Determination of Copy Number of Target Carbohydrase Genes in the Penicillium verruculosum fungus Recombinant Strains

2019 ◽  
Vol 35 (5) ◽  
pp. 51-57 ◽  
Author(s):  
V.Yu. Kislitsin ◽  
I.N. Zorov ◽  
A.P. Sinitsyn ◽  
A.M. Rozhkova
Keyword(s):  
Copy Number ◽  
Target Gene ◽  
Gene Copy Number ◽  
Gene Promoter ◽  
Gene Copy ◽  
Penicillium Verruculosum ◽  
Penicillium Canescens ◽  
Enzyme Preparations ◽  
Recombinant Strains

The protein profiles of the culture liquids of recombinant Penicillium verruculosum strains that secreted an Aspergillus niger β-glucosidase (BGL series) or a Penicillium canescens xylanase A (XYLA series) have been previously analyzed. It was shown that the producers were divided into two groups according to the target protein content: 75-85% (1) and no more than 15% (2). It was established in this work that the copy number of the target gene bgll in the recombinant strains correlated with the β-glucosidase content in the BGL-F10 and BGL-F12 enzyme preparations. However, this proportion was violated in the direction of decreasing the xylanase A relative amount: the target enzyme content of 50% and 17% in the XylA3 and XylA4 preparations corresponded to 20-22 and 6 copies, respectively, of the xylA gene. We believe that this effect is a result of titration of positive transcription factors in the P. verruculosum XylA3 strain containing 20 to 22 copies of the cbhl gene promoter. Penicillium verruculosum, real-time PCR, gene copy number This work was partially supported by the Russian Foundation for Bsic Research (Project number: 18-29-07070).

Solid-phase minisequencing confirmed by FISH analysis in determination of gene copy number

1995 ◽  
Vol 96 (3) ◽  
Author(s):  
Maris Laan ◽  
Kristiina Gr�n-Virta ◽  
Armi Salo ◽  
Pertti Aula ◽  
Leena Peltonen ◽  
...  
Keyword(s):  
Copy Number ◽  
Solid Phase ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Fish Analysis

scholarly journals Determination of CYP2D6 Gene Copy Number by Pyrosequencing

2005 ◽  
Vol 51 (3) ◽  
pp. 522-531 ◽  
Author(s):  
Erik Söderbäck ◽  
Anna-Lena Zackrisson ◽  
Bertil Lindblom ◽  
Anders Alderborn
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Relative Quantification ◽  
Sequence Context ◽  
Cyp2d6 Gene ◽  
Equivalent Region ◽  
Peak Pattern ◽  
Single Set

Abstract Background: Identification of CYP2D6 alleles *5 (deletion of the whole CYP2D6 gene) and *2xN (gene duplication) is very important because they are associated with decreased or increased metabolism of many drugs. The most commonly used method for analysis of these alleles is, however, considered to be laborious and unreliable. Methods: We developed a method to determine the copy number of the CYP2D6*5 and CYP2D6*2xN alleles by use of Pyrosequencing™ technology. A single set of PCR and sequencing primers was used to coamplify and sequence a region in the CYP2D6 gene and the equivalent region in the CYP2D8P pseudogene, and relative quantification between these fragments was performed. The CYP2D8P-specific Pyrosequencing peak heights were used as references for the CYP2D6-specific peak heights. Results: Analysis of 200 pregenotyped samples showed that this approach reliably resolved 0–4 genome copies of the CYP2D6 gene. In 15 of these samples, the peak pattern from one analyzed position was unexpected but could be solved by conclusive results from a second position. The method was verified on 270 other samples, of which 267 gave results that corresponded to the expected genotype. One of the samples could not be interpreted. The reproducibility of the method was high. Conclusions: CYP2D6 gene copy determination by Pyrosequencing is a reliable and rapid alternative to other methods. The use of an internal CYP2D8P control as well as generation of a sequence context ensures a robust method and hence facilitates method validation.

scholarly journals Digital PCR for determination of cytochrome P450 2D6 and sulfotransferase 1A1 gene copy number variations

2017 ◽  
Vol 11 (6) ◽  
pp. 336-341 ◽  
Author(s):  
Yutaro Motoi ◽  
Kazufumi Watanabe ◽  
Hiroyuki Honma ◽  
Yousuke Tadano ◽  
Hiroshi Hashimoto ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Copy Number ◽  
Gene Copy Number ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Gene Copy ◽  
Cytochrome P450 2D6 ◽  
Sulfotransferase 1A1 ◽  
P450 2D6

Determination of Foreign Gene Copy Number in Stably Transfected Cell Lines by Southern Transfer Analysis

2003 ◽  
pp. 381-396 ◽  
Author(s):  
Ronald M. Fourney ◽  
Rémy Aubin ◽  
Kevin D. Dietrich ◽  
Malcolm C. Paterson
Keyword(s):  
Cell Lines ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Foreign Gene

scholarly journals The Anticholinesterase Phenserine and Its Enantiomer Posiphen as 5′Untranslated-Region-Directed Translation Blockers of the Parkinson’s Alpha Synuclein Expression

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Sohan Mikkilineni ◽  
Ippolita Cantuti-Castelvetri ◽  
Catherine M. Cahill ◽  
Amelie Balliedier ◽  
Nigel H. Greig ◽  
...  
Keyword(s):  
Copy Number ◽  
Untranslated Region ◽  
Cholinesterase Inhibitors ◽  
Ex Vivo ◽  
Gene Copy Number ◽  
Gene Promoter ◽  
Mrna Translation ◽  
Alpha Synuclein ◽  
Gene Copy ◽  
Promoter Polymorphisms

There is compelling support for limiting expression of alpha-synuclein (α-syn) in the brains of Parkinson’s disease (PD) patients. An increase ofSNCAgene copy number can genetically cause familial PD where increased dose of this pathogenic protein correlates with severity of symptoms (triplication of theSNCAgene causes dementia in PD patients). Gene promoter polymorphisms were shown to increaseα-synuclein expression as a risk for PD. Cholinesterase inhibitors can clinically slow cognitive decline in the later stages of PD etiology similar to their widespread use in Alzheimer’s disease (AD). Pertinent to this, we identified that the well-tolerated anticholinesterase, phenserine, blocked neuralSNCAmRNA translation and tested for targeting via its 5′untranslated region (5′UTR) in a manner similar to its action to limit the expression of the AD-specific amyloid precursor protein (APP). Posiphen, its better-tolerated (+) enantiomer (devoid of anticholinesterase action), repressed neuralα-synuclein translation. Primary metabolic analogs of posiphen were, likewise, characterized using primary fetal neurons grownex vivofrom the brains of Parkinson’s transgenic mice expressing the humanSNCAgene.

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