)(21)(22)30). There are also corresponding phosphomonoesterases (23,33,36), and the inositol lipids mentioned above are thought to be in equilibrium with each other through "futile cycles" constituted by these kinases and esterases. The close relation between this signaling system and cell growth regulation was suggested by the fact that several growth factors could induce PI turnover (10,14,34). In addition, tumor-promoting phorbol esters can stimulate protein kinase C directly (5).The stimulation of this system in the transforming process of a tumor virus was first suggested by Diringer and Friis (9) PI. They suggested that various oncogene products possessing tyrosine kinase activity might also have PI kinase activity and that PI kinase activity is responsible for transformation.In this report, we asked two questions relevant to two important points of the above hypotheses. (i) If elevated PI turnover is a direct effect of the PI kinase activity of oncogene products, then what is the difference in the overall PI kinase activity between transformed and nontransfor...