RESUMEN
A new disease was found in September 2001 on greenhouse-produced onion transplants of cv. Colorado 6 grown in a field in Larimer County in northern Colorado. Symptoms included straw-colored, dry, tan, spindle- or diamond-shaped lesions on the leaves and scapes of onion plants. Infected plants were scattered (less than 5% incidence) throughout the outer perimeter of the sprinkler-irrigated field. Iris yellow spot virus (IYSV) in two collections each with 4 to 6 symptomatic onion plants was confirmed with western blot assays by James Moyer of North Carolina State University. Western blot showed a faint band from protein extracts of infected Nicotiana benethimiana. Western blot assay is the most definitive method of identification. IYSV can be mechanically transmitted to N. benethimiana, but it cannot be recovered and transmitted back to onion, and it is difficult to detect in infected onion plants. IYSV is a tospovirus that is transmitted by various species of thrips, including onion thrips and western flower thrips (1). The host range for this disease includes onion, leek, and iris. IYSV has been reported previously on onion in Israel, Brazil, and Idaho (2). There are no reports that this disease affects bulb quality or marketability; however, heavy losses of onion bulb production are reported (1). University and industry personnel in other onion-growing areas of the country are encouraged to monitor onion and other host fields for evidence and distribution of IYSV. References: (1) A. Kritzman et al. Plant Dis. 85:838, 2001. (2) L. Pozzer et al. Plant Dis. 83:345, 1999.
RESUMEN
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Linfocitos B/enzimología , Secuencia de Bases , Células CHO , Línea Celular , Cromosomas Artificiales de Levadura/genética , Secuencia Conservada , Cricetinae , Cartilla de ADN/genética , Reparación del ADN/genética , Reparación del ADN/fisiología , Proteína Quinasa Activada por ADN , Células Madre Hematopoyéticas/enzimología , Ratones , Ratones SCID , Mutagénesis Sitio-Dirigida , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de ProteínaRESUMEN
The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Células CHO , Línea Celular , Cromosomas Artificiales de Levadura , Cricetinae , ADN/metabolismo , Daño del ADN , ADN Nucleotidiltransferasas/metabolismo , Reparación del ADN , Proteína Quinasa Activada por ADN , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Biblioteca de Genes , Caballos , Humanos , Ratones , Ratones SCID , Proteínas Nucleares , Fosfatidilinositol 3-Quinasas/metabolismo , Reacción en Cadena de la Polimerasa , Transfección , VDJ RecombinasasRESUMEN
The gene product defective in radiosensitive CHO mutants belonging to ionizing radiation complementation group 5, which includes the extensively studied xrs mutants, has recently been identified as Ku80, a subunit of the Ku protein and a component of DNA-dependent protein kinase (DNA-PK). Several group 5 mutants, including xrs-5 and -6, lack double-stranded DNA end-binding and DNA-PK activities. In this study, we examined additional xrs mutants at the molecular and biochemical levels. All mutants examined have low or undetectable levels of Ku70 and Ku80 protein, end-binding, and DNA-PK activities. Only one mutant, xrs-6, has Ku80 transcript levels detectable by Northern hybridization, but Ku80 mRNA was detectable by reverse transcription-PCR in most other mutants. Two mutants, xrs-4 and -6, have altered Ku80 transcripts resulting from mutational changes in the genomic Ku80 sequence affecting RNA splicing, indicating that the defects in these mutants lie in the Ku80 gene rather than a gene controlling its expression. Neither of these two mutants has detectable wild-type Ku80 transcript. Since the mutation in both xrs-4 and xrs-6 cells results in severely truncated Ku80 protein, both are likely candidates to be null mutants. Azacytidine-induced revertants of xrs-4 and -6 carried both wild-type and mutant transcripts. The results with these revertants strongly support our model proposed earlier, that CHO-K1 cells carry a copy of the Ku80 gene (XRCC5) silenced by hypermethylation. Site-directed mutagenesis studies indicate that previously proposed ATP-binding and phosphorylation sites are not required for Ku80 activity, whereas N-terminal deletions of more than the first seven amino acids result in severe loss of activities.
