Proc. Natl. Acad. Sci. USA Vol. 80, pp. 7481-7485, December 1983 Biochemistry Cloning of cDNA sequences of human adenosine deaminase (adenosine deaminase mutants/leukemia lymphoblasts/differential screening/A phage) DAN A. WIGINTON*, GWENDOLYN S. ADRIAN*, RICHARD L. FRIEDMANt, D. PARKER SUTTLE*, AND JOHN J. HUTTON*: *Department of Medicine, University of Texas Health Science Center, and Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX 78284; and tDepartment of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Communicated by Eloise R. Giblett, August 22, 1983 ABSTRACT Cloned cDNA sequences of human adenosine de- amined by translation in vitro (7). Included was the interesting aminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been finding that two cell lines with mutations in ADA contained isolated from a cDNA library constructed in bacteriophage AgtlO. higher levels of translatable ADA mRNA than corresponding The cDNA for the library was prepared from poly(A)+ RNA iso- normal lines. In the present report, we describe isolation of lated from a human T-lymphoblast cell line, CCRF-CEM. The cloned cDNA sequences for human ADA. The molecular size library was initially screened by differential plaque hybridization and quantity of ADA mRNA in a number of normal and mutant to labeled cDNA prepared from human T- and B-lymphoblast cell human lymphoblast cell lines have been determined directly by lines with a 21-fold difference in levels of translatable ADA mRNA. to cDNA Two recombinants containing cloned cDNA sequences for ADA hybridization the ADA-specific probes. were identified by hybridization-selected translation. Both re- combinants contained approximately 1,600 base pairs of inserted METHODS human DNA. Restriction maps of the two inserts were not iden- RNA Isolation and Translation. Procedures for extraction tical. One contained approximately 40 base pairs of additional DNA and isolation of total cellular RNA, purification of poly(A)+ RNA, toward the center of the cDNA. The cloned cDNA specifically translation of RNA in a reticulocyte lysate system, and specific hybridized to five fragments generated by HindIll digestion of immunoprecipitation and quantitation of ADA have been de- human genomic DNA. It also hybridized to human lymphoblast scribed previously (7). RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast Cell Lines. The lymphoblast cell lines GM-130, GM-131, cell lines. ADA mRNA levels correlate closely with levels of ADA GM-2184, GM-3043, and GM-2606 are transformed B-cell lines catalytic activity and ADA protein in cell lines containing struc- obtainedfrom the Human Genetic MutantCellRepository (Cam- turally normal ADA. A leukemic T-lymphoblast line produced 6 den, NJ). GM-130, GM-131, and GM-2184 were established to 9 times as much ADA protein and ADA mRNA as transformed from individuals with normal ADA activity, whereas GM-3043 B-lymphoblast lines. Two mutant B-lymphoblast lines from pa- and GM-2606 are from ADA-deficient individuals (5). The CCRF- tients with hereditary ADA deficiency contained unstable ADA CEM cells were obtained from the American Type Culture protein but had 3 to 4 times the normal level of ADA mRNA. Collection. The HPB-ALL cells were a gift from Jun Mino- wada, Roswell Park Memorial Institute (Buffalo, NY). Both the Adenosine deaminase (ADA; adenosine aminohydrolase, EC CCRF-CEM and HPB-ALL are human leukemia T-lympho- 3.5.4.4), a part of the purine catabolic pathway, catalyzes the blast lines and have structurally normal ADA. Cells were grown irreversible deamination of adenosine and deoxyadenosine. in RPMI 1640 medium supplemented with 10-20% fetal calf Deficiency of adenosine deaminase activity in humans is as- serum. sociated with an autosomal recessive form of severe combined Construction of a Human T-Lymphoblast cDNA Library. immunodeficiency disease (1). The underlying specific bio- The cDNA cloning vector, Agt1O, was kindly provided by Thanh chemical and metabolic abnormalities involved and their effects Huynh and R. Davis (Stanford University). After addition of on the immune system have been the subject of intensive in- EcoRI linkers, cDNA was inserted into the EcoRI site of Agtl0 vestigation (2, 3). and recombinants were selected on Escherichia coli BNN 102 A number of different mutations have occurred that can cause as described by Young and Davis (9). Technical details of the human ADA deficiency (4-8). Both the clinical and biochem- preparation of cDNA libraries in AgtlO will be presented else- ical effects of the mutations are strikingly different among in- where. The phage library then was grown on E. coli C600. dividuals. Some mutations are associated with an unstable ADA Screening of the cDNA Library. The cDNA library was protein, total