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Query: UNIPROT:P06889 (Mol)
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The most common mutation in the cystinosis gene, CTNS, is a 65-kb deletion thought to have originated in Germany. Although homozygotes for this deletion are detectable by the absence of the D17S829 polymorphic marker, no method exists to identify heterozygotes. We identified the 65-kb deletion breakpoints and used flanking PCR primers to amplify a 423-bp fragment present only in the deletion alleles. Using this method, we determined that 121 of 216 (56%) cystinosis alleles examined bore the 65-kb deletion. We found no non-Europeans with the deletion, and the deletion size and breakpoints appeared identical in all patients studied, supporting the concept of a founder effect. The addition of D17S829 primers (266 bp apart) to the PCR created a multiplex PCR system useful for diagnosing cystinosis patients homozygous and heterozygous for the 65-kb deletion.
Mol Genet Metab 1999 Feb
PMID:Identification and detection of the common 65-kb deletion breakpoint in the nephropathic cystinosis gene (CTNS). 1006 13

Fanconi anemia (FA) is an autosomal recessive disease characterized by a variety of congenital abnormalities. Cells from FA patients show chromosomal instability and are hypersensitive to DNA cross-linking agents, though the basic cellular defect in FA is not known. The FANCA gene encodes a protein with an Mr of 162 kDa and with unknown function. The cellular localization of the FANCA protein has been controversial, and has been shown in different reports to be exclusively cytoplasmic and predominantly nuclear. In the present study, we further confirm that FANCA localizes primarily to the nucleus. Fusions of FANCA with the green fluorescent protein (GFP) showed a strong nuclear signal and a weak cytoplasmic signal in several cell types. Confocal laser microscopy confirmed that FANCA is evenly distributed throughout the nucleus. We also examined regions in FANCA that participate in its nuclear import. FANCA contains two bipartite nuclear localization signal (NLS) motifs at the extreme N-terminus. Deletion of amino acids N-terminal to the NLS motifs had no effect on the nuclear localization of FANCA or on its ability to correct mitomycin C sensitivity in an FA-A cell line, while deletion of both motifs impeded but did not prevent nuclear import. Deletions of 75, 90 and 150 residues from the N-terminus yielded a mixture of cells with only a cytoplasmic signal, and with both a nuclear and cytoplasmic signal. Deletion of the N-terminal 250 amino acids was required to block nuclear localization completely. Fusion of GFP to the N-terminal 250 amino acids showed a localization pattern similar to FANCA. Mutant forms of FANCA with deletions of the C-terminal 70 or 260 residues localized to the cytoplasm, although the C-terminal 260 amino acids alone lacked NLS activity. The results show that nuclear localization of FANCA involves several functional regions.
Hum Mol Genet 1999 Jun
PMID:Characterization of regions functional in the nuclear localization of the Fanconi anemia group A protein. 1033 32

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently cloned FANCG/XRCC9 protein is required for binding of the FANCA and FANCC proteins. Moreover, the FANCG protein is a component of a nuclear protein complex containing FANCA and FANCC. The amino-terminal region of the FANCA protein is required for FANCG binding, FANCC binding, nuclear localization, and functional activity of the complex. Our results demonstrate that the three cloned FA proteins cooperate in a large multisubunit complex. Disruption of this complex results in the specific cellular and clinical phenotype common to most FA complementation groups.
Mol Cell Biol 1999 Jul
PMID:Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. 1037 36

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.
Mol Cell Biol 1999 Aug
PMID:Interstrand cross-links induce DNA synthesis in damaged and undamaged plasmids in mammalian cell extracts. 1040 51

Genomic instability in its broadest sense is a feature of virtually all neoplastic cells. In addition to the mutations and/or gene amplifications that appear to be a prerequisite for the acquisition of a neoplastic phenotype, human cancers exhibit other "markers" of genomic instability--in particular, a high degree of aneuploidy. Indeed, many studies have shown that aneuploidy is an almost invariant feature of cancer cells, and it has been argued by some that the emergence of aneuploid cells is a necessary step during tumorigenesis. The functional link between genomic instability and cancer is strengthened by the existence of several "genetic instability" disorders of humans that are associated with a moderate to severe increase in the incidence of cancers. These disorders include ataxia telangiectasia, Bloom's syndrome, Fanconi anemia, xeroderma pigmentosum, and Nijmegen breakage syndrome, all of which are very rare and are inherited in a recessive manner. Analysis of the cells from such cancer-prone individuals is clearly a potentially fruitful approach for delineating the genetic basis for instability in the genome. It is assumed that by identifying the underlying cause of genetic instability in these disorders, one can derive valuable information not only about the basis of particular genetic diseases, but also about the underlying causes of genomic instability in sporadic cancers in the general population. In this article, we review the clinical and cellular properties of genetic instability disorders associated with cancer predisposition. In particular, we focus on the rapid advances made in our understanding of these disorders that have derived from the cloning of the genes mutated in each case. Because in many instances the affected genes have analogs in lower eukaryotic species, we shall discuss how studies in yeasts in particular have proved valuable in our understanding of human diseases and predisposition to cancer.
Prog Nucleic Acid Res Mol Biol 1999
PMID:Genetic disorders associated with cancer predisposition and genomic instability. 1050 32

