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1.
Pediatr Neurol ; 25(2): 156-61, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11551746

ABSTRACT

Hallervorden-Spatz syndrome (neurodegeneration with brain iron accumulation type 1; OMIM entry 234200) is a rare inherited neurodegenerative disease. In this article, evidence for a newly identified gene as a candidate for Hallervorden-Spatz syndrome is given. Previously Hallervorden-Spatz syndrome was mapped to a 4-cm region in 20p12.3-13. During positional cloning efforts a new member of the glial-derived neurotrophic factor receptor family was discovered in this region. Like other members of this receptor family, this new gene is predicted to be secreted and glycosyl-phosphatidylinositol linked, and it maintains conserved cysteine residues. However, cDNA and genomic studies in both humans and mice indicate that this gene lacks the sequence corresponding to exons 2 and 3 in other family members. In situ hybridization reveals that it is expressed primarily in the brain and bladder in the embryonic mouse. Mutation analysis of patients with Hallervorden-Spatz syndrome revealed two potentially significant amino acid changes in two patients but failed to identify mutations in the remaining 10 subjects. The implication of these findings for the relationship between this gene and Hallervorden-Spatz syndrome is discussed.


Subject(s)
Drosophila Proteins , Membrane Glycoproteins/genetics , Pantothenate Kinase-Associated Neurodegeneration/genetics , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Cell Surface/genetics , Receptors, Nerve Growth Factor , Amino Acid Sequence , Animals , Brain/metabolism , DNA Mutational Analysis , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , In Situ Hybridization , In Vitro Techniques , Iron/metabolism , Membrane Glycoproteins/metabolism , Mice , Molecular Sequence Data , Pantothenate Kinase-Associated Neurodegeneration/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cell Surface/metabolism
2.
J Biol Chem ; 260(11): 7015-22, 1985 Jun 10.
Article in English | MEDLINE | ID: mdl-2581962

ABSTRACT

We have isolated and sequenced overlapping cDNA clones containing the entire coding region of mouse ribonucleotide reductase subunit M1. The coding region comprises 2.4 kilobases and predicts a polypeptide of 792 amino acids (Mr 90,234) which shows striking homology with ribonucleotide reductases from Escherichia coli and the herpesviruses, Epstein-Barr virus and herpes simplex virus. The homologies reveal three domains: an N-terminal domain common to the mammalian and bacterial enzymes, a C-terminal domain common to the mammalian and viral ribonucleotide reductases, and a central domain common to all three. We speculate on the functional basis of this conservation.


Subject(s)
Cloning, Molecular , DNA/analysis , Escherichia coli/enzymology , Ribonucleotide Reductases/genetics , Simplexvirus/enzymology , Amino Acid Sequence , Animals , Cell Line , Herpesvirus 4, Human/enzymology , Macromolecular Substances , Mice , Poly A/metabolism , RNA/metabolism , RNA, Messenger
3.
J Mol Appl Genet ; 1(6): 527-38, 1982.
Article in English | MEDLINE | ID: mdl-6296255

ABSTRACT

Cloned bovine papilloma virus (BPV) DNA induces cellular transformation when introduced into mouse cells growing in culture; the transferred viral DNA replicates as an extrachromosomal, closed circular element. BPV DNA is therefore an inviting replicon to construct a "shuttle" vector that can replicate in both mammalian cells and E. coli. Although BPV DNA devoid of bacterial plasmid sequences has been successfully employed to reintroduce cloned genes into rodent cells, construction of a true shuttle plasmid has been hampered by a disruptive influence of bacterial plasmid sequences that appear to block cellular transformation when included on the transferred molecule. We constructed a molecule, pGP, containing the transforming region of the BPV genome, the rat growth hormone gene, and bacterial plasmid pBR 327, and have found unexpectedly that the intact molecule can induce cellular transformation of mouse cells at high efficiency despite the presence of bacterial sequences in the transferred plasmid. A similar plasmid without the growth hormone segment does not transform mouse cells. The pGP molecule replicates as a stable plasmid in both mouse cells, where there are 30 to 80 monomer episomes per cell, and E. coli, and may be shuttled back and forth unaltered between the two kinds of cells. The growth hormone gene is transcribed in the mouse cells and gives rise to a transcript that is longer than authentic rat growth hormone mRNA and does not appear to be regulated by glucocorticoids. When pGP is cotransferred into mouse L-cells with herpes simplex virus tk gene, it appears to integrate, and free monomer episomes are not observed.


Subject(s)
Bovine papillomavirus 1/genetics , Escherichia coli/genetics , Mice/genetics , Papillomaviridae/genetics , Plasmids , Animals , Cells, Cultured , DNA Replication , DNA, Recombinant , Gene Expression Regulation , Growth Hormone/genetics , Penicillin Resistance , Thymidine Kinase/genetics , Transformation, Genetic
6.
Adv Exp Med Biol ; 122A: 375-86, 1980.
Article in English | MEDLINE | ID: mdl-6158849

ABSTRACT

The isolation and characterization of a mutant mouse T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which accounts for its resistance to adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. Furthermore, the intact cells of this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in situ. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.


