Proc. Natl. Acad. Sci. USA Vol. 75, No. 12, pp. 6159-6162, December 1978 Genetics Argininosuccinic aciduria: Assignment of the argininosuccinate lyase gene to the pter--q22 region of human chromosome 7 by bioautography (somatic cell hybrids/aminoaciduria) S. L. NAYLOR*t, R. J. KLEBE*, AND T. B. SHOWSf *Division of Human Genetics, University of Texas Medical Branch, Galveston, Texas 77550; and *Biochemical Genetics Section, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 Communicated by Victor A. McKusick, September 21, 1978 ABSTRACT Argininosuccinic aciduria, an autosomal re- cessive disorder of the urea cycle in humans, is associated with a deficiency of argininosuccinate lyase (ASL; L-arginino- succinate arginine-lyase, EC 4.3.2.1). ASL activity was visualized on gels after electrophoresis by a new method, termed bioau- tography. Bioautography involves the use of mutant bacteria to visualize the location of mammalian enzymes after zone electrophoresis. By this technique, human ASL migrated to a position different from mouse ASL, while a survey of mouse strains, tissues, and tissue culture cell extracts demonstrated the same electrophoretic form and no genetic variants of mouse ASL. Identifying human ASL by bioautography in human- mouse somatic cell hybrids has made it possible to regionally locate the ASL gene on human chromosome 7. The human ASL phenotype segregated concordantly with the human enzyme ,-glucuronidase (GUS; jlD-glucuronide glucuronosohydrolase, EC 3.2.1.31) in cell hybrids, but showed discordant segregation with 32 other enzyme markers representing 23 linkage groups. The gene for GUS has been assigned to chromosome 7 in hu- mans, and cosegregation (synteny) of ASL and GUS demon- strates the assignment of ASL to chromosome 7. Regional lo- cation of ASL and GUS to the pter--q22 region of chromosome 7 was achieved in hybrids segregating a 7/9 translocation. Bioautography, a method of isozyme visualization, uses bac- terial growth to locate an enzyme after zone electrophoresis. The auxotrophic bacteria used require a nutrient product that is generated by the enzyme (1). Since there is a wide variety of genetically marked bacterial strains (2), it has been possible to use bioautography to visualize several isozymes after electro- phoresis that have not been amenable to study by standard procedures, such as branched chain aminotransferase (1) and aminoacylase-1 (3). These new enzyme markers, generated by bioautography, have immediate application in the detection of genetic electrophoretic polymorphisms. Bioautography should be instrumental in generating many new markers for gene mapping studies by somatic cell genetic techniques. Bioautography has been used to detect the human and rodent forms of argininosuccinate lyase in human-rodent somatic cell hybrids (Fig. 1). Argininosuccinate Iyase (ASL; L-argininosuccinate argi- nine-lyase, EC 4.3.2.1), an enzyme of the urea cycle, cleaves argininosuccinate to L-arginine and fumarate (4). Deficiency of ASL has been associated with argininosuccinic aciduria, an aminoaciduria that is characterized by mental retardation, seizures, ataxia, hepatomegaly, hyperammonemia, and large amounts of argininosuccinic acid in blood, urine, and cere- brospinal fluid (5). Although all patients with the disease show autosomal recessive inheritance, argininosuccinic aciduria appears to have several definable forms (6). Screening tests for argininosuccinate aciduria with auxotrophic bacteria have been devised by Murphey et al. (7). In this study we have investigated the expression of human ASL in human-mouse somatic cell hybrids. By the bioauto- graphic procedure for studying ASL (Fig. 1), evidence estab- lishing the chromosome assignment and location of the ASL gene has been gathered from human-mouse somatic cell hy- brids segregating human chromosomes. The human ASL structural gene is syntenic with the gene coding for ,B-glucu- ronidase (GUS; J3-D-glucuronide glucuronosohydrolase, EC 3.2.1.31), which has previously been assigned to chromosome 7 (8-10). A 7/9 chromosome translocation (11) has been used in cell hybrids to map ASL as well as GUS to the pter- q22 region of chromosome 7 in humans. MATERIALS AND METHODS Human and Rodent Parental Cells. Human primary cell strains for fusions were DUV [46,X,t(X;15)(pll;qll)] (12) and JoSt [46,XY,t(7;9)(q22;p24)] (11);,and CaVa [46,X,t(X;22p (q22;ql3)] (13) and AnLy [46,X,t(X;9)(q12;p24)] (13) leuko- cytes. Human fibroblasts were maintained on Eagle's basal medium (diploid) (GIBCO), 10% fetal calf serum, and antibi- otics. Rodent parental lines with selectable markers were RAG (HPRT-) (14), A9 (HPRT-) (15), and LM/TK- (16) main- tained on Dulbecco's modified Eagle's medium. Human-Rodent Somatic Cell Hybrids. Human and mouse cells were fused in suspension or as monolayers with inactivated Sendai virus (13) and the resulting hybrids were cloned and maintained as monolayers on HAT (hypoxanthine/aminop- terin/thymidine) selection medium (17) consisting of Dul- becco's modified Eagle's medium (GIBCO), 10% fetal calf serum, and antibiotics. Primary hybrid clones were established from four fusion experiments with four different human pa- rental cells. Human-mouse hybrid clones were designated DUA (DUV X A9) (18), JSR (JoSt X RAG) (11), ALR (AnLy X RAG) (13), and REX (CaVa X RAG) (13). Mouse Tissues and Strains. Bioautography of ASL was performed on homogenates of the following mouse tissues in C57BL/6J: liver, kidney, brain, heart, lung, stomach, skeletal muscle, testis, spleen, whole blood, salivary glands, and skin. In addition to the Tris/EDTA/borate buffer system (pH 8.6) (below), buffer systems at pH 7.2 [phosphate (1)] and pH 6.15 [4-morpholineethanesulfonic acid (Mes): 50 mM for electrode buffer and 60 mM for hydration of cellulose acetate plates] were tested. In these same electrophoretic systems, liver and kidney extracts of the following strains of mice were assayed for ASL The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6159 Abbreviations: ASL, argininosuccinate lyase; GUS, f3-glucuronidase. t Present address: Biochemical Genetics Section, Roswell Park Me- morial Institute, New York State Department of Health, Buffalo, NY 14263. Downloaded from https://www.pnas.org by 171.243.67.90 on May 23, 2023 from IP address 171.243.67.90.
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