The Locus for Human Adenine

Evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT) to human chromosome No. 16 is presented. Hybrids of APRT-deficient mouse cells and of human fibroblasts having normal APRT were isolated by fusing the parental cells with Sendai virus, blocking de nouo purine nucleotide synthesis with azaserine and selecting for hybrids that could use exogenous adenine. The hybrid clones that were studied had only APRT activity that was indistinguishable from human APRT with regard to electrophoretic migration and reaction with antibodies against the partially purified human enzyme. No. 16 was the only human chromosome consistently present in all of the clones, and in one clone, it was the only human chromosome detected. Selection against hybrid cells with 2,6-diaminopurine (DAP) yielded DAP-resistant survivors that lacked chromosome No. 16. One hybrid that originally had an intact No. 16 yielded adenine-utilizing subclones that lacked No. 16 but had a new submetacentric chromosome. The distribution of centromere-associated heterochromatin and the fluorescence pattern indicated that this chromosome consisted of a mouse telocentric chromosome and the long arm of NO. 16. Cells having the submetacentric chromosome had human APRT. Both the enzyme and the chromosome were absent in DAPresistant derivatives. These results suggest that the structure of APRT is defined by a locus on the long arm of human chromosome No. 16. UMAN genes can be mapped to their respective chromosomes in somatic cell hybrids by detecting concordant segregation patterns between specific phenotypic expressions and specific human chromosomes in interspecies hybrid cells. This principle is now well established, and one or more genes have already been assigned to most of the human chromosomes using this approach (RUDDLE 1973). We report here evidence for assigning the locus determining the structure of adenine phosphoribosyltransferase (APRT; E.C. 2.4.2.7) (ENZYME NOMENCLATURE: RECOMMENDATIONS OF 1964, 1965) , which converts adenine to adenosine-5’-monophosphate (AMP), to human chromosome No. 16. This Abbreviations: AG, 8-azaguanine; DAP, 2,6-diaminopurine; [AS], azaserine; APRT, adenine phosphoribosyltransferam; CS, calf serum; FCS, fetal calf serum; A[AS], adenine-azaserine selective medium; MNNG, N-methyl-N’-nitroN-nitrosogilanidine; HGPRT, hypoxanthine-guanine phosphoribosyltransferase. Present address: Universititskinderklinik, D-2000 Hamburg 20-Martinistr. 52, West Germany. This is paper no. 1737 from the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706. Gene:ic\ 78: 11+>115G December, 1974. 1144 B. H A H A N , K. R. HELD AND R. DE MARS enzyme is dispensible for growth under ordinary conditions, since cells can synthesize the nucleotide via a de novo pathway. If de nouo synthesis is blocked cells having APRT can utilize exogenously supplied adenine and grow but APRT-deficient cells are arrested. Therefore, when APRT-deficient mouse cells are fused with normal human cells and the de lrouo synthesis of AMP is blocked in adenine-containing medium, mouse parental cells are eliminated and hybrid cells containing human APRT form distinct colonies against the background of persistent human cells. KUSANO, LONG and GREEN (1971) used alanosine to block de nouo AMP synthesis in applying this principle and tentatively assigned genes for APRT to a human acrocentric chromosome. However, two ongoing research problems in this laboratory prompted us to independently determine the chromosomal location of APRT genes: (i) We have obtained variant diploid human fibroblasts that are resistant to 2,6-diaminopurine (DAP) . At least some of the variants have deficient APRT activity and it was necessary to determine the location of APRT genes in order to genetically analyze the DAP-resistant variants. (ii) We were attempting to detect derepression of the human inactive X chromosome by selecting for expression of the HGPRT gene on the inactive X in hybrids of female fibroblasts with HGPRT-deficient mouse cells. The principle of these experiments is described elsewhere (KAHAN and DEMARS 1973). As an alternative, we thought that initial selection for APRT in hybrid cells would permit a condition of gratuity, in which events necessary for X-chromosome reactivation could occur without cell proliferation being immediately dependent on expression of previously inactive X-chromosomal genes. Such hybrids could subsequently be checked for expression of selectable, or even unselectable alleles of genes that were on the inactive X in the human cell parent. In the course of these hybridization studies we were able to assign the structural gene for APRT to human autosome No. 16. The assignment is probably reliable since it agrees with that made independentdy by