Interaction of DNA repair proteins with damaged DNA in eukaryotic cells is influenced by the packaging of DNA into chromatin. The basic repeating unit of chromatin, the nucleosome, plays an important role in regulating accessibility of repair proteins to sites of damage in DNA. There are a number of different pathways fundamental to the DNA repair process. Elucidation of the proteins involved in these pathways and the mechanisms they utilize for interacting with damaged nucleosomal and nonnucleosomal DNA has been aided by studies of genetic diseases where there are defects in the DNA repair process. Two of these diseases are xeroderma pigmentosum (XP) and Fanconi anemia (FA). Cells from patients with these disorders are similar in that they have defects in the initial steps of the repair process. However, there are a number of important differences in the nature of these defects. One of these is in the ability of repair proteins from XP and FA cells to interact with damaged nucleosomal DNA. In XP complementation group A (XPA) cells, for example, endonucleases present in a chromatin-associated protein complex involved in the initial steps in the repair process are defective in their ability to incise damaged nucleosomal DNA, but, like the normal complexes, can incise damaged naked DNA. In contrast, in FA complementation group A (FA-A) cells, these complexes are equally deficient in their ability to incise damaged naked and similarly damaged nucleosomal DNA. This ability to interact with damaged nucleosomal DNA correlates with the mechanism of action these endonucleases use for locating sites of damage. Whereas the FA-A and normal endonucleases act by a processive mechanism of action, the XPA endonucleases locate sites of damage distributively. Thus the mechanism of action utilized by a DNA repair enzyme may be of critical importance in its ability to interact with damaged nucleosomal DNA.
Prog Nucleic Acid Res Mol Biol 1999
PMID:DNA repair and chromatin structure in genetic diseases. 1050 34

Infantile nephropathic cystinosis is a rare, autosomal recessive disease caused by a defect in the transport of cystine across the lysosomal membrane and characterized by early onset of renal proximal tubular dysfunction. Late-onset cystinosis, a rarer form of the disorder, is characterized by onset of symptoms between 12 and 15 years of age. We previously characterized the cystinosis gene, CTNS, and identified pathogenic mutations in patients with infantile nephropathic cystinosis, including a common, approximately 65 kb deletion which encompasses exons 1-10. Structure predictions suggested that the gene product, cystinosin, is a novel integral lysosomal membrane protein. We now examine the predicted effect of mutations on this model of cystinosin. In this study, we screened patients with infantile nephropathic cystinosis, those with late-onset cystinosis and patients whose phenotype does not fit the classical definitions. We found 23 different mutations in CTNS; 14 are novel mutations. Out of 25 patients with infantile nephropathic cystinosis, 12 have two severely truncating mutations, which is consistent with a loss of functional protein, and 13 have missense or in-frame deletions, which would result in disruption of transmembrane domains and loss of protein function. Mutations found in two late-onset patients affect functionally unimportant regions of cystinosin, which accounts for their milder phenotype. For three patients, the age of onset of cystinosis was <7 years but the course of the disease was milder than the infantile nephropathic form. This suggests that the missense mutations found in these individuals allow production of functional protein and may also indicate regions of cystinosin which are not functionally important.
Hum Mol Genet 1999 Dec
PMID:Severity of phenotype in cystinosis varies with mutations in the CTNS gene: predicted effect on the model of cystinosin. 1055 99

The X-linked form of the bone marrow failure syndrome Dyskeratosis congenital is caused by mutations in dyskerin, a 514 amino acid protein that is presumed to play a role in ribosome biogenesis. Here we report that dyskerin tagged with the human immunoglobulin epitope localizes to nuclei of transfected HeLa and COS-1 cells. A carboxyl-terminal domain consisting of amino acids 467-475 and encoding KKEKKKSKK is both necessary and sufficient to mediate nuclear entry. Immunoglobulin-tagged dyskerin did not interact with the Fanconi anemia group A protein, FANCA. These results suggest a nuclear role for dyskerin. Moreover, hematopoietic failure observed in both Dyskeratosis congenital and the most common type of Fanconi anemia is unlikely to have a common mechanism resulting from abnormal physical interactions between the respective gene products of these disorders.
Blood Cells Mol Dis
PMID:Analysis of epitope-tagged forms of the dyskeratosis congenital protein (dyskerin): identification of a nuclear localization signal. 1074 26

Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-gamma) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-gamma receptor alpha chain (IFN-gammaRalpha) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-gammaR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-gamma-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-gamma hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1(-/-) mice were resistant to IFN-gamma. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT(-/-) mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic growth and survival factor molecules, we reason that FA-C hematopoietic cells are excessively apoptotic because of an imbalance between survival cues (owing to a failure of STAT1 activation in FA-C cells) and apoptotic and mitogenic inhibitory cues (constitutively activated in FA-C cells in a STAT1-independent fashion).
Mol Cell Biol 2000 Jul
PMID:The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. 1084 98

Fanconi anemia (FA) is a hereditary chromosomal instability syndrome with cancer predisposition. Bone marrow failure resulting in pancytopenia is the main cause of death of FA patients. Diagnosis of FA is based on their cellular hypersensitivity to DNA crosslinking agents and chromosome breakages. Somatic complementation experiments suggest the involvement of at least eight genes in FA. The gene for complementation group A (FANCA) is defective in the majority of FA patients. We show here that mice deficient of FANCA: are viable and have no detectable developmental abnormalities. The hematological parameters showed a slightly decreased platelet count and a slightly increased erythrocyte mean cell volume in mice at young age, but this did not progress to anemia. Consistent with the clinical phenotype of FA patients, both male and female mice showed hypogonadism and impaired fertility. Furthermore, embryonic fibroblasts of the knock-out mice exhibited spontaneous chromosomal instability and were hyper-responsive to the clastogenic effect of the crosslinker mitomycin C.
Hum Mol Genet 2000 Jul 22
PMID:Mice with a targeted disruption of the Fanconi anemia homolog Fanca. 1091 69


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