Proc. Nat. Acad. Sci. USA Vol. 71, No. 4, pp. 1569-1573, April 1974

Human O-D-N-Acetylhexosaminidases A and B: Expression and Linkage Relationships in Somatic Cell Hybrids (GM2 /lysosomal /anti- sera/electrophoresis)

P. A. LALLEY*, M. C. RATTAZZIt, AND T. B. SHOWS* * Roswell Park Memorial Institute, New York State Department of Health, Buffalo, N.Y. 14203; and t Dept. of Pediatrics at Children's Hospital, State University of New York at Buffalo, Buffalo, N.Y. 14222 Communicated by James V. Neel, January 24, 1974

ABSTRACT Knowledge of the genetic relationships defects responsible for deficiencies involved in GM2 between fl-D-N-acetylhexosaminidases A and B (EC 3.2.1.30) may help in understanding the hexosaminidase gangliosidoses. deficiency associated with GM2 gangliosidosis, a fatal lipid Somatic cell hybrids provide an alternative system for storage disease in man. Through the use of man-mouse linkage studies since human , but not mouse somatic cell hybrids we have found that a involved in chromosomes, are preferentially lost from proliferating hybrid hexosaminidase A expression was linked to the cells; human enzymes can be distinguished electrophoretically coding for mannosephosphate isomerase and pyruvate kinase-3. The gene coding for hexosaminidase B was not or antigenically from homologous enzymes from the mouse; linked to any of the genes coding for 25 enzyme markers and concordant segregation of gene markers determines hu- tested. A combination of immunological and electrophore- man linkage groups. In addition, concordant segregation of tic techniques was employed to identify human hexos- gene markers and individual human chromosomes, which can aminidases A and B with certainty in cell hybrids. Dis- cordant segregation of hexosaminidase A and hexosamini- be identified by cytological techniques, determines gene dase B in 60 clones indicated that the genes coding for their assignments (5). expression were not linked. However, hexosaminidase A We have investigated the linkage relationships of genes was never expressed in cell hybrids in the absence of hexos- involved in the expression of HexA and HexB and their link- aminidase B. This suggests that the gene responsible for age to genes for other enzyme markers in man-mouse somatic the hexosaminidase A phenotype, linked to mannosephos- phate isomerase and pyruvate kinase-3, requires the cell hybrids. Discordant segregation of the HexA and HexB presence of the gene coding for hexosaminidase B for the phenotypes indicates that they are coded by separate genes expression of hexosaminidase A. These observations offer located on different chromosomes. However, expression of a genetic explanation for the biochemical and immunolog- the HexA phenotype in cell hybrids appeared to be dependent ical relationships between A and B and provide the framework for identifying the basic genetic on the presence of hexosaminidase B. Concordant segregation defects responsible for GM2 gangliosidosis. and thus gene linkage was observed for the HexA and man- nosephosphate isomerase phenotypes (MPI, EC 5.3.1.8). The human lysosomal enzyme O-D-N-acetylhexosaminidase Pyruvate kinase-3 (PK-3, EC 2.7.1.40), which has been pre- (2-acetamido-2-deoxy-j-D-glucoside acetamidodeoxyglucohy- viously reported to segregate concordantly (6) with MPI, drolase, EC 3.2.1.30) exists as two major molecular forms, also segregated concordantly with the HexA phenotype in hexosaminidases A (HexA) and B (HexB) (1). Deficiency of mouse L-cell X human hybrids. The mannosephosphate isom- hexosaminidase activity in man is associated with three lethal erase gene has been assigned to 7 in hybrid lipid storage diseases: Tay-Sachs disease, Sandhoff-Jatzke- studies (7), suggesting that a gene coding for the HexA pheno- witz disease, and juvenile GM2 gangliosidosis (2-4). Each type might also be located on chromosome 7. The HexB gene disease is inherited as an autosomal recessive trait and is could not be linked to genes coding