RESUMO
BACKGROUND & AIMS: Microsatellite instability (MSI) is the phenotype of colorectal carcinomas with defect mismatch repair. Genes with repetitive sequences within their coding regions are targets for mutations in these tumors. We have evaluated 2 novel candidate genes for potential involvement in development of MSI colorectal carcinomas and compared them with alterations in known target genes. METHODS: The MSI status was determined by multiplex polymerase chain reactions (PCRs) of 5-17 markers in a Norwegian series of 275 colorectal carcinomas. All MSI tumors were analyzed for gene mutations using fluorescence PCR followed by capillary electrophoresis. Two novel candidate genes, WNT1-inducible signaling pathway protein 3 (WISP-3) and caspase-1, and 9 known target genes were analyzed. RESULTS: Thirteen percent of the tumors were MSI-high (H) and 12% were MSI-low (L). Thirty-three of 37 MSI-H vs. 1 of 34 MSI-L tumors showed mutations in the target genes (P < 0.001). WISP-3 was mutated in 31% of the MSI-H tumors. The frequencies of frameshift mutations in the known target genes were comparable with other studies. CONCLUSIONS: The relative high frequency of mutation, higher than those seen for other known target genes, the predicted truncation of the protein product, and the homology with WISP-1 and WISP-2, 2 proteins induced downstream of WNT1 signaling, strongly suggest WISP-3 as a novel target in development of MSI-H colorectal carcinomas.
Assuntos
Carcinoma/genética , Neoplasias Colorretais/genética , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Idoso , Proteínas de Sinalização Intercelular CCN , Caspase 1/genética , Feminino , Frequência do Gene , Marcação de Genes , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Masculino , Mutação , Células Tumorais CultivadasRESUMO
The nucleotide sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and the Autographa californica MNPV were found. These include the presence in OpMNPV of three complete iap gene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses.
Assuntos
Genoma Viral , Mariposas/virologia , Nucleopoliedrovírus/genética , Análise de Sequência de DNA , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral , Genes Virais , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Pirofosfatases/genética , Sequências Repetitivas de Ácido Nucleico , Ribonucleotídeo Redutases/genética , Homologia de Sequência de AminoácidosRESUMO
Regions of the genome of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) containing the DNA polymerase and helicase genes were sequenced. The DNA polymerase and helicase genes encode predicted proteins of 985 (112.6 kDa) and 1223 (140.5 kDa) amino acids and exhibited 63 % and 59 % amino acid identity, respectively, with their homologues in the Autographa californica MNPV (AcMNPV). The influence of sequence variation between the OpMNPV and AcMNPV DNA polymerase and helicase was investigated by employing gene substitution experiments in transient replication assays in Lymantria dispar and Spodoptera frugiperda cells. The DNA polymerase gene appeared to be interchangeable in this assay; both the AcMNPV and OpMNPV DNA polymerase supported high levels of replication of an origin-containing reporter plasmid when substituted for their homologue and cotransfected with a set of heterologous essential and stimulatory replication genes into uninfected insect cells. However, the OpMNPV helicase failed to support replication when it replaced the AcMNPV helicase from the set of AcMNPV replication genes cotransfected into S. frugiperda cells. In contrast, the AcMNPV helicase gene supported about 50% of the level of replication when substituted for its homologue in the OpMNPV set of replication genes.
Assuntos
DNA Helicases/genética , DNA Polimerase Dirigida por DNA/genética , Genes Virais , Nucleopoliedrovírus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Dados de Sequência Molecular , Fases de Leitura Aberta , SpodopteraRESUMO
A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.
Assuntos
Replicação do DNA/genética , Genes de Insetos/genética , Nucleopoliedrovírus/fisiologia , Replicação Viral/genética , Sequência de Aminoácidos , Animais , Apoptose/genética , Sequência de Bases , Linhagem Celular , Regulação da Expressão Gênica , Insetos/virologia , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Fases de Leitura Aberta/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/biossíntese , RNA Viral/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Spodoptera , Transativadores/genética , Proteínas Virais/genéticaRESUMO
Using a transient complementation assay for DNA replication of an origin-containing reporter plasmid, a 2.4-kb region (m.u. 48.3-50.1) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was identified that contains a gene essential for OpMNPV DNA replication. This region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 3 (lef-3), as the essential replication gene. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that lef-3 was expressed as an early gene with a major transcript of 1.7 kb in length. OpMNPV lef-3 encodes a protein with a predicted molecular weight of 42.6 kDa (373 amino acids) and exhibits 41% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. A pattern of amino acids similar to that found in single-stranded DNA binding proteins is present in the amino-terminus of both OpMNPV and AcMNPV LEF-3.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Virais/genética , Nucleopoliedrovírus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Replicação do DNA/genética , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Nucleopoliedrovírus/fisiologia , Fases de Leitura Aberta/genética , RNA Mensageiro/análise , RNA Viral/análise , Origem de Replicação/genética , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNA , Replicação Viral/genéticaRESUMO
A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV HindIII-N fragment, we identified a subset of cosmids and plasmids from an OpMNPV cosmid library that was able to supply all the essential trans-acting factors and support replication of the origin-containing plasmid in uninfected Lymantria dispar cells. However, this limited set of DNA's was unable to support replication of a second origin-containing plasmid derived from a different region of the OpMNPV genome. Replication analysis of deletion clones of the HindIII-N fragment led to the identification of a gene, late expression factor 1 (lef-1), that is essential for the transactivation of DNA replication in this system. Transcriptional analysis of lef-1 mapped both early and late transcripts of about 1.75 kb. A motif characteristic of nucleoside triphosphate-binding sites present in the carboxy-terminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.
Assuntos
Replicação do DNA/genética , Genes Virais , Nucleopoliedrovírus/genética , Ativação Transcricional , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral/biossíntese , DNA Viral/genética , Genoma Viral , Insetos/virologia , Dados de Sequência Molecular , Nucleopoliedrovírus/metabolismo , Plasmídeos/genética , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas Virais/genéticaRESUMO
A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous regions from AcMNPV. In isolation, these repeated sequences were not sufficient for origin activity in OpMNPV-infected Lymantria dispar cells, but required an additional 1.3 kb of sequences located to the left of the repeats. Four regions of the OpMNPV genome that crosshybridize with the repeated region were also found to replicate in our infection-dependent DNA replication assay. A deletion clone of Op5 that replicates efficiently in OpMNPV-infected L. dispar cells, was found to replicate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infected Spodoptera frugiperda cells.
Assuntos
Replicação do DNA/genética , Nucleopoliedrovírus/genética , Origem de Replicação , Animais , Clonagem Molecular , Cosmídeos , Genoma Viral , Insetos/virologia , Nucleopoliedrovírus/metabolismo , SpodopteraRESUMO
By use of a transient replication assay, nine genes involved in DNA replication were identified in the genome of the Autographa californica baculovirus. Six genes encoding helicase, DNA polymerase, IE-1, LEF-1, LEF-2, and LEF-3 are essential for DNA replication while three genes encoding P35, IE-2, and PE38 stimulate DNA replication. No stimulation by the AcMNPV pcna gene, encoding a protein with sequence homology to proliferating-cell nuclear antigen, was observed. A pattern of amino acids found in a number of single-stranded-DNA-binding proteins was identified in the carboxyl-terminal region of IE-1.
