26 research outputs found
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Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: assignment of human asnS gene to chromosome 18.
We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39 degrees C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS, which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18
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Assignment of structural gene for asparagine synthetase to human chromosome 7.
Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5 degrees C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5 degrees C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent
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Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8.
The segregation of human cytosolic alanine aminotransferase (AAT1) and the individual human chromosomes has been studied in 27 secondary and tertiary rat hepatoma-human (liver) fibroblast hybrids. The staining solution used to visualize AAT activity on starch gels was specific for AAT since it was visualized only when all components of the stain were present. The locus for human AAT1 has been assigned to chromosome 8
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Assignment of the gene for cytosolic alanine aminotransferase (AAT1) to human chromosome 8.
The segregation of human cytosolic alanine aminotransferase (AAT1) and the individual human chromosomes has been studied in 27 secondary and tertiary rat hepatoma-human (liver) fibroblast hybrids. The staining solution used to visualize AAT activity on starch gels was specific for AAT since it was visualized only when all components of the stain were present. The locus for human AAT1 has been assigned to chromosome 8
Purine nucleoside phosphorylase deficiency in two unrelated Saudi patients
Purine nucleoside phosphorylase (PNP) deficiency is a rare autosomal recessive metabolic disorder that results in combined immunodeficiency, neurologic dysfunction and autoimmunity. PNP deficiency has never been reported from Saudi Arabia or in patients with an Arabic ethnic background. We report on two Saudi girls with PNP deficiency. Both showed severe lymphopenia and neurological involvement. Sequencing of the PNP gene of one girl revealed a novel missense mutation Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT. Expression of PNP (146L) cDNA in E coli indicated that the mutation greatly reduced, but did not completely eliminate PNP activity
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Assignment of structural gene for asparagine synthetase to human chromosome 7.
Somatic cell hybrids obtained from the fusion of human B lymphocytes and an asparagine synthetase-deficient Chinese hamster ovary cell line were isolated after growth in asparagine-free medium. The human and hamster forms of asparagine synthetase differ significantly in their rate of inactivation at 47.5 degrees C. The asparagine synthetase activity expressed in the hybrids was inactivated at 47.5 degrees C at the same rate as the human form of the enzyme. Karyotypic analysis and analysis for chromosome-specific enzyme markers showed that the structural gene for asparagine synthetase is located on chromosome 7 in humans. The heat-inactivation profile for asparagine synthetase in extracts of hybrids formed between human peripheral leukocytes and a hamster cell line expressing asparagine synthetase activity was intermediate between the two parental types when human chromosome 7 was present, but was identical to the hamster parent when chromosome 7 was absent
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Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive Chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: Assignment of humanasnS gene to chromosome 18
We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39 degrees C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS, which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18
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Regional assignment of the structural gene for human acid beta-glucosidase to q42 leads to qter on chromosome 1.
The structural gene for human acid beta-glucosidase (GBA) has been regionally assigned to a narrow region on chromosome 1 using somatic cell hybridization, specific immunoprecipitation, and assay with the natural substrate. A human fibroblast line, 46,XX,del(1)(pter leads to q42:), was fused with mouse RAG fibroblasts and the heterokaryons were subcloned. All hybrid subclones containing a normal chromosome 1 were positive for GBA. In contrast, subclones with a single del(1) were negative for GBA by both immunoprecipitation and natural substrate assays. These results were consistent with the previous assignment of GBA to the region 1p11 leads to qter and further localized the gene to the narrow region 1q42 leads to qter
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Chromosomal localization of the gene for Gaucher disease
Two isozymes with a - glucosidase activity have been
identified in normal human tissues using the artificial
substrate, 4-methylwnbelliferyl- S-D-glucopyranoside (4MUGlc).
The acid (EC 3.2.1. 45) and neutral (EC 3.2.1.21)
B- glucosidases (designated GBA and GBN, respectively) have
been differentiated by their relative pH optima (Beutler
and Kuhl, 1970; Ho, et al., 1972; Turner et al., 1977;
Mueller and Rosenberg, 1977), subcellular localizations
(Ho, 1972; Peters et al., 1973), substrate specificities
(Patrick, 1965; Ho, 1972; Peters et al., 1976), sensitivities
to anionic detergents and acidic phospholipids
(Peters et al., 1976), affinity for concanavalin A (Beutler
et al., 1975; Shafit- Zagardo et al . , 1980) and most recently
by their differential electrophoretic migration on
cellulose acetate gels (Shafit- Zagardo et al., 1980). The
acid isozyme, a membrane bound activity, has been shown to
be deficient in the various subtypes of Gaucher disease,
lysosomal storage diseases characterized by the accumulation
of glucosyl ceramide (Brady, 1978). To date, the
chromosomal assignment of the structural gene for either of
the human 8 -glucosidase isozymes has not been determined .
We report here .the regional assignment of the structural
gene for human GBA using human-rodent somatic cell
hybrids . A sensitive immunoprecipitation assay was developed
for the selective detection of the human enzyme in the
presence of mouse 6 -glucosidase activity. Initial data for the regional assignment of the locus on chromosome 1 was
obtained from a hybrid clone containing a 11\\)USe-hwuan
chromosome 1 rearrangement. Further regional localization
of the locus for GBA near lqter was obtained using an
informative hybrid clone carrying a human chromosome 1
deletion. In addition to the immunoprecipitation assay,
use of the specific natural substrate further supported the
assignment of the structural gene for GBA to this region