We previously isolated a partial cDNA fragment of a novel gene, Elm1 (expressed in low-metastatic cells), that is expressed in low-metastatic but not in high-metastatic K-1735 mouse melanoma cells. Here we determined the full-length cDNA structure of Elm1 and investigated the effect of Elm1 expression on growth and metastatic potential of K-1735 cells. The Elm1 gene encodes a predicted protein of 367 amino acids showing ∼40% amino acid identity with the CCN (connective tissue growth factor [CTGF], Cyr61/Cef10, neuroblastoma overexpressed gene [Nov]) family proteins, which consist of secreted cysteine-rich proteins with growth regulatory functions. Elm1 is also a cysteine-rich protein and contains a signal peptide and four domains conserved in the CCN family proteins. Elm1 was highly conserved, expressed ubiquitously in diverse organs, and mapped to mouse chromosome 15. High-metastatic K-1735 M-2 cells, which did not express Elm1, were transfected with an Elm1 expression vector, and several stable clones with Elm1 expression were established. The in vivo growth rates of cells expressing a high level of Elm1 were remarkably slower than those of cells expressing a low level of Elm1. Metastatic potential of transfectants was reduced in proportion to the level of Elm1 expression. Thus, Elm1 is a novel gene of CCN family that can suppress the in vivo growth and metastatic potential of K-1735 mouse melanoma cells.

Transfer ribonucleic acid (tRNA) guanine transglycosylase (guanine insertion enzyme) was isolated from rat liver and extensively purified. The enzyme catalyzes an exchange of queuine (the base of queuosine, Q) as well as its precursors and guanine for guanine originally located in the first position of the anticodon of "undermodified" tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from an Escherichia coli mutant or rat ascites hepatoma cells. This is in contrast to the previous observation that E. coli tRNA-guanine transglycosylase catalyzes the exchange of queuine precursors, such as 7-(aminoethyl)-7-deazaguanine and 7-cyano-7-deazaguanine, but not of queuine itself [Okada, N., Noguchi, S. Kasai, H., Shindo-Okada, N., Ohgi, T., Goto, T., & Nishimura, S. (1979) J. Biol. Chem. 254, 3067-3073]. The Km value for queuine of the rat liver enzyme is 9.2 X 10(-7) M, much lower than the values for the bases of queuosine precursors or guanine. Thus, the actual substrate for tRNA-guanine transglycosylase in queuosine biosynthesis in vivo in rat liver may not be 7-(aminomethyl)-7-deazaguanine, which is thought to be an actual substrate guanine, the E. coli system. Queuine or some queuine derivative may be the actual substrate for the tRNA-guanine transglycosylase reaction in the biosynthesis of Q in tRNA of mammalian cells. 6-Thioguanine and 8-azaguanine are also found to be good substrates.