Asunto(s)
Antígenos Nucleares , Células CHO , ADN Helicasas , Proteínas de Unión al ADN/genética , Mutación , Proteínas Nucleares/genética , Tolerancia a Radiación/genética , Animales , Azacitidina/farmacología , Células CHO/efectos de la radiación , Cricetinae , ADN/metabolismo , ADN Complementario/genética , Proteína Quinasa Activada por ADN , Proteínas de Unión al ADN/metabolismo , Rayos gamma , Dosificación de Gen , Prueba de Complementación Genética , Autoantígeno Ku , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse.
Asunto(s)
Proteínas de Unión al ADN , Mutación , Proteínas Serina-Treonina Quinasas/genética , Células 3T3 , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Línea Celular , Cricetinae , Cartilla de ADN , Proteína Quinasa Activada por ADN , Evolución Molecular , Exones , Biblioteca de Genes , Humanos , Ratones , Ratones SCID/genética , Datos de Secuencia Molecular , Proteínas Nucleares , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/biosíntesis , Roedores , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
DNA-dependent protein kinase is a nuclear serine/threonine kinase whose catalytic properties are expressed only when the enzyme is bound to DNA ends or other discontinuities in the DNA. DNA-PK comprises two components: one mediates binding to DNA and corresponds to the heterodimeric human autoimmune antigen Ku; the other, DNA-PK catalytic subunit (DNA-PKcs), is a polypeptide of approximately 450 kDa. DNA-PK deficiencies are associated with certain mutant rodent cell lines that display defects in DNA double strand break repair and V(D)J recombination. Specifically, hamster xrs-6 cells lack Ku function, whereas murine scid and hamster V3 cells lack functional DNA-PKcs. Furthermore, the phenotypes of xrs-6 and V3 cells can be corrected by the expression of the genes encoding the 80 kDa component of Ku or DNA-PKcs, respectively. These results imply that DNA-PK is an important component of the DNA double strand break repair/recombination apparatus. Possible roles for DNA-PK in these processes are discussed.
Asunto(s)
ADN Nucleotidiltransferasas/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN , Reordenamiento Génico de Linfocito T/fisiología , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteína Quinasa Activada por ADN , VDJ RecombinasasRESUMEN
The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.
Asunto(s)
Antígenos Nucleares , Clonación Molecular , ADN Helicasas , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Prueba de Complementación Genética , Proteínas Nucleares/genética , Animales , Secuencia de Bases , Células CHO , Cromosomas Artificiales de Levadura , Cricetinae , Cartilla de ADN , Humanos , Células Híbridas , Autoantígeno Ku , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Protoplastos/metabolismoRESUMEN
Murine cells homozygous for the severe combined immune deficiency mutation (scid) and V3 mutant hamster cells fall into the same complementation group and show similar defects in V(D)J recombination and DNA double-stranded break repair. Here we show that both cell types lack DNA-dependent protein kinase (DNA-PK) activity owing to defects in DNA-PKcs, the catalytic subunit of this enzyme. Furthermore, we demonstrate that yeast artificial chromosomes containing the DNA-PKcs gene complement both the DNA repair and recombination deficiencies of V3 cells, and we conclude that DNA-PKcs is encoded by the XRCC7 gene. As DNA-PK binds to DNA ends and is activated by these structures, our findings provide novel insights into V(D)J recombination and DNA repair processes.
Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética/genética , Inmunodeficiencia Combinada Grave/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO/efectos de la radiación , Extractos Celulares/química , Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Cricetinae , ADN/metabolismo , Proteína Quinasa Activada por ADN , Rayos gamma , Prueba de Complementación Genética , Región Variable de Inmunoglobulina/genética , Ratones , Ratones SCID , Datos de Secuencia Molecular , Tolerancia a Radiación/genéticaRESUMEN
DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit called p350 and a DNA binding component termed Ku. Ku consists of two tightly associated polypeptides of approximately 70 kDa and 80 kDa (Ku80). An intriguing feature of DNA-PK is that it binds to DNA ends and other discontinuities in DNA and requires these structures for its activation. This suggests that DNA-PK may function in DNA repair and/or recombination. Consistent with this, Ku DNA binding activity was shown recently to be absent in extracts of hamster xrs-6 cells, which are defective in DNA double-strand (ds) break repair and V(D)J recombination. Furthermore, xrs-6 cells are complemented by expression of the Ku80 cDNA. To date, DNA-PK activity has been demonstrated unequivocally only in extracts of primate cells. Here, we describe an assay that can detect DNA-PK activity in extracts of mouse, hamster, Xenopus, and Drosophila cells. Using this assay, we find that xrs-6 cells completely lack DNA-PK activity. By contrast, xrs-6 derivatives complemented by human chromosome fragments bearing the Ku80 gene have restored both the DNA end binding and kinase activities associated with DNA-PK. Finally, we show that xrs-6 extracts are complemented biochemically by purified Ku. Our findings indicate that the xrs-6 defects are direct consequences of the mutation in Ku80 and implicate DNA-PK in recombination and DNA repair processes.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Proteína Quinasa Activada por ADN , Drosophila , Prueba de Complementación Genética , Células HeLa , Humanos , Cinética , Mamíferos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares , Proteínas Serina-Treonina Quinasas/análisis , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Xenopus laevisRESUMEN
The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cells, is defective in DNA double-strand break repair and in ability to undergo V(D)J recombination. The human XRCC5 DNA repair gene, which complements this mutant, is shown here through genetic and biochemical evidence to be the 80-kilodalton subunit of the Ku protein. Ku binds to free double-stranded DNA ends and is the DNA-binding component of the DNA-dependent protein kinase. Thus, the Ku protein is involved in DNA repair and in V(D)J recombination, and these results may also indicate a role for the Ku-DNA-dependent protein kinase complex in those same processes.
Asunto(s)
Antígenos Nucleares , ADN Helicasas , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Genes de Inmunoglobulinas , Proteínas Nucleares/genética , Receptores de Antígenos de Linfocitos T/genética , Recombinación Genética , Animales , Secuencia de Bases , Células CHO , Supervivencia Celular/efectos de la radiación , Clonación Molecular , Cricetinae , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Prueba de Complementación Genética , Humanos , Células Híbridas , Autoantígeno Ku , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , TransfecciónRESUMEN
We have studied the RH genes in donors with the RhD-negative haplotype dCes. In contrast to the usual arrangement of genes in RhD-negative individuals, where the lack of antigen expression is due to deletion of the entire RHD gene, we find that the dCes haplotype includes an RHD gene with an internal deletion. Moreover, there appear to be no 5' sequences characteristic of RHC suggesting that the RhC antigen may be encoded by a truncated RHD or a recombinant RHD/CE gene in these dCes/dce genomes.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/genética , Eliminación de Secuencia , Secuencia de Bases , Población Negra/genética , Tipificación y Pruebas Cruzadas Sanguíneas , Análisis Mutacional de ADN , Haplotipos/genética , Humanos , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
We have investigated the arrangement of genes in the rare Rh (Rhesus) partial null condition D--. Southern blot and PCR studies under conditions which distinguish the highly homologous RH D and RH C/E genes show that in an Icelandic family with the D-- haplotype at least 85% of the RH C/E gene is deleted. This finding is in contrast to one other published case of this phenotype, where intact RH D and C/E genes were found, and also to the full amorph Rhnull phenotype, where an intact RH C/E gene was found, accompanied by the deletion of the RH D gene typical of Rh D-negative individuals.
Asunto(s)
Eliminación de Gen , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Bases , Exones , Heterocigoto , Homocigoto , Humanos , Islandia , Intrones , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Alu element-primed PCR was performed on genomic clones containing human RH blood group genes. When used as a probe, the Alu PCR product detected a restriction fragment-length polymorphism which is in complete linkage disequilibrium with the Rh C/c serological polymorphism, irrespective of the Rh D or E serological type it is coupled with. This provides the opportunity to type individuals for their RH C gene directly at the DNA level. RFLP analysis of two individuals with the amorph Rh null phenotype revealed that in one case this phenotype occurred on an RH C background, whereas in the other it was on an RH c background. Taken together these results indicate that the Rh C/c polymorphism has arisen only once, but that the amorph Rh null phenotype, although exceedingly rare, is the result of at least two independent mutations.