Subject(s)
Adenylosuccinate Synthase/deficiency , Ligases/deficiency , Lymphoma/metabolism , Purines/metabolism , Animals , Cell Line , Cell Survival/drug effects , Mice , Mutation , Neoplasms, Experimental/metabolism , Phosphoribosyl Pyrophosphate/metabolism , Purines/pharmacology , Ribonucleotides/metabolism , T-Lymphocytes/metabolism
7.
J Biol Chem ; 254(18): 8736-9, 1979 Sep 25.
Article in English | MEDLINE | ID: mdl-225312

ABSTRACT

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.


Subject(s)
Lymphoma/enzymology , Phosphotransferases/deficiency , Uridine Kinase/deficiency , Animals , Cell Line , Cytidine , Genetic Variation , Methylnitronitrosoguanidine/pharmacology , Mice , Mutation , Neoplasms, Experimental/enzymology
9.
J Cell Physiol ; 99(1): 139-51, 1979 Apr.
Article in English | MEDLINE | ID: mdl-222775

ABSTRACT

The isolation and characterization of a mutant murine T-cell lymphoma (S49) with altered purine metabolism is described. This mutant, AU-100, was isolated from a mutagenized population of S49 cells by virtue of its resistance to 0.1 mM 6-azauridine in semisolid agarose. The AU-100 cells are resistant to adenosine mediated cytotoxicity but are extraordinarily sensitive to killing by guanosine. High performance liquid chromatography of AU-100 cell extracts has demonstrated that intracellular levels of GTP, IMP, and GMP are all elevated about 3-fold over those levels found in wild type cells. The AU-100 cells also contain an elevated intracellular level of pyrophosphoribosylphosphate (PPriboseP), which as in wild type cells is diminished by incubation of AU-100 cells with adenosine. However AU-100 cells synthesize purines de novo at a rate less than 35% of that found in wild type cells. In other growth rate experiments, the AU-100 cell line was shown to be resistant to 6-thioguanine and 6-mercaptopurine. Levels of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) measured in AU-100 cell extracts, however, are 50-66% greater than those levels of HGPRTase found in wild type cell extracts. Nevertheless this mutant S49 cell line cannot efficiently incorporate labeled hypoxanthine into nucleotides since the salvage enzyme HGPRTase is inhibited in vivo. The AU-100 cell line was found to be 80% deficient in adenylosuccinate synthetase, but these cells are not auxotrophic for adenosine or other purines. The significant alterations in the control of purine de novo and salvage metabolism caused by the defect in adenylosuccinate synthetase are mediated by the resulting increased levels of guanosine nucleotides.


Subject(s)
Adenylosuccinate Synthase/deficiency , Ligases/deficiency , Purines/biosynthesis , Animals , Clone Cells , Diphosphates/metabolism , Leukemia, Experimental/genetics , Lymphoma/genetics , Mice , Mutation , T-Lymphocytes
10.
J Biol Chem ; 252(21): 7834-41, 1977 Nov 10.
Article in English | MEDLINE | ID: mdl-21184

ABSTRACT

Upon removal of the nucleus from rat hepatoma tissue culture cells, levels of the enzyme tyrosine aminotransferase no longer change in response to withdrawal of glucocorticoids. The rate of tyrosine aminotransferase degradation is drastically reduced in rat hepatoma tissue culture cytoplasts leading to stabilization of pre-existing levels of tyrosine aminotransferase. Moreover, the rate of synthesis of the enzyme in cytoplasts is very low near that observed in uninduced whole cells. These effects of enucleation occur very rapidly and appear to be specific for tyrosine aminotransferase and a small number of other unstable hepatoma proteins. A nuclear effect is thus directly involved in the control of tyrosine aminotransferase degradation and synthesis.


Subject(s)
Carcinoma, Hepatocellular/enzymology , Cell Nucleus/physiology , Tyrosine Transaminase/metabolism , Cell Line , Cytoplasm/enzymology , Kinetics , Liver Neoplasms/enzymology , Molecular Weight , Precipitin Tests
11.
Cell ; 10(2): 297-304, 1977 Feb.
Article in English | MEDLINE | ID: mdl-189937

ABSTRACT

Changes in plasminogen activator activity have been examined as a clonal line of mouse embryonal carcinoma cells aggregate and differentiate to form cystic embryoid bodies in vitro. Within the first 10 days of study, the pluripotent embryonal carcinoma cells aggregate; a layer of endodermal cells appears on the outside of the aggregate forming an embryoid body; a basement membrane forms between the outer layer of endodermal cells and the internal cells; a cyst forms within the embryoid body; and the internal cells assume a columnar appearance along the inner portion of the basement membrane. After the formation of the endodermal layer, there is a rise in intracellular plasminogen activator activity. This rise continues for up to 25 days in culture, providing that the three-dimensional integrity of the embryoid bodies is maintained by culturing them on bacterial petri dishes. Selective removal of the outer endodermal layer of cells reduces the plasminogen activatory activity of the resulting embryoid body cores. Intracellular and secreted plasminogen activator activity of simple embryoid bodies composed of only two cell types can be increased by culturing the embryoid bodies in dbcAMP, theophylline, or cholera toxin. These results suggest that the embryoid body endodermal cells are the source of a cAMP-inducible plasminogen activator activity.


