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1.
Yeast ; 13(12): 1195-7, 1997 Sep 30.
Article in English | MEDLINE | ID: mdl-9301025

ABSTRACT

The following urease genes of the fission yeast Schizosaccharomyces pombe have been mapped by induced haploidization and tetrad analysis--ure1: chromosome are III-L; ure2 and ure3: chromosome are I-R. The previously determined tps19-rad1 interval (11-12 cM) has been increased to 18 cM. A convenient medium for rapidly scoring the ure gene markers of fission yeast was developed.


Subject(s)
Chromosome Mapping , Schizosaccharomyces/genetics , Urease/genetics
2.
Can J Microbiol ; 42(2): 132-40, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8742356

ABSTRACT

The urease from the ascomycetous fission yeast Schizosaccharomyces pombe was purified about 4000-fold (34% yield) to homogeneity by acetone precipitation, ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column chromatography, and if required, Mono-Q ion-exchange fast protein liquid chromatography. The enzyme was intracellular and only one species of urease was detected by nondenaturing polyacrylamide gel electrophoresis (PAGE). The native enzyme had a M(r) of 212 kDa (Sepharose CL6B-200 gel filtration) and a single subunit was detected with a M(r) of 102 kDa (PAGE with sodium dodecyl sulfate). The subunit stoichiometry was not specifically determined, but the molecular mass estimations indicate that the undissociated enzyme may be a dimer of identical subunits. The specific activity was 700-800 micromols urea.min-1.mg protein-1, the optimum pH for activity was 8.0, and the Km for urea was 1.03 mM. The sequence of the amino terminus was Met-Gln-Pro-Arg-Glu-Leu-His-Lys-Leu-Thr-Leu-His-Gln-Leu-Gly-Ser-Leu-Ala and the sequence of two tryptic peptides of the enzyme were Phe-Ile-Glu-Thr-Asn-Glu-Lys and Leu-Tyr-Ala-Pro-Glu-Asn-Ser-Pro-Gly-Phe-Val-Glu-Val-Leu-Glu-Gly-Glu-Ile- Glu- Leu-Leu-Pro-Asn-Leu-Pro. The N-terminal sequence and physical and kinetic properties indicated that S. pombe urease was more like the plant enzymes than the bacterial ureases.


Subject(s)
Schizosaccharomyces/enzymology , Urease/isolation & purification , Urease/metabolism , Amino Acid Sequence , Bacteria/enzymology , Chromatography, Gel , Chromatography, Ion Exchange , Hydrogen-Ion Concentration , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Macromolecular Substances , Manganese/pharmacology , Molecular Sequence Data , Molecular Weight , Nickel/pharmacology , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Schizosaccharomyces/growth & development , Sequence Homology, Amino Acid , Urease/chemistry
3.
J Biol Chem ; 261(2): 733-8, 1986 Jan 15.
Article in English | MEDLINE | ID: mdl-3001079

ABSTRACT

The primary defect responsible for mucolipidosis III is a deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine 1-phosphotransferase activity (GlcNAc phosphotransferase). Genetic complementation analysis of cultured fibroblasts derived from 12 patients with mucolipidosis III identified complementation groups A, B, and C (Honey, N. K., Mueller, O. T., Little, L. E., Miller, A. L., and Shows, T. B. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7420-7424). The GlcNAc phosphotransferase activity present in the cell lines comprising the complementation groups was characterized with respect to endogenous substrates and two exogenous acceptors, alpha-methyl-D-mannoside and high mannose glycopeptides. All group C cell lines and one group A cell line were found to have normal GlcNAc phosphotransferase activity levels at 37 degrees C when screened with these exogenous acceptors. The enzyme activity in group A cell lines was within normal range when assayed at 23 degrees C. Inhibition of the phosphorylation of alpha-methyl-D-mannoside in the presence of increasing amounts of endogenous substrate N-acetyl-beta-D-hexosaminidase B was demonstrated in normal cell lines at 23 and 37 degrees C and in group A cells at 23 degrees C. However, group C cell lines did not show any inhibition at either temperature. This suggests that the alteration of the GlcNAc phosphotransferase from individuals in group C affects the recognition site for the protein portion of lysosomal enzymes, whereas group A individuals have mutations which result in a temperature-sensitive enzyme.