deficiency of ADA activity in erythrocytes, partial screened by a variation of the Benton and Davis in situ plaque deficiency of ADA activity in peripheral lymphocytes, and ab- hybridization method (10, 11). Recombinant bacteriophage were sence of clinical disease (5). Other mutations have resulted in plated on 9 X 9 cm square plates and grown to produce plaques the virtual absence of ADA activity in erythrocytes and lym- approximately 1 mm in diameter. Duplicate replicas from each phocytes and cause severe combined immunodeficiency dis- plate were prepared on nitrocellulose filters, and these were ease (2-7). Within the second group, there are a number of dis- screened by differential hybridization to labeled cDNA pre- tinct mutations, as indicated by the amount of mutant ADA pared from mRNA preparations from lines HPB-ALL and GM- protein detectable by radioimmunoassay in extracts of cell lines 2184. These mRNA preparations contained 0.051% and 0.0025% carrying those mutations (4, 6). In a recent report, ADA mRNA ADA-specific sequences, respectively, as determined by in vi- in a number of these human lymphoblast cell lines was ex- tro translation and immunoprecipitation (7). The mRNA used to prepare cDNA for screening was first The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: ADA, adenosine deaminase. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 7481 Downloaded by guest on September 29, 2021 7482 Biochemistry: Wiginton et al. Proc. Natl. Acad. Sci. USA 80 (1983) partially purified by agarose/urea gel electrophoresis (12). Gel cDNAs were isolated from the A recombinants and recloned in slices were frozen, homogenized, and extracted with 0.04 M pBR325. Inserts were then isolated from the plasmids and di- Tris acetate buffer, pH 7.4. The RNA samples were extracted gested with restriction enzymes. Fragments were separated on with phenol and chloroform, precipitated, and assayed. The 5% polyacrylamide gels (14). To distinguish fragments at ends relative ADA mRNA contents of the HPB-ALL and GM-2184 from fragments located centrally, approximately 8,000 cpm of preparations were increased approximately 6-fold to 0.33% and insert DNA that had been 32P-labeled at both ends was in- 0.016%, respectively. Aliquots (0.5 Ag) of these partially pu- cluded in each digest. rified mRNAs were used for preparation of 32P-labeled cDNA Transfer of Denatured RNA to Nitrocellulose Paper and (8 x 108 cpm/,ug) by a variation of the method described by Hybridization with cDNA. Poly(A)+ RNA samples were glyoxy- Maniatis et al. (11), with oligo(dT) as primer for avian myelo- lated, electrophoresed in a 1.1% agarose gel, and transferred blastosis virus reverse transcriptase (J. Beard, Life Sciences) at to nitrocellulose paper (15). The filters were probed with nick- 100-150 units per Ag of RNA. translated (16) phage DNA (2 x 108 cpm/Ag). Each of the two labeled cDNA preparations (5 X 105 cpm Dot-Blot Hybridization. Relative amounts of ADA mRNA in per filter) was hybridized to one of the sets of replica filters poly(A)+ RNA preparations from lymphoblast cell lines were described earlier. The hybridization was carried out as de- estimated by hybridization of 32P-labeled cDNA to RNA spot- scribed by Maniatis et al. (11), with agitation at 420C for 72 hr. ted on nitrocellulose paper (15, 17). A 0.25-Ag sample of DNA The filters were autoradiographed for 18 hr at -70TC. Plaques from each of the two recombinant bacteriophages that con- producing a hybridization signal significantly stronger with the tained ADA sequences (2-11 and 3-3) was labeled by nick trans- HPB-ALL cDNA than with the GM-2184 cDNA were picked lation (16). A molar excess of the nick-translated recombinant with sterile toothpicks and replated. The resulting plaque ar- phage DNA (108 cpm/,ug) was hybridized with the bound RNA rays were transferred to duplicate nitrocellulose filters and re- at 420C for 20 hr (15). The filters were washed and autoradio- screened with the differential probes. graphed for 18 hr at -700C. Relative amounts of ADA mRNA The group of plaques that demonstrated a "strong" differ- were estimated by measuring the optical absorbance of auto- ential signal was screened further. Labeled cDNA was pre- radiographic spots, using a Dynatech micro ELISA minireader pared from mRNA derived from T-lymphoblast lines HPB-ALL MR590. and CCRF-CEM and B-lymphoblast lines GM-2606 and GM- Southern Blot Analysis. DNA (15 jig) was digested to com- 2184. The mRNA preparations from the paired T-cell lines and pletion with HindIII, separated in an 0.8% agarose gel, trans- paired B-cell lines differed 4-fold in ADA mRNA by in vitro ferred to a nitrocellulose filter, and hybridized to 3 P-labeled translation (HPB-ALL greater than CCRF-CEM; GM-2606 probe ADA 3-3 (11).