for 25 other enzyme characterized by the massive accumulation of the complex markers. lipid GM2 in the central nervous system. Each disorder, however, is clinically and biochemically distinguish- MATERIALS AND METHODS able, and apparently the result of a different . Tay- Parental Cells. The human cells were karyotypically normal Sachs disease is characterized by the deficient activity of WI-38 fibroblasts (8); AnLy fibroblasts prossessing an X/9 HexA (2); Sandhoff-Jatzkewitz disease by the deficient activ- reciprocal translocation (9); and JoVa fibroblasts and white ity of HexA and HexB (3); and juvenile GM2 gangliosidosis blood cells possessing a 2/1 translocation (10). Human fibro- by the partial deficiency of HexA activity (4). A knowledge blasts were maintained on Eagle's basal medium (Diploid) of the genetic relationships between HexA and HexB and (Gibco), 10% fetal-calf serum, and antibiotics. Mouse lines their linkage to other human genes would contribute to a were LTP, a thymidine kinase (TK-), and hypoxanthine- better understanding of structural relationships suggested guanine phosphoribosyl transferase (HGPRT-) deficient cell by their biochemical characteristics (1, 3) and of the primary line (11) and RAG, an HGPRT- line (12). Hybrid Cells. Human and mouse parental cells were fused Abbreviations: HexA and HexB, hexosaminidases A and B; with inactivated Sendai virus (11-13). The JoVa white blood MPI, mannosephosphate isomerase; PK-3, pyruvate kinase-3. cell X RAG fusion was performed in suspension (14). Hy- 1569 Downloaded by guest on September 29, 2021 1570 Genetics: Lalley et al. Proc. Nat. Acad. Sci. USA 71 (1974)

'l /1 e/ C 0 C-/ 4i- 4'i Io I -">OV

- MOUSE

- HexA

4- MOUSE

- HexB

FIG. 1. Reaction of human, mouse, and hybrid cell extracts with anti-human hexosaminidase antisera. Pattern A: Reaction FIG. 2. Starch-gel electrophoresis of 8-D-N-acetylhexosamini- of specific anti-human HexA with partially purified HexA (1); dase demonstrates HexA and HexB in human parental cells human AnLy (2); mouse LTP (3); partially purified HexB (4); (channel 6); mouse isozymes in RAG parental cells (channel 5); Tay-Sachs fibroblasts (5); and human WI-38 (6). Pattern B: HexB in fibroblasts of a Tay-Sachs disease patient (channel 1); Reaction of specific anti-human HexA with partially purified the HexA+/HexB + cell hybrid phenotype (channel 2); the HexA (1); cell hybrid JVR-16 (2); mouse RAG (3); cell hybrid HexA-/HexB+ cell hybrid phenotype (channel 3); and the WIL-lOR (4); human WI-38 (6); and cell hybrid JVR-13 (6). HexA /HexB- cell hybrid phenotype (channel 4). Mouse hexos- Pattern C: Reaction of nonspecific anti-human hexosaminidase aminidase exhibits 2 zones of activity, one which migrates with partially purified HexA (1); mouse LTP (2); cell hybrid anodal to human HexA (see in channels 2-4) and is unstable on WII1OR (3); mouse RAG (4); partially purified HexB (5); storing (channel 5), and one which remains at the origin and is and cell hybrid JVR-13 (6). Immune complexes are visualized by often only faintly visible in some hybrids (channel 4). HexC, a specific staining for hexosaminidase enzymatic activity. minor human component, migrates anodal to the anodal mouse band. In samples where HexA activity was low, HexB was not easily visualized, necessitating the use of immunological assays brid cells were selected and maintained in HAT (15) (hy- to determine its presence or absence. poxanthine, aminopterin, thymidine) medium consisting of Dulbecco's modified Eagle's medium (Gibco), HAT, 10% HCl buffer, pH 8.6, containing 0.01% NaN3. Diffusion pro- fetal-calf serum, and antibiotics. Mouse LTP and RAG cells ceeded for 64 hr at 270 in humid chambers and gels were die in HAT medium, while human cells in low dilution pro- washed for 6 hr with phosphate-buffered saline at room tem- liferate slowly. Sixty independent primary hybrid clones were perature. isolated (16) from five fusion experiments. Mannosephosphate Isomerase