The guanine insertion enzyme from Escherichia coli catalyzes exchange of guanine located at the first position of the anticodon of tRNA with radioactive guanine (N. Okada and S. Nishimura, unpublished data). tRNA isolated from various tumors, including slowly growing Morris hepatoma 7794A, incorporated considerable guanine with E. coli guanine insertion enzyme, whereas tRNA isolated from all normal tissues so far tested, except regenerating rat liver, incororated scarcely any. In the rat ascites hepatoma AH7974, the guanine was mostly incorporated into minor isoaccepting species of tRNA^sP that contained the guanine residue instead of Q base in the first position of the anticodon. This is a sensitive and easy method for identifying unique tRNA species in tumor tissues.Many new isoaccepting tRNA species have been found in particular tissues, cells at various stages of differentiation, tumor tissues, transformed cells, and cells grown under different culture conditions (for reviews, see refs. 1-3). Various techniques have been used in attempts to detect unique tRNA species in tumor cells, and it has been found that the amount of methylated nucleosides in tRNA and the activities of tRNA methylases are generally high in tumor tissues (3, 4). However, analyses of modified nucleosides using total unfractionated tRNA have given results dependent upon the tumor tissues examined (5). Recently, Kuchino and Borek (6) demonstrated that the tRNAPhe that specifically appeared in Novikoff hepatoma and Ehrlich ascites tumor cells contains 1-methylguanine, unlike tRNAPhe in normal tissues. The new tRNAPhe species that appears in some tumor tissues is due to lack of modified base Y in the position next to the anticodon (7,8). A new isoaccepting species of tRNA has often been detected in tumor cells by analyzing changes in the chromatographic profile of amino acid acceptor activity (3). However, new tRNA cannot always be detected in this way because the elution position of tRNA may be influenced by several factors. In addition, most methods used previously require a large quantity of material and pure species of tRNA, which are difficult to obtain from tumor tissues.In this paper, we report a method for detecting unique tRNA species specifically present in tumor cells. We previously reported that the guanine insertion enzyme from rabbit reticulocytes, discovered by Farkas (9), specifically catalyzes the exchange of modified base Q in Escherichia coli tRNA with guanine without breaking the polynucleotide chain (10). A guanine insertion enzyme has also been found in other organisms, such as E. coli (N. Okada and S. Nishimura, unpublished data) and Ehrlich ascites tumor cells (11), and an extensively purified preparation from E. coli catalyzed exchange of guanine with guanine, but not of Q base in tRNA with guanine (N.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fac...

We previously reported that mouse NIH 3T3 cells transformed by transfection of activated human c-Ha-ras become apparently normal upon treatment with the antibiotic azatyrosine. The revertant cells maintain their normal phenotype during prolonged culture in the absence of azatyrosine, although activated p21 is still expressed. The normal phenotype induced by azatyrosine could be due to activation of expression of some cellular gene(s) in the cells that results in suppression of ras function. To identify the genes with increased expression in the revertant cells, we adopted differential screening of recombinants from a phage cDNA library made from mRNA of the revertant cells, hybridized with 32P-labeled cDNAs made from mRNAs of the rs-transformed NIH 3T3 cells and the revertant cells. Two clones thus isolated were found to be almost identical to the ras recision gene (rrg), the clones were found to contain a sequence corresponding to that of the murine retrovirus-like intracisternal A particle. We speculate that azatyrosine activates several cellular genes in the ras-transformed cells and that some of these genes, including prg, act cooperatively to counteract ras function, resulting in reversion of the ras-transformed cells to the normal phenotype.Mouse NIH 3T3 cells that have been transformed by insertion of activated c-Ha-ras, c-Ki-ras, N-ras, c-raf, or c-erbB-2 (neu) can be converted to an apparently normal phenotype by incubation for 6 days in culture medium containing the antibiotic azatyrosine [L-P-(5-hydroxy-2-pyridyl)alanine] (ref. 1 and unpublished results). The apparently normal flat revertant cells show contact inhibition, cannot proliferate in soft agar, and scarcely form tumors in nude mice, although the activated oncogenes inserted are still expressed in the revertant cells. The reversion induced by azatyrosine is permanent, and the phenotype of the revertant cells does not change during prolonged culture in the absence of azatyrosine. Since the reversion efficiency is quite high (more than 85% of the cells showed reversion, and the other 15% were killed on treatment with azatyrosine), it is likely that an epigenetic change takes place in the revertant cells, and the gene(s) specifically expressed in the revertant cells counteracts ras, raf, or erbB-2 function, acting as a tumor-suppressor gene. To determine which genes are activated in the revertant cells, we prepared a AgtlO cDNA library from the revertant cells and screened it by differential hybridization with 32p-labeled cDNAs from the original ras-transformed NIH 3T3 cells and the revertant cells. Among 30 clones thus isolated, two were found to be nearly identical with the ras recision gene (rrg), § which was isolated as a tumor-suppressor gene by Contente et al. (2) and subsequently identified as the gene for lysyl oxidase (3). The other clones thus far identified were those of collagen type III and rhoB. They were not expressed in normal NIH 3T3 cells or in ras-transformed NIH 3T3 cells. Approximately half the 30 clones were found t...