TISCHFIELD and RUDDLE (1974) using different strains of cells and a different selection system. In addition, we have observed translocated chromosomes in the hybrid cells that lead us to assign the APRT gene to the long arm of No. 16. MATERIALS AND METHODS Details of cell propagation methods are presented in DEMARS and HELD (1972). Cell lines: The human fibroblasts were derived from a skin biopsy of a female having normal APRT who was heterozygous for the A and B alleles of the X-chromosomal glucose-6-phosphate dehydrogenase (G6PD) locus. For purposes of X-chromosome derepression studies an HGPRTdeficient subclone was produced by a mutagenic exposure of strain No. 129 cells to 2.5 &ml MNNG for 24 hours. Clone 129.22 was isolated in 3 x l O r j M 8-azaguanine (AG) in F10 medium lacking hypoxanthine (HAM 1963) and containing 15% calf serum (CSF10 medium). Clone 129.22 is resistant to AG due to a deficiency in HGPRT enzyme activity, having 5% of the normal specific activity, and is unable to grow in HAT selective medium (SZYBALSKI, SZYBALSKA and RAGNI 1962), modified as previously described (DEMARS and HELD 1972). Diploid cells of the clone have a normal fluorescence karyotype. The mouse line D7 was derived by AG selection (8 x 10-6M) from ID LM(TK-) cells (DUBBS and KIT 1964) and lacks detectable HGPRT activity. APRT-deficient cells of the 1D line were found to spontaneously exist as about 20% of the population. D7 is an APRT-deficient HUMAN GENE MAPPING 1145 subclone of ID cells, and is therefore an APRT-, HGPRT-, TK-, triply deficient cell h e . The modal chromosome number for Di is 52, including 7 large metacentric chromosomes and one large marker chromosome having the appearance of a dicentric chromosome due to a prominent secondary constriction. The remaining chromowmes are acrocentrics. Dl l is an APRT+ subclone of ID. Formation and selection of hybrids: Hybrid cells were isolated and maintained in selective medium consisting of CS-F10 medium containing 1W M azaserine ([AS]) to block the de nouo purine pathway, and 3 x 10-5 M adenine (ACAS] medium). Normal human fibroblasts and I D mouse lines not deficient in APRT grow well in A[AS], whereas growth of APRTdeficient mouse Di cells is completely inhibited. Cells from 129.22 were fused with Di cells following a modification of the monolayer technique of KLEBE, CHEN and RUDDLE (1970). On day 0, 106 cells of each parental type were mixed in suspension and plated in a 60 mm diameter Falcon plastic petri dish (P60) in a total of 5 ml F10 medium supplemented with 15% fetal calf serum (FCSFIO). The following day, the medium was aspirated and the dish was chilled at 4" for 10 minutes. 20W H A V S of p-propiolactone inactivated Sendai virus in one ml of chilled 10% F E F 1 0 was added, and the dish was incubated at 4" for 20 minutes. The cells were then returned to 37" and 4 ml warm 10% FCSF10 was added. After 2 hours, the dish was rinsed twice and renewed with 5 ml volumes of FCSFIO medium. On day 2, the cells were suspended by trypsinization and distributed into 100 P60's containing FCSFlO. On day 6, selection of hybrid cells was begun. The seven hybrid colonies that appeared were isolated 3 weeks later by encircling the colonies with 7 mm I.D. stainless steel cylinderc and removing the trypsinized cells with a Pasteur pipette. In experiments in which loss of APRT activity in hybrid cells was correlated with loss of a particular chromosome, hybrid cells resistant to the adenine analog 2,6-diaminopurine (DAP) were isolated in CSFIO medium containing 10-4 M DAP. The incidence of DAP-resistant colony formers was between 10-4 and 10-2 in two independent hybrids. Chrcmosome analysis: Slides of metaphase chromosomes were prepared by the air-dry method of ROTHFELS and SIMINOVITCH (1958). For quinacrine banding patterns (CASPERSSON et al. ISTO), slides were stained for 30 minutes in an aqueous 0.5% Atebrin solution, rinsed three times with distilled water, and mounted in a 0.1 M KH,PO, buffer, pH 5.5, 20% ( w h ) sucrose solution. Photographs were obtained with a Zeiss fluorescence microscope fitted with a Planachromat 1OOx oil immersion objective with iris diaphragm (.8/1.25 N.A.) and dark field ultracondensor, 1.2/1.4 N.A. Light was transmitted by an HBO 200W/4* mercury bulb through exciter filter BG12 and barrier filter 53. Kodak High Contrast cbpy Film and Kodabromide F3-F5 paper were used for prints. Constitutive heterochromatin patterns were obtained by treating I-8-week-old slides with 90-95% hot formamide for 15-30 minutes, then staining with a 1% Giemsa solution for 30-60 minutes (DEV et al. 1972). Enzyme assays: Cell monolayers containing about 1 to 3 x 106 cells per P60 were rinsed twice with cold 0.155 RI KC1 and were collected in .2 ml of 0.155 M KC1 per P60 by