Electrophoresis. Human and Hexosaminidase Electrophoresis. Starch and agar gel elec- mouse MPI and PK-3 phenotypes were separated by starch trophoresis were employed to separate hexosaminidases A and gel electrophoresis in a Tris-ethylenediaminetetraacetate- B. Starch gel electrophoresis was performed in a Tris- citrate, borate, pH 8.6, buffer system (21) and visualized as described pH 7.0, system (17). Cell homogenates were prepared from (6, 22). confluent monolayers at concentrations of 0.75 to 1.0 X 108 RESULTS ml For cells were cells per (11). agar gel electrophoresis, Cell hybridization was demonstrated by the presence of hu- washed three times with cold phosphate-buffered saline, man and mouse enzymes and chromosomes (11). Sixty in- suspended in 3 volumes of cold 0.05 M citric acid-phosphate five dependent hybrid clones from five sets of man-mouse hybrids buffer, pH 5.5, frozen and thawed times, and centrifuged were tested for the presence or absence of human HexA and at 40 for 30 min at 40,000 g. Supernatants (10 Al) were applied HexB and 24 other human enzymes. The presence of HexA 2 mm of to gels (8 cm X 10 cm X thick) 1% agar (Difco and HexB in the hybrid cells was determined by immunodiffu- Bacto) in 0.025 M trisodium citrate-citric acid buffer, pH sion, immunoelectrophoresis, starch-gel, and agar-gel elec- 7.2. Electrophoresis was performed for 31/2 hr at 15 mA per trophoresis. gel and 90 V on a cooling plate at 4°. Hexosaminidase activity im- incubation Rabbit anti-human hexosaminidase antibodies form was observed with long wave ultraviolet light after mune with human which of 4 complexes hexosaminidases retain (370) of gels in a solution methylumbelliferyl-o3-D-N- to allow visualization Chem. in 0.1 M sufficient enzymatic activity for by acetylglucosaminide (Pierce Co.) (0.1 mg/ml) specific enzyme staining (19). It was established by immuno- 4.5. Fluorescence was en- citric acid-phosphate buffer, pH electrophoresis in the agar-gel system, which separated mouse hanced with 0.25 M Na carbonate- buffer, pH 10 (18). (anodal) and human (cathodal) hexosaminidase A and B, Immunological Identification of Hexosaminidase. Rabbit that anti-human hexosaminidase rabbit sera did not react with anti-human hexosaminidase sera and specific anti-human mouse hexosaminidase in mouse and hybrid cell extracts HexA sera were prepared as previously described (19). Im- under the experimental conditions used. As expected, non- munoelectrophoresis was carried out on agar gels (above) specific anti-human hexosaminidase reacted with HexA and (20), allowing diffusion of the antisera to take place at 270 for HexB, and specific anti-human HexA reacted only with hu- 24 hr. Precipitin arcs possessing hexosaminidase activity man HexA in human and hybrid cell extracts. Immunodif- were observed by the staining procedure above. Double im- fusion tests (Fig. 1) on cellulose acetate gel showed that the munodiffusion tests were carried out on cellulose acetate gels specific anti-human HexA antiserum gave a reaction of (Cellogel-Reeve Angel) equilibrated in 0.04 M Na barbital- identity between partially purified HexA and HexA from the Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Human Hexosaminidases in Cell Hybrids 1571 TABLE 1. Segregation of hexosaminidase A (HexA) and mannosephosphate isomerase (MPI) in independent hybrid clones M-~~~~~ HexA/ HexA/ HexA/ HexA/ MPI MPI MPI MPI Hybrid sets +/+ +/- -/+ -/- WIL 6 1 0 6 2 3 4 5 6 7 8 9 REW 20 0 0 6 FIG 3. Mannosephosphate isomerase (MPI) zymogram of JVR 2 0 0 7 human (H), mouse (M), and hybrid cell enzymes. Human MPI JWR 5 0 0 5 in fibroblasts (channels 8 and 9), kidney (channel 1), and white ALR 1 0 1 0 blood cells (channel 2), migrates cathodal to mouse MPI in Total 34 1 1 24 parental cells (channel 6) and tissues. An artificial mixture of human and mouse cell extracts is in channel 7. Hybrid cell ex- tracts positive for human MPI are in channels 4 and 5, and a Independent hybrid clones from each