Subject(s)
Cell Differentiation , Plasminogen Activators/metabolism , Teratoma , Alkaline Phosphatase/metabolism , Bucladesine/pharmacology , Cell Line , Endoderm/enzymology , Enzyme Induction , Plasminogen Activators/biosynthesis , Teratoma/enzymology , Theophylline/pharmacology , Toxins, Biological/pharmacology , Vibrio cholerae
12.
Proc Natl Acad Sci U S A ; 72(6): 2007-11, 1975 Jun.
Article in English | MEDLINE | ID: mdl-237268

ABSTRACT

The specific rate of synthesis of tyrosine aminotransferase (EC 2.6.1.5; L-tyrosine:2-oxoglutarate aminotransferase) is used as a measure of the level of functional, cytoplasmic, tyrosine aminotransferase-specific mRNA in cultured rat hepatoma cells. An analysis of the kinetics of change in this rate after the addition or withdrawal of glucocorticosteroids sets an upper limit on the half-life of the enzyme-specific mRNA of 1-1.5 hr, whether or not steroid is present. The inactivation rate of the enzyme mRNA is independent of the growth condition of the cells, occuring equally rapidly in the presence or absence of serum or insulin, both of which induce tyrosine aminotransferase in these cells. The implications of these results for the mechanism of steroid induction are discussed.


Subject(s)
Carcinoma, Hepatocellular/enzymology , Dexamethasone/pharmacology , Tyrosine Transaminase/biosynthesis , Animals , Blood , Carcinoma, Hepatocellular/metabolism , Cattle , Cells, Cultured , Computers , Enzyme Induction/drug effects , Insulin/pharmacology , Kinetics , Leucine/metabolism , Liver Neoplasms , Models, Chemical , Precipitin Tests , RNA, Messenger/metabolism , Rats
13.
Cell ; 5(1): 29-35, 1975 May.
Article in English | MEDLINE | ID: mdl-236835

ABSTRACT

Reexamination of the effects of actinomycin D (AMD) on the intracellular level and rate of synthesis of tyrosine aminotransferase (TAT) in hepatoma tissue culture (HTC) cells reveals that much apparent controversy can be resolved with acknowledgment of the multi-faceted nature of this inhibitor's action. AMD can slow overall protein synthesis and inhibit the degradation of both TAT and its mRNA as well as block the synthesis of RNA. The extent of these secondary actions of the inhibitor depend somewhat upon the growth condition of the cells. The effects of cordycepin (3'-deoxyadenosine) on the metabolism of TAT and its mRNA are also complex, but differ in several respects from those of AMD.


Subject(s)
Dactinomycin/pharmacology , Tyrosine Transaminase/biosynthesis , Animals , Carcinoma, Hepatocellular , Cell Division , Cell Line , Cell-Free System , DNA, Neoplasm/biosynthesis , Deoxyadenosines/pharmacology , Dexamethasone/pharmacology , Enzyme Induction , Fluorometry , Liver Neoplasms , Neoplasm Proteins/biosynthesis , RNA, Messenger/metabolism , Rats , Serum Albumin, Bovine , Tyrosine Transaminase/metabolism
15.
Science ; 175(4018): 189-90, 1972 Jan 14.
Article in English | MEDLINE | ID: mdl-4332760

ABSTRACT

The cellular site of binding of dexamethasone by specific glucocorticoid receptors in cultured hepatoma cells was investigated with the use of certain mercurials. p-Chloromercuribenzene sulfonate and p-chloromercuribenzoate inhibit the binding of steroid by receptors in cell-free extracts, but they allow the steroid-receptor complex to form in whole cells. In contrast, HgCl(2) inhibits binding both in extracts and cells. Since both organic mercury compounds, unlike HgCl(2), do not readily enter intact cells, it appears that the specific steroid binding occurs inside the cell rather than at the cell membrane.


Subject(s)
Binding Sites , Dexamethasone/metabolism , Receptors, Drug , Animals , Autoradiography , Binding Sites/drug effects , Carcinoma, Hepatocellular , Cell-Free System , Cells, Cultured , Chlorides/pharmacology , Chloromercuribenzoates/pharmacology , Liver Neoplasms , Mercury/pharmacology , Rats , Sulfonic Acids/pharmacology , Tritium
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