ISOLATION AND CHARACTERIZATION OF A NOVEL MITOGENIC REGULATORY GENE, 322, WHICH IS TRANSCRIPTIONALLY SUPPRESSED IN CELLS TRANSFORMED BY SRCAND RAS

In an attempt to isolate novel regulatory and/or tumor suppressor gene s, we identified cDNAS whose abundance is low in NIH 3T3 cells and fur ther decreased following the expression of the activated oncogene, v-s rc. The transcription of one such gene, 322, is suppressed at least 15 -fold in src-, ras-, and fos-transformed cells and 3-fold in myc-trans formed cells but is unaffected in raf-, mos-, or neu-transformed cells . Activation of a ts-v-src allele in confluent 3Y1 fibroblasts resulte d in an initial increase in 322 mRNA levels after 1 to 2 h followed by a rapid decrease to suppressed levels after 4 to 8 h. Morphological t ransformation was not detected until 12 h later, indicating that the a ccumulation of 322 transcripts is regulated by v-src and not as a cons equence of transformation. Addition of fetal calf serum to starved sub confluent NIH 3T3 or 3Y1 fibroblasts resulted in a similar biphasic re gulation of 322, indicating that 322 transcription is responsive to mi togenic factors, Sequence analysis of a putative full-length 322 cDNA clone (5.4 kb) identified a large open reading frame (ORF) encoding a 148.1-kDa product. In vitro transcription and translation of the 322 c DNA from a T7 promoter resulted in a 207-kDa product whose electrophor etic mobility on a sodium dodecyl sulfate-polyacrylamide gel electroph oresis gel was unaffected by digestion with endoglycosidase F. The dis crepancy in predicted versus measured molecular weights may result fro m the high percentage of acidic residues (roughly 20% Glu or Asp) in t he 322 ORF product, Comparison of the 322 cDNA ORF with sequences in d ata banks indicates that this gene is novel. The 322 ORF product conta ins a potential Cys-1-His-3 Zn finger, at least Eve nuclear localizati on signals of the adenovirus E1a motif K(R/K)X(R/K), and alternating a cidic and basic domains. Overexpression of the 322 cDNA from retrovira l vectors resulted in significantly decreased cell proliferation rates compared with those of controls in both untransformed and src-transfo rmed NIH 3T3 cells, Continued passage of the 322 cells resulted in the selection of rapidly growing cells which had lost the transduced 322 cDNA. Thus, 322 represents a novel src- and ras-regulated gene which e ncodes a potential regulator of mitogenesis and/or tumor suppressor.