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Gao S.-J.,Fujian Agriculture and forestry University | Damaj M.B.,Texas AgriLife Research Center | Park J.-W.,Texas AgriLife Research Center | Beyene G.,Donald Danforth Plant Science Center | And 9 more authors.
PLoS ONE | Year: 2013

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane. © 2013 Gao et al.


Kulyyassov A.,French National Center for Scientific Research | Kulyyassov A.,National Center for Biotechnology of the Republic of Kazakhstan | Shoaib M.,French National Center for Scientific Research | Ogryzko V.,French National Center for Scientific Research
Current Protocols in Cell Biology | Year: 2011

This unit describes a system for expression of biotinylated proteins in mammalian cells in vivo, and its application to chromatin immunoprecipitation (ChIP). The system is based on co-expression of the target protein fused to a short biotin acceptor domain, together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction in the modified ChIP protocol presented here allows one to employ more stringent washing conditions, resulting in a better signal/noise ratio. Methods for interpreting the data obtained from ChIP samples analyzed by qPCR, and methods for testing the efficiency of biotinylation using a streptavidin gel-shift are also presented. In addition, a complementary method, based on isothermal multiple strand displacement amplification (IMDA) of circular concatemers generated from the DNA fragments obtained after ChIP, is described. This method helps to decrease bias in DNA amplification and is useful for the analysis of complex mixtures of DNA fragments typically generated in miniscale ChIP experiments. © 2011 by John Wiley & Sons, Inc.


Tarlykov P.V.,National Center for Biotechnology of the Republic of Kazakhstan | Zholdybayeva E.V.,National Center for Biotechnology of the Republic of Kazakhstan | Akilzhanova A.R.,National Center for Biotechnology of the Republic of Kazakhstan | Nurkina Z.M.,National Center for Biotechnology of the Republic of Kazakhstan | And 3 more authors.
Croatian Medical Journal | Year: 2013

Aim To study the genetic relationship of Kazakhs from East Kazakhstan to other Eurasian populations by examining paternal and maternal DNA lineages. Methods Whole blood samples were collected in 2010 from 160 unrelated healthy Kazakhs residing in East Kazakhstan. Genomic DNA was extracted with Wizard® genomic DNA Purification Kit. Nucleotide sequence of hypervariable segment I of mitochondrial DNA (mtDNA) was determined and analyzed. Seventeen Y-short tandem repeat (STR) loci were studied in 67 samples with the Amp- FiSTR Y-filer PCR Amplification Kit. In addition, mtDNA data for 2701 individuals and Y-STR data for 677 individuals were retrieved from the literature for comparison. Results There was a high degree of genetic differentiation on the level of mitochondrial DNA. The majority of maternal lineages belonged to haplogroups common in Central Asia. In contrast, Y-STR data showed very low genetic diversity, with the relative frequency of the predominant haplotype of 0.612. Conclusion The results revealed different migration patterns in the population sample, showing there had been more migration among women. mtDNA genetic diversity in this population was equivalent to that in other Central Asian populations. Genetic evidence suggests the existence of a single paternal founder lineage in the population of East Kazakhstan, which is consistent with verbal genealogical data of the local tribes.


Akhmetova A.,National Center for Biotechnology of the Republic of Kazakhstan | Kozhamkulov U.,National Center for Biotechnology of the Republic of Kazakhstan | Bismilda V.,National Center for Tuberculosis Problems of the Republic of Kazakhstan | Chingissova L.,National Center for Tuberculosis Problems of the Republic of Kazakhstan | And 4 more authors.
International Journal of Tuberculosis and Lung Disease | Year: 2015

SETTING: Pyrazinamide (PZA), an important first-line drug for anti-tuberculosis treatment, demonstrates potent activity against semi-dormant bacilli in acidic environments. However, the diagnosis of PZA resistance is often impeded by technical difficulties. OBJECTIVE: To characterise mutations in the pncA and rpsA genes among PZA-resistant and PZA-susceptible clinical Mycobacterium tuberculosis isolates circulating in Kazakhstan. The potential use of genotyping to identify PZA resistance was also investigated. DESIGN: PZA drug susceptibility testing and pncA and rpsA gene sequencing were performed on 77 clinical M. tuberculosis isolates; mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRUVNTR) typing was performed on 74 clinical M. tuberculosis isolates. RESULTS: Of the 77 clinical M. tuberculosis isolates, 41 (53.2%) were phenotypically resistant to PZA, whereas 36 (46.7%) were susceptible; 48 (62.3%) of these isolates were also multidrug-resistant (MDR). Furthermore, 38 (49.3%) clinical isolates showed mutations in the pncA gene and its flanking region; the majority of these isolates (n = 36, 94.7%) were also MDR. Gene sequencing showed that only synonymous substitutions affecting rpsA occurred. MIRU-VNTR typing revealed that 78.4% of isolates were of the Beijing genotype. CONCLUSIONS: Sequencing revealed that mutations in pncA, but not in rpsA, occurred in PZA-resistant M. tuberculosis isolates circulating in the territory of Kazakhstan. © 2015 The Union.


Sotnikov D.V.,RAS A.N. Bach Institute of Biochemistry | Byzova N.A.,RAS A.N. Bach Institute of Biochemistry | Zherdev A.V.,RAS A.N. Bach Institute of Biochemistry | Eskendirova S.Z.,National Center for Biotechnology of the Republic of Kazakhstan | And 5 more authors.
Journal of Immunoassay and Immunochemistry | Year: 2015

An immunochromatographic test system was developed for rapid detection of the levels of specific IgG antibodies to Brucella abortus lipopolysaccharide, as a tool for diagnosis of brucellosis in cattle. The pilot test strips were examined using blood sera from sick (78 samples) and healthy (35 samples) cows. The results obtained by immunochromatographic assay, using a portable optical densitometer for digital video detection, correlate well with the results obtained by immunoenzyme assay and are in agreement with the results of the disease diagnosis. The new test system allows detection of antibodies within 10 min and can be proposed as an alternative to the methods available for serodiagnosis of brucellosis. Copyright © Taylor & Francis Group, LLC.

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