Subject(s)
Mucolipidoses/enzymology , Phosphotransferases/metabolism , Transferases (Other Substituted Phosphate Groups) , Cell Line , Genetic Complementation Test , Hexosaminidases/metabolism , Humans , Kinetics , Methylmannosides/metabolism , Phosphorylation , Placenta/enzymology , Sucrose/pharmacology , Temperature , beta-N-Acetylhexosaminidases
4.
Hum Genet ; 72(1): 27-31, 1986 Jan.
Article in English | MEDLINE | ID: mdl-3455919

ABSTRACT

A rat cDNA probe for preprocarboxypeptidase A was used to follow the segregation of the human gene for carboxypeptidase A (CPA) in 49 human X mouse somatic cell hybrids using Southern filter hybridization techniques. CPA was assigned to human chromosome 7q22----qter. Similarly, the probe was used to follow the segregation of the mouse gene for carboxypeptidase A (Cpa) in 19 mouse X Chinese hamster somatic cell hybrids. Cpa was assigned to mouse chromosome 6. The gene for carboxypeptidase A forms part of a syntenic group that is conserved in man and mouse.


Subject(s)
Carboxypeptidases/genetics , Chromosome Mapping , Chromosomes, Human, 6-12 and X , Animals , Carboxypeptidases A , Cricetinae , Cricetulus , DNA/genetics , Electrophoresis, Agar Gel , Genetic Markers , Humans , Hybrid Cells , Mice
5.
Somat Cell Mol Genet ; 10(4): 369-76, 1984 Jul.
Article in English | MEDLINE | ID: mdl-6589790

ABSTRACT

The genes for the serine proteases trypsin, chymotrypsin B, and elastase were chromosomally assigned in man using cDNA probes that have been isolated from a rat pancreatic cDNA library. DNA from human X rodent somatic cell hybrids was cleaved with BamHI or EcoRI and analyzed by Southern filter hybridization methods for the segregation of the genes for trypsin-1 (TRY1), chymotrypsin B (CTRB), and elastase-1 (ELA1). TRY1 was assigned to human chromosome 7q22----qter, CTRB to chromosome 16, and ELA1 to chromosome 12. Although the three genes are members of the same gene family, they are dispersed over different chromosomes.


Subject(s)
Chromosomes, Human, 16-18 , Chromosomes, Human, 6-12 and X , Chymotrypsin/genetics , Pancreatic Elastase/genetics , Trypsin/genetics , Biological Evolution , Chromosome Mapping , DNA/genetics , Genes , Humans
6.
Somat Cell Mol Genet ; 10(4): 377-83, 1984 Jul.
Article in English | MEDLINE | ID: mdl-6589791

ABSTRACT

The mouse genes for the serine proteases trypsin (Try-1), chymotrypsin B (Ctrb), and elastase (Ela-1) were chromosomally assigned using Southern blot hybridization of mouse X Chinese hamster cell hybrid DNA. cDNA probes for the three genes were hybridized to cell hybrid DNA cleaved with BamHI or HindIII and the segregation of Try-1, Ctrb, and Ela-1 was correlated with the segregation of mouse chromosomes. Try-1 is located on chromosome 6, Ctrb is on chromosome 8, and Ela-1 is on chromosome 15. The three genes fall into three syntenic groups that are conserved in the mouse and human genomes.


Subject(s)
Mice/genetics , Animals , Biological Evolution , Chromosome Mapping , Chymotrypsin/genetics , DNA/genetics , Genes , Pancreatic Elastase/genetics , Trypsin/genetics
7.
Cancer Genet Cytogenet ; 10(3): 287-310, 1983 Nov.
Article in English | MEDLINE | ID: mdl-6354430

ABSTRACT

The tumor phenotype is associated with the rearrangement of genetic information and the altered expression of many gene products. In this review, genes associated with the tumor phenotype have been arranged on the human gene map and indicate the extent to which the tumor phenotype involves the human genome. Nonrandom chromosomal aberrations that are frequently observed in tumors are presented. Altered metabolic demands of the tumor cell are reflected in altered gene expressions of a wide range of enzymes and other proteins, and these changed enzyme patterns are described. The study of oncogenes increasingly suggests that they may be significant in certain cancers, and the assignment of these genes has been tabulated. The biochemical and metabolic changes observed in tumors are complex; studying the patterns and interactions of these changes will aid our genetic understanding of the origins and development of tumors.