scraping with a rubber policeperson. The cells were disrupted by 3 cycles of freezing and thawing and were then centrifuged at 4" for 20 minutes at 20,000 x g. 6-19 pg of cell protein was present in 50 pl of reaction mixture containing 5 x 10-2 M Tris HC1, pH 7.4, 5 x 10-3 M MgSO,, M PRPP, 2.0 x IO-" M [S-lC] adenine (0.235 mc/mmole, Schwarz-Mann), and 50 pg bovine serum albumin. Reactions proceeded for 30 minutes at 37". Detection of converted purines was as described in RAPPAPORT and DEMARS (1973). APRT electrophoresis: Two considerations motivated the choice of electrophoretic system: (1) for optimal separation of human and mouse forms of the enzyme, a buffer system near the isoelectric point of one of the species was sought; (2) for comparison purposes, a slab gel allowing side-by-side multiple runs is preferable. 'Iherefore, electrophoresis was conducted in acrylamide slabs at p H 6 0 using an ORTEC electrophoresis system (ORTEC, Inc., Oak Ridge, Tenn ) (But see TISCHFIELD, BERNHARD and RUDDLE [1973] for what appears to be a better method ) 1146 B. KAHAN, K. R . HELD A N D R. DE MARS Gels were cast as suggested by ORTEC (ORTEC Inc., 1973). An acrylamide gradient separating gel of 8% (51 mm), 6% (9 mm) and 31/2% (5 mm) acrylamide, and a well-forming gel of 8% (20 mm) acrylamide were used. The buffer system within the gel was potassium acetate (pH 6.0). The tank buffer was potassium cacodylate (pH 6.0). The following buffer solutions were prepared: (1) Separating gel buffer: 0.48 M acetate, adjusted to pH 6.0 with 1N KOH, and containing 0.5 ml TEMED per 100 ml. The final separating gel contained 0.12 M acetate. (2) Well and cap buffer: 0.12 M acetate, adjusted to pH 6.0 with I N KOH, and containing 0.5 ml TEMED per 100 ml. The final gel contained 0.3 M acetate. (3) Tank buffer: 0.0325 M cacodylate, adjusted to pH 6.0 with I N KOH. (4) Extraction (EX) buffer: 0.3 M acetate adjusted to pH 6.0 with I N KOH. (5) EX buffer containing 80% sucrose and a drop of .I % bromphenol blue. The cell samples were prepared as described above for APRT enzyme assays, using EX buffer instead of C.155 M KC1. 65 pl containing 15 to 40 pl of cell extract and at least 20% sucrose was placed in each well formed by an 8-place well former. Electrophoresis was conducted at 4'00 v and 0.5 mF capacitance at 4". The pulse settings during the run were as follows: 75 Hz (25 mA); t,,-,07 125 Hz (55 mA); t,,,-,,,,,in,,tes, 225 Hz (82 mA). The migration position of the enzyme was determined by a modification of the method of DER KALOUSTIAN et al. (1969), in which the reaction product is selectively adsorbed on an anion exchange paper and visualized by autoradiography. After electrophoresis, a piece of Whatman DE-81 (DEAE-cellulose) paper was tightly apposed to the gel saturated with reaction mixture containing 50 mM Tris buffer (pH 7.4), 5 mM MgCl,, 1.0 mM PRPP, and 2.5 pc/nil (8-14 C) adenine (Schwarz-Mann; specific activity = 46.9 mc/mmole). The gel and paper were wrapped in Saran Wrap and incubated at 37" for 2 hours. The paper was then placed in a perforated container and rinsed with three changes of 2 liters 0.1 M Tris.HC1, pH 9.5. The paper was dried and taped to x-ray film (Kodak XO MAT). The autoradiograph was developed after 3-7 days. Immunologicul methods-preparation of cell extracts: Dishes containing monolayers of cultured fibroblasts were drained. The cells were scraped into cold saline and washed 3 times with saline using centrifugation at 1000 x g for 15 min at 4". The pellet was taken up in an equal volume of double distilled water and the cells were lysed by three cycles of rapid freezing and thawing using an acetone-dry ice bath. The lysates were centrifuged at 30,000 X g for 20 min and the supernatant fluids were kept on ice prior to assay. Immunoprecipitatiorx Immunoprecipitation reactions* were performed in 6 x 50 mm KIMAX tubes. The 100 pl reaction mixture contained various amounts of rabbit anti-APRTSerum supplemented with non-specific rabbit serum to a total of 6.25 pl, 25,pl of cell lysate, IP4 M PRPP and 3% polyethylene glycol 6000 (HARRINGTON, FENTON and PERT 1971) in hemagglutination buffer (DIFCO). All reaction mixtures were incubated for 4 hours at room temperature (20 & 2") followed by centrifugation at 2000 x g for 60 min at 4". Unprecipitated APRT activities were determined by incubating 20 p1 aliquots of the supernatant fluids for 2 hours at 37". Activity was determined essentially as described by RAPPAPORT and DEMARS (1973) but 0.01 M Tris-HC1, pH 8.4, was used in the reaction mixture and bovine Serum albumin was omitted. Cell lysates were preassayed and used at concentrations that converted 50-80% of the substrate in determinations of unprecipitated APRT activity after incubation with serum from unimmunized rabbits.