Nucleic Acids Research A new member of the raj gene superfamily identified in a rat liver cell line

A new member of the ras genes superfamily was isolated from a cDNA library derived from a rat liver cell line (BRL-3A) . The predicted 201 amino acids ras-like protein shows 30-35% homology with other members of the ras and ras-related gene products so far described. Conserved features include the GTP-binding and hydrolysis domains and the carboxyl terminal cysteine residues. A protein of the expected size (Mr 23,000) was synthesized in an in vitro transcription-translation system. The BRL-ras gene is present in single copy in the rat genome and is ubiquitously expressed at high levels in all tissues and cell lines examined. INTRODUCTION Recent evidence which has accumulated in the past few years has indicated that ras and ras-related genes code for a superfamily of similar proteins sharing limited (30-50%) overall homology (1). This homology is usually due to the preservation of a few defined domains, in the proteins, which have been shown to be involved in the binding and hydrolysis of GTP (1-4) . Members belonging to the same family, as well as homologous counterparts from different species even quite removed evolutionary, share a much higher degree of similarity and possibly have comparable functions (5). Some of these genes were fortuitously discovered (6,7) and an high number of ras-related genes was isolated through screening of cDNA and genomic libraries, either with synthetic oligonucleotides corresponding to conserved domains or by using low stringency hybridization conditions (8-11). This fact suggests that the ras genes superfamily might comprise many more members than those currently identified. Although the physiological function of these proteins is still poorly © IRL Press Limited, Oxford, England. 9 9 7 9 Downloaded from https://academic.oup. com/nar/article-abstract/16/21/9979/2388285/A-new-member-of-the-ras-gene-superfamily by guest on 15 September 2017 Nucleic Acids Research understood it is reasonable to assume that the large number of different, but structurally related, guanine nucleotide-binding proteins, might regulate various cellular activities. In the present work we report the identification and characterization in rat of a novel ras-related gene (BRL-ras) which appears to encode for a member of the family of the ras-like proteins and which is transcribed with high efficiency at different ages and in many tissues. MATERIALS AND METHODS Chemicals and enzymes Restriction endonucleases, DNA polymerases, T4 DNA ligase, T4 polynucleotide kinase, reverse transcriptase and other enzymes were purchased from commercial suppliers (Boehringer Mannheim, Promega, Amershara Corp. and Bethesda Research Laboratories). All deoxyribonucleotide triphosphates, dideoxynucleotide triphosphates and ribonucleotide triphosphates were from Pharmacia and all radionuclides were obtained from Amersham Corp. Isolation of cDNA clones A cDNA library was constructed from 12S poly (A) RNA from BRL-3A cells using the procedure of Land e_t a_l. (12) with modifications, as previously described (13). The resulting tetracycline resistant clones were screened using a mixture of tetradecamers 5'-d(AARCARCAYTCYTC)-3' that have been 5' labeled with T4 polynucleotide kinase and [yP]ATP. An additional cDNA library was constructed from BRL-3A polyadenylated RNA. Complementary DNA was prepared with a cDNA synthesis kit (Amersham Corp.) following recomended procedures, except that methylation of double stranded cDNA before the addition of linkers was omitted. The XgtlO vector was cleaved with restriction endonuclease EcoRI and the cDNA was ligated to the phage arms after addition of EcoRI linkers. Recombinant phage DNA was packaged jLri vitro using the Gigapack kit supplied by Stratagene. The library was screened by the iri situ plaque hybridization technique (14). A cDNA 650 bp PstI restriction fragment was labeled to a 9 specific activity of 2x10 cpm/pg and used as a probe to screen the library. The filters were washed under stringent conditions