hybrid set were scored hybrid cell extract negative for human MPI is demonstrated in for the presence (+) or absence (-) of HexA and MPI. The channel S. A minor component of mouse MPI (channel 6) mi- parental cells used for the five hybrid sets were WI-38 and LTP grates to the same location as human MP1. The weak activity of JoVa (WIL); WI-38 and RAG (REW); JoVa X RAG (JVR); this minor band does not interfere with scoring human MPI white blood cells X RAG (JWR); and AnLy X RAG (ALR). (compare channels 3-5). TS-408 fibroblasts (channel 9) were de- rived from a patient with Tay-Sachs disease. human parental fibroblasts. No reaction was observed between specific anti-human HexA and partially purified HexB, ex- expression of human HexA in man-mouse hybrids (Table 1). tracts of fibroblasts from patients with Tay-Sachs disease HexA and MPI were expressed together in 34 primary clones (HexA deficient), or LTP and RAG mouse cells. Thus, hybrid and were absent together in 24 primary clones. If the two clones were scored for the presence of HexA by the presence genes were unlinked they would be expected to segregate in- of an enzymatically active immunoprecipitate, after reacting dependently. Only two discordant clones were observed (3%) with specific anti-human HexA rabbit serum. Since non-spe- which is most probably due to chromosome breakage. An cific anti-human hexosaminidase reacted with both human exception rate of 4-6% has been observed for several linkages HexA and HexB (Fig. 1, Pattern Cl and C5), human HexB determined with cell hybrids (6). could be determined immunologically iA cell hybrids by a posi- The presence of a third marker in this linkage group was tive reaction with non-specific anti-human hexosaminidase important for interpreting one discordant clone. Pyruvate and a negative reaction with specific anti-human HexA (Fig. kinase-3 has been linked to MPI in L-cell X human hybrids 1, compare B4 and C3). (6). If MPI and PK-3 are linked, then PK-3 and HexA should The presence of human HexA in hybrid cells was indepen- also segregate concordantly. Linkage of MPI, HexA, and dently determined by starch-gel electrophoresis (Fig. 2). The PK-3 was in fact demonstrated in WIL hybrids (Table 2). presence or absence of human HexA analyzed by electro- Twelve primary clones segregated concordantly for the three phoretic or immunological procedures coincided. An apparent markers and one clone (WIL-15) did not. WIL-15 was nega- alteration of the human HexB phenotype was detected in tive for MPI (Table 1), but positive for HexA and PK-3 some cell hybrid extracts when subjected to different electro- (Table 2). This indicates the retention of a chromosome frag- phoretic systems (23). However, the presence of HexB in ment containing the HexA and PK-3 loci in this discordant hybrid clones could be unambiguously identified by a combi- clone. nation of starch and agar-gel electrophoresis and immunodif- Twenty-three other enzymes representing markers for at fusion against anti-human hexosaminidase. In contrast to least 17 of the 23 human linkage groups (5) were tested for agar-gel electrophoresis in which only one mouse hexosamini- linkage with HexA (Table 2). Concordant segregation with dase band (anodal) was observed, starch-gel electrophoresis any of the other markers was not observed. separated mouse hexosaminidase activity into two major Discordant segregation of the genes responsible for the zones (Fig. 2). Hybrid extracts which were positive for human HexA and HexB phenotypes in cell hybrids was observed in HexB demonstrated an additional band of activity cathodal sixty primary clones (Table 2). Thirty-four clones were to the origin and between the mouse slowly migrating com- HexA+/HexB+, 12 clones were HexA-/HexB-, and 14 ponent and human HexB (Fig. 2, channels 2 and 3). Artificial clones were HexA-/HexB+, indicating that the two genes mixtures did not demonstrate this band but a composite of involved are not located on the same