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1.
Proc Natl Acad Sci U S A ; 88(4): 1531-5, 1991 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-1996353

RESUMEN

Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent Km of 1.0 microM for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.


Asunto(s)
Desoxicitidina Quinasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Línea Celular , Clonación Molecular , ADN de Neoplasias/genética , ADN de Neoplasias/aislamiento & purificación , Desoxicitidina Quinasa/aislamiento & purificación , Escherichia coli/genética , Expresión Génica , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Neoplásico/genética , ARN Neoplásico/aislamiento & purificación
2.
Blood ; 74(1): 448-53, 1989 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-2752123

RESUMEN

A marked tissue-specific increase in erythrocyte adenosine deaminase (ADA) activity is associated with an autosomal dominantly inherited hemolytic anemia. We investigated the molecular basis of ADA overproduction by studying reticulocyte ADA mRNA from affected individuals. Analysis of proband reticulocyte ADA cDNA clones revealed normal sequence. RNase mapping demonstrated that the amount of ADA mRNA in affected reticulocytes was greater than the amount in normal B lymphoblasts, whereas ADA mRNA was undetectable in normal reticulocytes. The 5'- and 3'-untranslated regions of reticulocyte and B-lymphoblast ADA mRNAs from affected individuals were structurally indistinguishable from those of normal B lymphoblasts. Northern blot analysis performed under stringent hybridization and washing conditions confirmed a markedly increased amount of reticulocyte ADA mRNA in affected individuals as compared with controls. We conclude that the RBC-specific overexpression of ADA in this disorder occurs at the mRNA level.


Asunto(s)
Adenosina Desaminasa/sangre , Anemia Hemolítica Congénita/enzimología , Nucleósido Desaminasas/sangre , Adenosina Desaminasa/genética , Northern Blotting , Clonación Molecular , ADN/genética , Sondas de ADN , Regulación de la Expresión Génica , Humanos , ARN Mensajero/genética , Ribonucleasas/farmacología
3.
Prog Clin Biol Res ; 319: 55-64; discussion 65-8, 1989.
Artículo en Inglés | MEDLINE | ID: mdl-2622927

RESUMEN

A kindred with an autosomal dominant form of chronic hemolytic anemia has been found to have a 40- to 70-fold elevation in erythrocyte adenosine deaminase (ADA) activity in association with depletion of red blood cell (RBC) ATP pools. ADA activities in B lymphoblasts, skin fibroblasts, and granulocytes were normal. There were no alterations in the kinetic properties of partially purified proband ADA. We have shown by Western blot analysis that the elevation in ADA activity is accompanied by a corresponding increase in the amount of immunoreactive ADA protein. Southern blot analysis of proband DNA ruled out gene amplification and revealed no gross insertions, deletions, or rearrangements in the ADA gene. Northern blot analysis demonstrated a marked increase in the amount of ADA mRNA in proband and sibling reticulocytes compared to high reticulocyte controls. ADA mRNA levels in B lymphoblasts from the proband, sibling, and GM558 cell line were normal. Cloning and sequencing of proband reticulocyte cDNA revealed normal ADA mRNA sequence. No polymorphisms were detected among the seven clones studied. RNase mapping of the 5'- and 3'-non-coding sequences confirmed the quantitative increase in reticulocyte ADA mRNA and verified that these regions were normal in length and sequence. Southern blot analysis of DNA from four affected and three unaffected family members revealed two restriction fragment length polymorphisms (RFLPs) which segregate with the ADA allele from the unaffected grandfather. Both RFLPs are present in the unaffected grandchild and absent in the affected grandchild. These findings are consistent with a cis- mutation within the ADA gene, but they do not rule out a trans- mutation affecting some non-ADA regulatory factor. We conclude that erythrocyte-specific ADA overproduction is associated with increased amounts of structurally normal ADA mRNA. This increase may result from either increased transcription of the ADA gene or altered post-transcriptional processing resulting in increased stability of the RNA transcript. Further elucidation of the defect should provide valuable insights into the normal tissue-specific regulation of the ADA gene and the mechanisms by which erythroid cells regulate gene expression during differentiation.


Asunto(s)
Adenosina Desaminasa/sangre , Eritrocitos/enzimología , Regulación Enzimológica de la Expresión Génica , Nucleósido Desaminasas/sangre , Adenosina Desaminasa/biosíntesis , Adenosina Desaminasa/genética , Northern Blotting , Southern Blotting , Clonación Molecular , ADN/genética , Femenino , Humanos , Linaje , Ribonucleasas/metabolismo
5.
J Clin Invest ; 79(3): 1001-5, 1987 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-3029177

RESUMEN

We have investigated the molecular basis of the marked elevation in erythrocyte adenosine deaminase (ADA) activity in a kindred with hereditary hemolytic anemia. Red cell ADA-specific activity was verified to be 70- to 100-fold normal levels. Western blots demonstrated a corresponding increase in erythrocyte ADA-specific immunoreactive protein. Analysis of genomic DNA revealed no evidence for amplification or major structural changes in the ADA gene. ADA-specific messenger RNA (mRNA) from proband reticulocytes was comparable in size and amount to mRNA from control reticulocytes. Translation of proband poly A+ reticulocyte mRNA in a rabbit reticulocyte lysate system and immunoprecipitation of 35S-labeled protein products with anti-ADA antibody yielded a band of approximately 42,000 apparent mol wt that was absent in translation products from control reticulocyte mRNAs. These data suggest that the increased ADA activity in red cells in this disorder results from the increased translation of an aberrant ADA mRNA.


Asunto(s)
Adenosina Desaminasa/sangre , Anemia Hemolítica Congénita/enzimología , Eritrocitos/enzimología , Nucleósido Desaminasas/sangre , Biosíntesis de Proteínas , Adenosina Desaminasa/genética , Anemia Hemolítica Congénita/genética , ADN/genética , Enzimas de Restricción del ADN , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas para Inmunoenzimas , Linfocitos/análisis , Hibridación de Ácido Nucleico , ARN Mensajero/sangre , Reticulocitos/análisis
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