Subject(s)
Neoplasms/genetics , Oncogenes , Carcinoembryonic Antigen/analysis , Cell Transformation, Neoplastic , Chromosome Aberrations , Chromosome Disorders , Chromosome Mapping , Enzymes/genetics , Genes , Hormones/genetics , Humans , Hybridomas , Membrane Proteins/genetics , Phenotype
8.
J Clin Invest ; 72(3): 1016-23, 1983 Sep.
Article in English | MEDLINE | ID: mdl-6309902

ABSTRACT

The genetic relationships between the multiple variants of mucolipidosis II (I-cell disease) and mucolipidosis III (pseudo-Hurler polydystrophy) were investigated with a sensitive genetic complementation analysis procedure. These clinically distinct disorders have defects in the synthesis of a recognition marker necessary for the intracellular transport of acid hydrolases into lysosomes. Both disorders are associated with an inherited deficiency of a uridine diphosphate-N-acetyl-glucosamine: lysosomal enzyme precursor N-acetyl-glucosamine-phosphate transferase activity. We had previously shown that both disorders are genetically heterogeneous. Complementation analysis between mucolipidosis II and III fibroblasts indicated an identity of mucolipidosis II with one of the three mucolipidosis III complementation groups (ML IIIA), suggesting a close genetic relationship between these groups. The presence of several instances of complementation within this group suggested an intragenic complementation mechanism. Genetic complementation in heterokaryons resulted in increases in N-acetyl-glucosamine-phosphate transferase activity, as well as in the correction of lysosomal enzyme transport. This resulted in increases in the intracellular levels of several lysosomal enzymes and in the correction of the abnormal electrophoretic mobility pattern of intracellular beta-hexosaminidase. The findings demonstrate that a high degree of genetic heterogeneity exists within these disorders. N-acetyl-glucosamine-phosphate transferase is apparently a multicomponent enzyme with a key role in the biosynthesis and targeting of lysosomal enzymes.


Subject(s)
Lysosomes/enzymology , Mucolipidoses/genetics , Phosphotransferases/genetics , Transferases (Other Substituted Phosphate Groups) , Cell Fusion , Cells, Cultured , Fibroblasts/enzymology , Genetic Complementation Test , Humans , Mucolipidoses/classification , Mucolipidoses/enzymology , Mutation , Phosphotransferases/deficiency
9.
Proc Natl Acad Sci U S A ; 79(23): 7420-4, 1982 Dec.
Article in English | MEDLINE | ID: mdl-6961420

ABSTRACT

Mucolipidosis III (ML III), or pseudo-Hurler polydystrophy, is an inherited childhood disorder characterized biochemically by low activities and abnormal electrophoretic patterns of multiple lysosomal enzymes in fibroblasts. The primary deficiency of ML III has been proposed to be in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. However, variation in this enzyme and in other biochemical properties of different ML III lines has been observed. Therefore, we investigated genetic heterogeneity within the disorder by complementation analysis. Heterokaryon cell fractions were generated by fusing together ML III fibroblast lines. When pairs of cells complemented, correction of lysosomal enzyme activities and electrophoretic patterns was observed. Twelve fibroblast lines from 10 sibships were analyzed and three distinct complementation groups were characterized. One complementation group represents the classical ML III disorder. A single cell line identifies a second complementation group. The cell lines comprising a third complementation group have a number of biochemical characteristics different from classical ML III and may represent a genetically distinct disorder.


Subject(s)
Mucolipidoses/genetics , Cell Line , Genetic Complementation Test , Humans , Hybrid Cells , Hydrolases/genetics , Lysosomes/enzymology , Phenotype
10.
Am J Med Genet ; 12(3): 343-53, 1982 Jul.
Article in English | MEDLINE | ID: mdl-6287841