chromosome. However, both parental patterns. The significance of this band has not the fourth possible combination (HexA+/HexB-), expected been determined. for two unlinked genes with independent phenotypic expres- Human mannosephosphate isomerase (MPI) was sepa- sions, was never observed. This indicates that the expression rated from mouse MPI in hybrid cell extracts by starch-gel of HexA is dependent on the presence of HexB in cell hybrids. electrophoresis (Fig. 3). No intermediate enzyme band Concordant segregation of HexB with 25 enzyme markers (heteropolymer) was detected, suggesting that MPI is a (Table 2) was not observed. monomer. Human MPI migrated to the same position in fibroblasts and all tissues examined. The enzyme was not ex- DISCUSSION pressed in plasma or erythrocytes. The assignment of a human gene to a linkage group or chro- A positive correlation was observed for the segregation of mosome by the somatic cell hybrid technique is now an estab- the gene coding for human MPI and the gene controlling the lished and reliable alternative to Mendelian linkage proce- Downloaded by guest on September 29, 2021 1572 Genetics: Lalley et al. Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 2. Segregation of HexA, HexB, and 24 other The finding of concordant segregation of HexA and MPI enzyme markers phenotypes in 60 independent man-mouse somatic cell hy- brids (Tables 1 and 2) demonstrates linkage between the HexA HexB genes coding for human MPI and for the expression of hexos- segregation segregation aminidase A. Since linkage between the genes for human MPI Chromo- Con- Dis- Con- Dis- and fibroblasts pyruvate kinase (PK-3) has been previously some cordant cordant cordant cordant described (6), the gene coding for the expression of human HexA should also be linked to the gene coding for PK-3. In HexA - 46 14 HexB - 46 14 WIL hybrids (WI-38 X LTP cells) this linkage was in fact PGM-1/Pep-C 1 39 21 47 13 demonstrated (Table 2). This demonstration was restricted IDH/MDH 2 35 25 47 13 to mouse Icell X human fibroblasts cell hybrids, since ex- ME 6 30 24 37 17 pression of human PK-3 was suppressed in all mouse RAG X MPI* 7 58 2 46 14 human fibroblast hybrids tested (6). Mannosephosphate isom- PK-3* - 13 0 8 5 erase and PK-3 are expressed in fibroblasts of patients with GOT 10 35 23 49 11 Tay-Sachs disease (HexA deficiency) (Fig. 3), suggesting that LDH-A/Es-A4 11 38 22 47 13 genetic variants of these two enzymes could be used for con- LDH-B/Pep-B 12 39 19 38 20 firmation of the linkage in family studies, and possibly in NP 14 35 25 44 16 APRTt 16 5 8 6 7 genetic counseling. Mannosephosphate isomerase variants Pep-A 18 37 23 46 14 have been described in non-human primates (25). PHI 19 36 22 43 15 The gene for human MPI has been assigned to chromosome ADA 20? 30 18 36 12 7 by man-mouse somatic cell hybrid studies (7). Pending con- IPO-A 21 31 19 37 13 firmation of this assignment, our data indicate that the gene HGPRT/ coding for the expression of human HexA, linked to the genes PGK/G6PD X 34 27 48 12 for MPI and PK-3, may also be located on chromosome 7. AcP-2- 19 9 20 8 Independent cytological confirmation is necessary to substan- AK-1§ 14 16 13 17 tiate this tentative assignment. AK-2 1 18 12 11 19 The discordant segregation of HexB and of 25 enzyme markers coded for by genes assigned to 14 human chromo- Segregation analysis of human HexA, HexB, and 24 other somes (5) and studies on X/9 and X/22 translocations (T. B. human enzyme markers. The gene/gene linkages and chromo- Shows and J. A. Brown, unpublished) restricts the somes assigned are as previously summarized (5). The concordant assign- column gives the number of clones in which human HexA and a ment of the gene coding for HexB to either chromosome 3, 4, particular marker were both present or absent together; the dis- 5, 8, 13, 15, or 17. cordant column gives the number of clones in which only HexA The discordant segregation of human HexA and HexB or the enzyme