The nudeotide sequence of the gpt gene coding for the enzyme xanthine guanine phosphoribosyl transferase has been determined. The gene codes for a protein of molecular weight 16,950. The construction of deletions in the gpt

The nudeotide sequence of the gpt gene coding for the enzyme xanthine guanine phosphoribosyl transferase has been determined. The gene codes for a protein of molecular weight 16,950. The construction of deletions in the gpt gene which can be used for the genetic analysis of mutations in the gpt gene, is described. INTRODUCTION The ability to clone and sequence unique DNA fragments permits the detailed study of the types of nudeotide alterations that occur during spontaneous and induced mutagenesis. Analyses of the types of sequence changes following mutation of procaryotic DNA have been reported (1,2,3). A logical extension of the work being done in procaryotes is to carry out the analogous study in mammalian cells. An excellent target gene for this type of study is the pSV2-gpt gene complex constructed by Mulligen and Berg (4,5,6). This consists of an E. coli DNA fragment containing the gpt gene which codes for the enzyme xanthine guanine phosphoribosyl transferase (XGPRT) which is flanked by SV40 DNA carrying the sequences necessary for mRNA initiation and processing in mammalian cells. This complex is expressed both in mammalian cells and in E. coli, and selective conditions exist for the isolation of cells with and without gpt function. Thackar et al.(7) and Tindall et al.(8) have described mutation systems in Chinese hamster ovary cells using the gpt gene complex of Mulligan and Berg. A necessary starting point in the analysis of induced mutations is knowledge of the wild type sequence of the gpt gene. Single-strand sequence data for a section of the fragment containing gpt has been obtained by Mulligan and Berg (6). We present here the complete nudeotide sequence of the E. coli fragment in pSV2-gpt, which contains the gpt coding region, and describe the construction of deletion mutants in the gene. © IRL Press Limited, Oxford, England. 8809 Nucleic Acids Research MATERIALS AND METHODS Bacterial strains E. coli strain 33694 (leu B6, pro A2, rec A13, thi-1, ara-14, lac Yl, gal K2, xyl-5, mtl-1, rps L20, lambda-, sup E44, hsd S20), strain 33572 (supE, supF, thyA, metE), and strain 33526 (met Bl, lac-3 or lac Yl, gal K2, gal T22, lambda-, sup E44, hsd R2) were obtained from American Type Culture Collection (Rockville, KD). E. coli strain GP120 (pur E, delta (lac pro gpt)) was the generous gift of J. Gots (University of Pennsylvania, Philadelphia, PA). Bacteria were grown routinely in brain-heart infusion (Difco). The gpt activity of plasmid derivatives in GP120 was tested by growing the bacteria in medium containing 0.2 mg/ml MgSOWHp, 2.0 mg/ml citric acid-H-O, 10 mg/ml K2HP04, 3.5 mg/ml NaNH4HP04-4H20, 20 mg/ml glucose, O.lmg/ml thiamine, 10 mg/ml casamino acids, and 25 pg/ml guanine. Nucleic Acid Plasmid pSV2-gpt (Figure 1) was the generous gift of R. Mulligan (MIT, Cambridge, MA). Plasmid gpt2Eco (Figure 1), a derivative of SV2-gpt, was constructed by first protecting the existing EcoRl site with EcoRl methylase, cutting with PvuII, adding EcoRl linkers, and transfecting into E. coli 33694. In gpt2Eco, the gpt gene and surrounding SV40 sequences, and derivatives of this complex, can be excised by cutting with EcoRl for future transfer to other vehicles. EcoRl, Hindlll, and BamHl linkers were purchased from Bethesda Research Laboratories (Gaithersburg, MD). Enzymes EcoRl, Hindlll, Bglll, Kpnl, BamHl, PvuII, EcoRl methylase, T4 polynudeotide kinase, T4 DNA polymerase, T4 DNA ligase, exonuclease III, bacterial alkaline phosphatase and SI nuclease, were purchased from Bethesda 32 Research laboratories (Gaithersburg, M0). T4 DNA ligase used for P labelling was purchased from New England Nuclear (Boston, MA). E. coli DNA polymerase I was purchased from Boehringer Mannheim (Indianapolis, IN). Reaction buffers used were those specified by the vendor. Chemicals Chemicals were obtained from the following sources: deoxyadenosine 5 op o? triphosphate (a P, 2000 Ci/mnole) and adenosine 5' triphosphate (gamma P, 5000 Ci/mmole)-Amershara (Arlington Heights, IL); adenosine triphosphate, piperidine, forroamide, ampiciilin, agarose, ammonium persulfate, cesium chloride, guanine, hypoxanthine, agarose-Sigma Chemical Co. (St. Louis, MO); deoxynucleotide triphosphates-P-L Biochemicals, Inc. (Milwaukee, WI); hydrazine, Eastman Kodak Co.— (Rochester, NY); diraethylsulfate, formic acid,