NotI clones in the analysis of the human genome

Zabarovsky, Eugene R.; Gizatullin, Rinat; Podowski, Raf M.; Zabarovska, Veronika I.; Xie, Li; Muravenko, Olga V.; Козырев, С. В.; Petrenko, Lev; Skobeleva, Natalia; Li, Jing‐Feng; Protopopov, Alexei; Kashuba, Vladimir I.; Ernberg, Ingemar; Winberg, Gösta; Wahlestedt, Claes

doi:10.1093/nar/28.7.1635

Cited by 25 publications

(18 citation statements)

References 17 publications

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“…Analysis of the human SREBP1c gene promoter A human genomic clone (NR1-B022) which contains NotI flanking regions corresponding to the SREBP1c promoter was obtained from Zabarovsky et al [29] and subcloned into the luciferase reporter gene vector pGL3-Enhancer (Promega, Charbonnières, France). HEK293 cells were maintained in serum-free medium for 18-24 h before transfection using Exgen 500 reagent (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions.…”

Section: Quantification Of Messenger Rnasmentioning

confidence: 99%

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells

et al. 2006

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Aims/hypothesis: The aim of this study was to investigate the effects of liver X receptor (LXR) activation on lipid metabolism and insulin action in human skeletal muscle cells prepared from control subjects and from patients with type 2 diabetes. Subjects and methods: Cultured myotubes were obtained from muscle biopsies of 11 lean, healthy control subjects and ten patients with type 2 diabetes. The mRNA levels of LXR isoforms and lipogenic genes were estimated by RT-quantitative PCR, and the effects of LXR agonists on insulin action were evaluated by assays of protein kinase B serine 473 phosphorylation and glycogen synthesis. Results: Both LXRα and LXRβ were expressed in human skeletal muscle and adipose tissue and there was no difference in their mRNA abundance in tissues from patients with type 2 diabetes compared with control subjects. In cultured muscle cells, LXR activation by T0901317 strongly increased expression of the genes encoding lipogenic enzymes, including sterol regulatory element binding protein 1c, fatty acid synthase and stearoyl-CoA desaturase 1, and also promoted triglyceride accumulation in the presence of a high glucose concentration. Importantly, these effects on lipid metabolism did not affect protein kinase B activation by insulin. Furthermore, LXR agonists did not modify insulin action in muscle cells from patients with type 2 diabetes. Conclusions/interpretation: These data suggest that LXR agonists may lead to increased utilisation of lipids and glucose in muscle cells without affecting the mechanism of action of insulin. However, the long-term consequences of triglyceride accumulation in muscle should be evaluated before the development of effective LXR-based therapeutic agents.

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Section: Quantification Of Messenger Rnasmentioning

confidence: 99%

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells

et al. 2006

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“…We generated more than 100,000 NotI flanking sequences and identified, among them, approximately 22,000 unique NotI sequences containing 17 Mb of information. 26,27 Using this information, we identified and mapped numerous new genes.…”

Section: Introductionmentioning

confidence: 99%

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

et al. 2012

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“…A standard protocol was used to prepare nylon filter replicas of the gridded NotI linking clones. Nylon filters contained 90 mapped chromosome-3-specific NotI linking clones (6) and five random unmapped human NotI linking clones (8). For hybridization to the nylon filters, the NotI representations (NRs; see below) probes were 32 P-labeled by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…It has been suggested and shown (5)(6)(7)(8) that NotI sites are almost exclusively located in CpG islands and are closely associated with functional genes. Therefore, NotI sites can serve as very useful markers for both physical and genetic mapping.…”

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confidence: 99%

Not I subtraction and Not I-specific microarrays to detect copy number and methylation changes in whole genomes

Li¹,

Protopopov²,

Wang³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Methylation, deletions, and amplifications of cancer genes constitute important mechanisms in carcinogenesis. For genome-wide analysis of these changes, we propose the use of NotI clone microarrays and genomic subtraction, because NotI recognition sites are closely associated with CpG islands and genes. We show here that the CODE (Cloning Of DEleted sequences) genomic subtraction procedure can be adapted to NotI flanking sequences and to CpG islands. Because the sequence complexity of this procedure is greatly reduced, only two cycles of subtraction are required. A NotI-CODE procedure can be used to prepare NotI representations (NRs) containing 0.1-0.5% of the total DNA. The NRs contain, on average, 10-fold less repetitive sequences than the whole human genome and can be used as probes for hybridization to NotI microarrays. These microarrays, when probed with NRs, can simultaneously detect copy number changes and methylation. NotI microarrays offer a powerful tool with which to study carcinogenesis. R epresentational difference analysis (RDA) (1) and restriction fragment length polymorphism subtraction (2) were reproducibly successful in cloning deleted sequences. However, these methods are sensitive to minor impurities, are laborious, and suffer from a number of limitations (e.g., the inability to detect differences due to point mutations, small deletions, or insertions). Furthermore, the PCR amplification after the first hybridization step and before the nuclease treatment may give rise to artifacts. Excess driver DNA can result in reduced efficiency in amplification of the tester-tester duplexes because of the potential formation of residual driver-driver and drivertester duplexes that act as competitors. As RDA is based mainly on the specific amplification of the desired products and requires 95-110 PCR cycles, it suffers from a ''plateau effect'' that is characterized by a decline in the exponential rate of accumulation of amplification products. However, the major problem results from the inefficiency of the multiple restriction digestion and ligation reactions that are used in this method, and which lead to the generation of false positives. Furthermore, these experiments result in the cloning of products that usually do not represent functional genes. Similarly, the methylation-sensitive representational difference analysis (3), aimed at CpG-rich sequences, suffers from the same limitations as the original RDA.Recently, we developed a procedure for cloning deleted sequences (Cloning Of DEleted sequences, CODE) (4) that is free from some of the limitations inherent in the RDA and restriction fragment length polymorphism subtraction protocols. Our major objective was to improve the subtractive enrichment, thereby avoiding excessive PCR kinetic enrichment steps that often generate small DNA products.

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NotI clones in the analysis of the human genome

Cited by 25 publications

References 17 publications

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells

Genetic and epigenetic analysis of non-small cell lung cancer with NotI-microarrays

Not I subtraction and Not I-specific microarrays to detect copy number and methylation changes in whole genomes

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