marker was present. The enzyme markers, analyzed phenotypes observed in a total of 60 primary clones indicates by gel electrophoresis, were: lactate dehydrogenase A and B that the genes coding for the expression of the two enzymes (LDH); esterase-A4 (Es-A4); peptidase A, B, and C (Pep-A, B, are not located on the same chromosome. This discordant C); phosphoglucomutase (PGM-1); glucose-6-phosphate dehy- segregation, however, was not complete, since the HexA drogenase (G6PD); malate dehydrogenase (MDH); malic en- phenotype was never present in the absence of HexB, indicat- zyme (ME); phosphohexose isomerase (PHI); glutamate oxaloace- ing dependence of the expression of HexA on the presence tate transaminase (GOT); adenylate kinase 1 and 2 (AK-1, -2); It should be noted that the of HexB indophenol oxidase (IPO-A); and isocitrate dehydrogenase of HexB (26). expression (IDH) (11, 32). Hypoxanthine-guanine phosphoribosyl trans- was independent of the expression of HexA. These observa- ferase (HGPRT) (33); phosphoglycerate kinase (PGK) (34); tions are at variance with the reported complete independence adenosine deaminase (ADA) (35); nucleoside phosphorylase of the phenotypic expression of human HexA and HexB in (NP) (36); adenine phosphoribosyl transferase (APRT) (37); Chinese hamster-human cell hybrids (30). Our results, how- pyruvate kinase (PK-3) (6); and acid phosphatase (AcP-2) (38) ever, have been obtained by the combined use of immunologi- were also determined. cal and electrophoretic techniques affording a greater re- * In WIL hybrids HexA, MPI, and PK-3 segregated together liability in the scoring of HexA and HexB than the coin- in 12 hybrid clones; 6 clones were positive for all three markers cidence of electrophoretic mobility of hexosaminidase bands. and 6 clones did not express the three markers. There was one This may account for the apparent discrepancy, since the exception which expressed HexA and PK-3 but not MP1. PK-3 of HexB can be altered in cell was only observed in WILhybrids (See Discussion). electrophoretic mobility hy- t Only in WIL hybrids. brids (23). t Only in WIL and some REW hybrids. The observed dependence of HexA expression on the pres- § Only in WIL, JVR, and JWR hybrids. ence of HexB shows that the somatic cell hybrid technique can yield data which go beyond the assignment of genes to dures (5). This approach is particularly useful when genetic linkage groups, and bear directly on the interaction between markers are not available, as in the case of human hexosamini- genes or gene products. We interpret these data as evidence in dase, in which enzyme deficiency is relatively rare and no elec- support of the concept of a structural relationship between trophoretic variants have been observed in over 2500 individ- human HexA and HexB. Structural relationships have been uals tested (24). More importantly, this technique offers suggested by biochemical and immunological data, and by the possibility of understanding the genetic structure of a the deficiency of both HexA and HexB activity in Sandhoff- complex enzyme system, such as human hexosaminidase, by Jatzkewitz disease, presumably due to a single mutation (1, 3, isolating the contribution of its individual components. 19, 27, 29). Our data do not allow distinction between dif- Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Human Hexosaminidases in Cell Hybrids 1573

