NOVEL MITOGENIC REGULATORY GENE

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We have identified a novel cellular gene, 322, which potentially encodes a regulatory or tumor suppressor function. This gene was isolated from a cDNA library representing genes whose transcription is low in untransformed cells and even lower (i.e.- suppressed) in cells transformed by the activated oncogene, v-src. We assumed that these genes might include candidate regulators of mitogenesis, and that the loss of their expression might contribute to the transformed phenotype. The steady-state level of 322 RNA is depressed >15-fold in src-, ras- and fos-transformed fibroblasts in comparison to untransformed controls, but not in raf- neu- or mos-transformed cells. This down-regulation is not the result of gross loss or damage to the 322 gene allele, as determined by southern blotting. Activation of a ts-src allele or addition of fetal calf serum to starved cells results in a rapid increase in 322 mRNA levels (1-2 hours) followed by a rapid decrease (after another 4-6 hours) to suppressed levels, indicating that 322 transcription is directly controlled by v-src and mitogenic stimuli. Over-expression of the 322 cDNA results in a significant decrease in the proliferation rates of untransformed and transformed cells, and significantly suppresses src- and fos-induced morphological transformation. Preliminary sequence analysis of the putative full-length 322 cDNA (5.4Kb) indicates that it is novel. Although the largest open-reading frame would encode a 170kD product, in vitro transcription/translation of this cDNA yields a 207kD polypeptide whose increased molecular weight cannot be attributed to N- linked glycosylation. Two glycine/arginine/lysine-rich regions, homologous to Epstein-Barr virus nuclear antigens, are found in the N- terminal domain of the putative 322 product. These data strongly suggest that 322 encodes a novel regulator of mitogenesis. Our overall aim is to characterize the function of this gene product. Specifically, we will i) characterize the mechanism of src- and ras-induced transcription suppression of 322 (initiation vs. meassage stability), ii) determine the effects of 322 over-expression in transformed and untransformed cells, iii) express the 322 protein product in vitro and raise specific anti- sera to this product, and iv) identify cellular proteins which specifically interact with the 322 product using either the yeast two- hybrid system or co-immunoprecipitation analysis. Our long-term goal is to elucidate pathways or sites of action of the 322 product. We envision that these data will help characterize the involvement of 322 in mitogenic control in untransformed cells and in tumor suppression in transformed cells. These data would also increase our knowledge of how oncogenes subvert cellular pathways in the process of inducing morphological transformation and tumorigenesis.