Characterization of heteropolymeric hexosaminidase A in human X mouse hybrid cells.

Abstract

(19,20) suggested that there was no structural relationship between these two isozymes. We have therefore considered the possibility that the "Hex A" that occurs in mouse X human hybrids in the absence of human Hex B is, in point of fact, not human Hex A. Accordingly, we have investigated the "Hex A" activity that is found in the absence of Hex B in a human X mouse hybrid cell line that contains human chromosome 15 but not human chromosome 5. Immunological studies indicate that this "Hex A" material contains a normal human a subunit and is most probably a hybrid molecule containing human and mouse components. The genetic control of Tay-Sachs and Sandhoff diseases is discussed in the light of our findings. (23) and the genes for mannosephosphate isomerase, pyruvate kinase, and "Hex A" have been assigned to chromosome 15 (16-19). Five of the resulting subclones were analyzed for the continued expression of the human enzyme markers.Electrophoresis. Cells for Hex electrophoresis were grown in McCoy's 5A medium with 10% fetal calf serum added. They were harvested with 0.25% trypsin, centrifuged down, and resuspended in a 0.02 M citrate buffer, pH 5.2. The cell suspension was frozen and thawed five times and centrifuged at 14,500 X g for 15 min. The supernatant was concentrated to dryness by lyophilization. The final samples were dissolved in small volumes of 0.02 M citrate buffer, pH 5.2 (24). Cells for mannosephosphate isomerase, pyruvate kinase, and glucose-6-phosphate dehydrogenase electrophoresis were prepared as reported before (18).Starch gel electrophoresis was used to identify the human isozymes of mannosephosphate isomerase, pyruvate kinase, and glucose-6-phosphate dehydrogenase in the hybrid cell samples as described (18). Cellulose acetate gel [Cellogel; (17 cm X 17 cm X 0.5 mm) Kalex Scientific Co., Manhasset, N.Y.] was used to identify Hex A and B using modifications of the method of Okada and O'Brien (11). The sample solution (approximately 0.4 Al) was applied to the Cellogel with the aid of a multiple sample applicator (Shandon Scientific Co., Inc., Sewickley, Pa.). Electrophoresis was performed at 250 V for 2 hr, and the gel was incubated with 4-methylumbelliferyl-f.-D-glucosami iide (Sigma Chemical Co., St. Louis, Mo.), 0.2 mg/ml of 0.5 M citrate-phosphate buffer, pH 4.5. Hex activity was visualized as fluorescence under long wave UV light. Column Chromatography. Ion exchange chroma-. tography of Hex was carried out as described by Beutler et al. (8), except that the maximal NaCI concentration in the gradient was increased from 0.4 to 0.6 M.Immunological Studies. Rabbit antisera against human Hex

Somatic cell genetic analysis of the galactocerebrosidase gene: Lack of complementation in human Krabbe disease/twitcher mouse cell hybrids

“…Complementation refers to a partial or complete correction of a defective function when the products of two mutant genotypes are brought together {Reuser, 1984). In many complementation studies involving human genetic diseases, cells from patients exhibiting different clinical forms of the same disease are crossed or fused in order to determine whether different gene mutations are responsible for the clinical heterogeneity seen in the disorder (Chern et al, 1976;Champion and Shows, 1977). In this way, where complementation was observed, it was shown among the sphingolipidoses that Tay-Sachs and Sandhoff GM2 gangliosidoses were due to separate genes encoding different subunits of the P-hexosaminidase enzyme (Rattazzi et al, 1976), and that sphingolipidoses can be due to structural gene or activator protein deficiencies (Kihara et al, 1984).…”

Mechanism of interrupted saccades in patients with late-onset Tay–Sachs disease

“…LOTS saccades are interrupted by transient decelerations, not always coming to a standstill, after which a new saccade occurs. In a mouse model of LOTS, GM2 storage is prominent in the motor cortex and cerebellum (granular cells and Purkinje cells), along with atrophy in the brainstem (Chern et al, 1976). Recent neuropathological studies of LOTS (Rucker et al, this volume) have demonstrated inclusions and reduction in metabolism in omnipause neurons (OPN), inclusions in fastigial nuclei (FN), and complete loss of Purkinje cells in the dorsal cerebellar vermis.…”