The protein truncation test: A review

Dunnen, Johan T. den; Ommen, Gert‐Jan B. van

doi:10.1002/(sici)1098-1004(1999)14:2<95::aid-humu1>3.0.co;2-g

Cited by 52 publications

(9 citation statements)

References 44 publications

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“…Therefore, we used RT -PCR to amplify IkBa cDNA from RC-K8 cells, and included a T7 promoter element on the 5' PCR primer to allow us to subject the pooled cDNA to in vitro transcription/translation (Den Dunnen and van Ommen, 1999). As a control, we also amplified IkBa cDNA from EBV-transformed cells, which synthesize wild-type IkBa (see Figure 4).…”

Section: Rel-nrg Homodimers Are Resistant To Inhibition By Ikbamentioning

confidence: 99%

The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-κB signal transduction pathway

et al. 2002

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The human large B-cell lymphoma cell line RC-K8 has a rearranged REL locus that directs the production of a chimeric protein, termed REL-NRG (Non-Rel Gene). In this study, we show that RC-K8 cells have constitutively nuclear heterodimeric and homodimeric DNA-binding complexes that consist of p50, REL, and REL-NRG. In vitro, IkBa can block the DNA-binding activity of wildtype REL homodimers but not REL-NRG homodimers. In vivo, REL-NRG cannot activate transcription of a kB site reporter plasmid, suggesting that it is a transcription repressing or blocking REL protein. By Western blotting, no IkBa protein can be detected in extracts of RC-K8 cells. The absence of IkBa protein in RC-K8 cells appears to be due to mutations that cause premature termination of translation in three of the four copies of the IKBA gene in RC-K8 cells. Re-expression of wild-type IkBa or a super-repressor form of IkBa in RC-K8 cells is cytotoxic; in contrast, expression of a dominant-negative form of IkB kinase does not affect the growth of RC-K8 cells. By cDNA microarray analysis, a number of previously identified Rel/NF-kB target genes are overexpressed in RC-K8 cells, consistent with there being transcriptionally active REL complexes. Taken together, our results suggest that the growth of RC-K8 cells is dependent on the activity of nuclear wild-type REL dimers, while the contribution of REL-NRG to the transformed state of RC-K8 cells is less clear. Nevertheless, the RC-K8 cell line is the first tumor cell line identified with mutations in genes encoding multiple proteins in the Rel/NF-kB signal transduction pathway.

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Section: Rel-nrg Homodimers Are Resistant To Inhibition By Ikbamentioning

confidence: 99%

The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-κB signal transduction pathway

et al. 2002

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“…Mutations are scattered over all exons; therefore, genetic tests for this gene require a mutation scanning analysis of the entire coding region of the BRCA1 gene. In most diagnostic laboratories, this procedure is currently performed using direct sequence analysis, often preceded by denaturing gradient gel electrophoresis (DGGE) [Beck et al, 1993;Guldberg and Guttler, 1993], protein truncation test (PTT) [den Dunnen and van Ommen, 1999;Ozcelik et al, 1996], or denaturing high-performance liquid chromatography (DHPLC) [Gross et al, 1999;Liu et al, 1998]. However, all these techniques are time-consuming and/or expensive.…”

Section: Introductionmentioning

confidence: 99%

Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation ofBRCA1mutation scanning using the 96-well LightScanner™

et al. 2009

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Genetic analysis of BRCA1 by sequencing is often preceded by a scanning method like denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT) or DHPLC. High-resolution melting curve (HRM) analysis is a promising and economical method for high-throughput mutation scanning. The EuroGentest network (www.eurogentest.org) aims to assist with the introduction of novel technologies in the diagnostic setting. Therefore, we have performed a thorough and highstandard interlaboratory evaluation and validation of HRM, in collaboration with Idaho Technology, the manufacturer of the LightScanner TM (LS). Through this detailed study of 170 variants, we have generated guidelines for easy setup and implementation of HRM as a scanning technique for new genes, which are adaptable to the quality system of an individual diagnostic laboratory. This validation study includes the description of a BRCA1-specific mutation screening test using the 96-well LS. This assay comprises 40 amplicons and was evaluated using a statistically significant elaborate panel of variants and control DNA samples. All heterozygous variants were detected. Moreover, genotype analysis for nine common polymorphisms created a fast screening and detection method for these frequently occurring nonpathogenic variants. A blind study using a total of 28 patient-derived DNA samples resulted also in 100% detection and showed an average specificity of 98%, indicating a low incidence of false positives (FPs).

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“…21 IVTT enables to pinpoint the site of a mutation, offers good sensitivity, and a low false-positive rate. 22 Further tight junction proteins (occludin and ZO-1) were co-investigated in this study. Interestingly, the up-regulated expression of l-CaD resulting from CALD1 missplicing was coincident with the down-regulation of occludin and ZO-1, causing tight junction (TJ) breakdown of the endothelial cells in glioma microvasculature.…”

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confidence: 99%

Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

Zheng

Sieuwerts

Luider

et al. 2004

The American Journal of Pathology

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Caldesmon is a cytoskeleton-associated protein whichhas not yet been related to neoplastic angiogenesis. In this study we investigated the expression of the caldesmon gene (CALD1) splicing variants and the protein expression level in glioma microvessels versus normal brain microvasculature. To exclude sources of splice variant expression from non-vascular components all possible cellular components present in control and glioma samples were prescreened by laser-capture microdissection followed by RT-PCR before the cohort study. We discovered differential expression of the splicing variants of CALD1 in the tumor microvessels in contrast to normal brain microvasculature. Missplicing of exons 1, 1 ؉ 4, and 1 ؉ 4 of the gene is exclusively found in glioma microvessels. To exclude the possibility that this missplicing results from splice-site mutations, Genome-wide analyses have revealed that 40 to 60% of human genes undergo alternative splicing. 1 Alternative splicing, therefore, seems to contribute considerably to enable the highly complex and diverse functions encoded by the human genome. Alternative splicing permits vertebrate pre-mRNA to be processed into multiple mRNAs differing in their precise combination of exon sequences, resulting in the encoding of different protein isoforms. 2 Multiple modes of alternative splicing exist, such as alternative 5Ј or 3Јsplice-site usage, differential inclusion or skipping of particular exons, mutually exclusion of exons, and more. 3 Importantly, alternative splicing is often tightly regulated in a cell type-or developmental stage-specific mode. 3 The essential nature of this process is underscored by the fact that misregulation (missplicing events) is often related to human disease. 4 -6

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The protein truncation test: A review

Cited by 52 publications

References 44 publications

The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-κB signal transduction pathway

The human B-cell lymphoma cell line RC-K8 has multiple genetic alterations that dysregulate the Rel/NF-κB signal transduction pathway

Diagnostic guidelines for high-resolution melting curve (HRM) analysis: An interlaboratory validation ofBRCA1mutation scanning using the 96-well LightScanner™

Differential Expression of Splicing Variants of the Human Caldesmon Gene (CALD1) in Glioma Neovascularization versus Normal Brain Microvasculature

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