ferent hypotheses on the structural relationship between the 13. Klebe, R. J., Chen, T. R. & Ruddle, F. H. (1970) Proc. Nat. two enzymes, as, for example, the proposed existence of a Acad. Sci. USA 66, 1220-1227. 14. Miggiano, V., Nabholtz, M. & Bodmer, W. (1969) "Hetero- common subunit shared by HexA and HexB (28) or the pro- specific Genome Interaction," Wistar Inst. Symp. Monogr. posed conversion of HexB to HexA by the action of glycosyl 9, 61-76. transferases (29). However, our data indicate that a gene 15. Littlefield, J. W. (1964) Science 145, 709-710. exists that is responsible for the expression of HexA, possibly 16. Puck, T. T., Marcus, P. T. & Cieciura, S. J. (1956) J. Exp. Med. 273-281. either a B or 103, through modification of hexosaminidase through 17. Shows, T. B., Ruddle, F. H. & Roderick, T. H. (1969) Bio- an interaction with a HexB gene product. chem. Genet. 3, 25-35. Expression of a lysosomal enzyme, such as hexosaminidase, 18. Leeback, D. H. & Walker, P. G. (1961) Biochem. J. 78, 151- most likely depends on an interaction of structural, regulatory, 156. and "architectural" genes (31). The mutation responsible 19. Bartholomew, W. R. & Rattazzi, M. C. (1974) Int. Arch. Allergy Appl. Immunol. 46, 512-524. for HexA deficiency in Tay-Sachs disease, therefore, may not 20. Grabar, P. (1964) in Immunoelectrophoretic Analysis, eds. necessarily involve the same gene responsible for HexA ex- Grabar, P. & Burtin, P. (Am. Elsevier, New York), p. 3. pression which we describe as linked to MPI and PK-3. 21. Shows, T. B. & Ruddle, F. H. (1968) Proc. Nat. Acad. Sci. Nevertheless, our results present a genetic framework for de- USA 61, 574-581. 22. Nichols, E. A., Chapman, V. M. & Ruddle, F. H. (1973) termining the basic defects responsible for hexosaminidase Biochem. Genet. 8, 47-53. deficiency in GM2 gangliosidosis. 23. Rattazzi, M. C., Lalley, P. A., Carmody, P. J. & Shows, We thank Ms. Linda Haley, Ms. Andrea Goggin, Ms. Ida Hill, T. B. (1973) Amer. J. Hum. Genet. 25, 63A. and Mr. Roger Eddy for excellent technical assistance. This work 24. Swallow, D. M. Stokes, D, C., Corney, G. & Harris, H. was supported in part by NIH Grants HD-05196, 06321and GM- (1974) Ann. Hum. Genet. 37, 287-302; Rattazzi, M. C., un- 20454 and NSF Grant HD-GM-GB-39273. P.A.L. is a recipient published data. of a New York State Department of Health predoctoral fellow- 25. Ritter, H. & Schmitt, J. (1973) Humangenetik 19, 325-326. ship. M.C.R. is a recipient of a Career Development Award 26. Lalley, P. A., Rattazzi, M. C. & Shows, T. B. (1973) Amer. J. (5-K04-GM-70638-02) from the National Institute of General Hum. Genet. 25, 44A. Medical Sciences. 27. Carroll, M. & Robinson, D. (1973) Biochem. J. 131, 91-96. 28. Srivastava, S. K. & Beutler, E. (1973) Nature 241, 463. 1. Robinson, D. & Stirling, J. L. (1968) Biochem. J. 107, 321- 29. Goldstone, A., Konecny, P. & Koenig, H. (1971) FEBS 327. Lett. 13, 68-72. 2. Okada, S. & O'Brien, J. S. (1969) Science 165, 698-700. 30. van Someren, H. & van Henegouwen, H. B. (1973) Hu- 3. Sandhoff, K. Harzer, K., Whssle, W. Jatzkewitz, H. (1971) mangenetik 18, 1-4. J. Neurochem. 18, 2469-2489. 31. Paigen, K. (1971) in Enzyme Synthesis and Degradation in 4. Suzuki, Y. & Suzuki, K. (1970) Neurology 20, 848-851. Mammalian Systems, ed. Recheigl, M. (University Park 5. Ruddle, F. H. (1973) Nature 242, 165-169. Press, Baltimore, Md.), pp. 1-46. 6. Shows, T. B. (1974) in Somatic Cell Hybridization, eds. 32. Shows, T. B. (1972) Biochem. Genet. 7, 193-204. Davidson, R. and de la Cruz, F. (Raven Press, New York), 33. Shin, S., Kahn, M. & Cook, P. R. (1971) Biochem. Genet. pp. 15-23. 5, 91-99. 7. McMorris, F. A., Chen, T. R., Ricciuti, F., Tischfield, J., 34. Chen, S. H., Malcolm, L. A., Yoshida, A. & Giblett, E. R. Creagen, R. & Ruddle, F. H. (1973) Science 179, 1129-1131. (1971) Amer. J. Hum. Genet. 23, 87-91. 8. Hayflick, L. & Moorhead, P. S. (1961) Exp. Cell Res. 25, 35. Spencer, N., Hopkinson, D. A. & Harris, H. (1968) Ann. 585-621. Hum. Genet. 32, 9-14. 9. Cohen, M. AM., Lin, C. C., Sybert, V. & Orecchio, E. J. 36. Edwards, Y. H., Hopkinson, D. A. & Harris, H. (1971) (1972) Amer. J. Hum. Genet. 24, 583-597. Ann. Hum. Genet. 34, 395-408. 10. Brown, J. A., unpublished data. 37. Mowbray, S., Watson, B. & Harris, H. (1972) Ann. Hum. 11. Shows, T. B. (1972) Proc. Nat. Acad. Sci. USA 69, 348-352. Genet. 36, 153-162. 12. Klebe, R. J., Chen, T. R. & Ruddle, F. H. (1970) J. Cell 38. Shows, T. B. & Lalley, P. A. (1974) Biochem. Genet. 11, Biol. 45, 74-82. 123-141. Downloaded by guest on September 29, 2021