ABSTRACT

I-cell disease (mucolipidosis II) is a fatal childhood disorder affecting the expression of multiple lysosomal acid hydrolases. The disorder is characterized by clinical and biochemical heterogeneity which may reflect different mutants with a similar phenotype. Genetic complementation studies demonstrating genetic heterogeneity within this disorder are described utilizing cultured fibroblasts from 11 different patients. Fibroblasts from I-cell disease (ICD) and from five different lysosomal storage diseases with single structural gene enzyme deficiencies were fused in different combinations, and fractions enriched for multinucleated heterokaryons were isolated and tested for acid hydrolase activity and electrophoretic mobility. In fusions of ICD fibroblasts and various single lysosomal enzyme-deficient fibroblasts, the activity of the deficient enzyme and of the other ICD hydrolases were restored, demonstrating that ICD is not a lysosomal enzyme structural gene defect and that the ICD defect, and not just the single enzyme deficiency, is corrected. In fusions involving only I-cell fibroblasts, at least two complementation groups were identified by the recovery of activities of all lysosomal enzymes tested in heterokaryons. These results demonstrate the existence of genetic heterogeneity within the disorder and suggest that different mutations can result in the I-cell clinical and biochemical phenotype. The data support an altered post-translational processing of lysosomal enzymes as the cause of ICD and suggest that at least two genes participate in this pathway.


Subject(s)
Hydrolases/deficiency , Lysosomes/enzymology , Mucolipidoses/genetics , Mutation , Cell Fusion , Fabry Disease , Fibroblasts/enzymology , Genes , Genetic Complementation Test , Hexosaminidases/deficiency , Humans , In Vitro Techniques , Lactose Intolerance , Mannosidases/deficiency , Mucolipidoses/enzymology , Phenotype , alpha-L-Fucosidase/deficiency , beta-Galactosidase
11.
Am J Med Genet ; 9(3): 239-53, 1981.
Article in English | MEDLINE | ID: mdl-7282783

ABSTRACT

The human mucolipidoses (ML) are characterized by abnormal activities and abnormal electrophoretic patterns of fibroblast lysosomal hydrolases. These altered mobility patterns can be used to confirm the clinical diagnosis of the four mucolipidoses. The mobility patterns of one nonlysosomal and seven lysosomal enzymes were tested in fibroblasts from two ML I (sialidosis type 2, infantile), fifteen ML II (I-cell disease), eight ML III (pseudohurler polydystrophy), and one ML IV patients. A single sialidosis type 2, juvenile, line was also examined. Characteristic mobility patterns were found which identify each of the four mucolipidoses. Both the ML I and sialidosis type 2 juvenile lines displayed anodal mobility patterns, but distinct differences between the two disorders were observed. Lysosomal hydrolases from ML II lines demonstrated reduced activities or had altered mobilities. Differing electrophoretic patterns demonstrated the presence of at least two groups within the clinical phenotype diagnosed as ML II, indicating heterogeneity. The ML III lines showed normal electrophoretic patterns for most lysosomal hydrolases. The ML IV line expressed normal mobilities for every enzyme studied, with a single exception. The electrophoretic patterns of only beta-hexosaminidase, acid phosphatase-2, alpha-galactosidase, and esterase A4 were sufficient to identify and distinguish the different mucolipidosis types. Electrophoretic variation was also seen in liver but not kidney extracts from three ML II patients. beta-Hexosaminidase and alpha-mannosidase B secreted into the medium by ML II and ML III fibroblasts had mobility patterns different from normal and from their intracellular patterns. These data suggest that the mucolipidoses are genetically distinct with heterogeneity within them.


Subject(s)
Hydrolases/metabolism , Lysosomes/enzymology , Mucolipidoses/diagnosis , Diagnosis, Differential , Electrophoresis , Genetic Variation , Humans , Hydrolases/genetics , Mucolipidoses/enzymology
12.
Hum Genet ; 58(4): 358-61, 1981.
Article in English | MEDLINE | ID: mdl-7327557

ABSTRACT

The human and rodent forms of glyoxalase II (Hydroxyacylglutathione hydrolase, HAGH) can readily be separated by starch gel electrophoretic procedures. Fifty-one human-rodent somatic cell hybrid clones were examined for their human HAGH and for human enzyme markers whose genes are encoded on each autosome and the X chromosome. Sixteen clones were also examined for their human karyotypes. Human glyoxalase II segregated only with chromosome 16, demonstrating that the gene is located on this chromosome.


Subject(s)
Chromosomes, Human, 16-18/ultrastructure , Genes , Thiolester Hydrolases/genetics , Animals , Cell Line , Chromosome Mapping , Cricetinae , Cricetulus , Electrophoresis, Starch Gel , Humans , Hybrid Cells/ultrastructure , Lung , Mice , Mice, Inbred C3H , Skin
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