Fluorescent tagging the NS1 protein in yellow fever virus: Replication-capable viruses which produce the secretory GFP-NS1 fusion protein.

Yellow fever virus, the prototype in the genus Flavivirus, was used to develop viruses in which the nonstructural protein NS1 is genetically fused to GFP in the context of viruses capable of autonomous replication. The GFP-tagging of NS1 at the amino-terminus appeared possible despite the presence of a small and functionally important domain at the NS1's amino-terminus which can be distorted by such fusing. GFP-tagged NS1 viruses were rescued from DNA-launched molecular clones. The initially produced GFP-tagged NS1 virus was capable of only poor replication. Sequential passages of the virus in cell cultures resulted in the appearance of mutations in GFP, NS4A, NS4B and NS5. The mutations which change amino acid sequences of GFP, NS4A and NS5 have the adaptive effect on the replication of GFP-tagged NS1 viruses. The pattern of GFP-fluorescence indicates that the GFP-NS1 fusion protein is produced into the endoplasmic reticulum. The intracellular GFP-NS1 fusion protein colocalizes with dsRNA. The discovered forms of extracellular GFP-NS1 possibly include tetramers and hexamers.

Towards development of plasmacytoma cells-based expression systems utilizing alphavirus vectors: An NS0-VEE model.

Plasmacytoma (myeloma) cells have a large protein expression capacity, although their industrial use is confined to stable expression systems. Vectors derived from genomes of viruses from the genus Alphavirus allow obtaining of high yields of target proteins but their use is limited to transient expression. Little information has been published to date on attempts to combine the myeloma cells as hosts with alphaviruses as expression vectors. A plasmid construct which allows rescue of a model alphavirus Venezuelan equine encephalitis virus (VEE) upon transfection of a cell culture was created. Mutations in the capsid and nsP2 genes allow for less cytopathogenic propagation of the virus. A cDNA-copy of the genome was placed in a plasmid under the control of the CMV promoter for virus rescue following DNA transfection. Parameters for the virus rescue by electroporating of the infectious clone in murine myeloma cells (NS0) were optimized. The highest FFU counts (1.2 × 105 FFU per 10 ug DNA) were produced with 2 pulses (voltage 250 V, capacitance 960 u F) and the best electroporation buffer was selected from eight buffers. Self-sustained VEE infection was established in NS0 cultures with high titers (8 × 108 FFU/ml) of the virus, despite a fraction of infected cells dying during 5-days observation. Further development of the NS0-VEE expression system may require addressing of apoptosis induced by VEE.

Recombinant Vaccinia virus-coded interferon inhibitor B18R: Expression, refolding and a use in a mammalian expression system with a RNA-vector.

B18R protein of Vaccinia virus binds to type I interferons and inhibits activation of interferon-mediated signal transduction. Cells which have unimpaired interferon signaling such as primary cell cultures or some industrially important cell lines are capable of development of an antiviral state. An establishment of the antiviral state limits replication of RNA-viruses and can suppress replication of RNA vectors. The interferon inhibitor B18R effectively prevents the establishment of the antiviral state. For this reason, B18R has become a ubiquitous component of protocols for epigenetic reprogramming which use transfections of RNA replicons or mRNA. Despite wide practical applicability, commercially available B18R is predominantly produced in cell cultures and little information has been published on a production and use of bacterially expressed B18R. Objectives of this study were to produce B18R in an E.coli expression system and to confirm the product's biological activity by using it to maintain RNA-vectors in cell cultures capable of the antiviral state. The described method allows the expression and efficient refolding to obtain 10-100 mg of B18R from a small-scale culture and the production process is economically attractive compared to a use of an eukaryotic expression. To check for a presence of the biological activity of bacterially-expressed B18R the protein was used to support persistence of an autonomously replicating RNA-vector in a cell culture which is capable of the antiviral state. A RNA-containing virus, Venezuelan equine encephalitis virus (VEE) can serve as an efficient vector for heterologous expression in cell cultures, although its replication is sensitive to the effects of type I interferons which limit a range of cell lines for a use with this vector. The VEE replicon was utilized to direct an expression of recombinant human granulocyte colony stimulating factor (G-CSF). The producing replicon could persist in HEK293 cells for sufficiently long time only in presence of B18R, whereas addition of B18R not only allowed persistence of the replicon but also increased production from the replicon. A model product granulocyte colony stimulating factor accumulated to 35.5 µg/ml during a 7 day experiment. This work describes efficacious expression and refolding of the viral cytokine inhibitor and demonstrates a utility of bacterially-expressed B18R.

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis.

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine-pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html , and its online version is located at http://primerdigital.com/tools/pcr.html .

Polymorphisms in genes involved in the absorption, distribution, metabolism, and excretion of drugs in the Kazakhs of Kazakhstan.

BACKGROUND: Studies of genes involved in the absorption, distribution, metabolism, and excretion (ADME) of drugs are crucial to the development of therapeutics in clinical medicine. Such data provide information that may improve our understanding of individual differences in sensitivity or resistance to certain drugs, thereby helping to avoid adverse drug reactions (ADRs) in patients and improve the quality of therapies. Here, we aimed to analyse single nucleotide polymorphisms (SNPs) involved in the ADME of multiple drugs in Kazakhs from Kazakhstan. RESULTS: A total of 158 SNPs involved in the ADME of various drugs were studied. We analysed 320 Kazakh DNA samples using OpenArray genotyping. Of the 158 SNPs, 75 were not found in heterozygous or homozygous variants. Comparative analysis among Kazakhs and world populations showed a fairly high percentage of population differentiation. CONCLUSION: These results provide further information for pharmacogenetic databases and may contribute to the development of personalized approaches and safer therapies for the Kazakh population. Moreover, these data provide insights into the different racial groups that may have contributed to the Kazakh population.

Mitochondrial and Y-chromosomal profile of the Kazakh population from East Kazakhstan.

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 AmpFiSTR 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.

BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast Cancer Patients In Kazakhstan.

BACKGROUND: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Kazakhstan women. AIM: To evaluate the role of BRCA1/2 mutations in Kazakhstan women presenting with sporadic breast cancer. METHODS: We investigated the distribution and nature of polymorphisms in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic breast cancer cases and 112 age-matched controls using automatic direct sequencing. RESULTS: We identified 22 distinct variants, including 16 missense mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7 missense mutations and 3 polymorphisms were detected. There was a higher prevalence of observed mutations in Caucasian breast cancer cases compared to Asian cases (p<0.05); higher frequencies of sequence variants were observed in Asian controls. No recurrent or founder mutations were observed in BRCA1/2 genes. There were no statistically significant differences in age at diagnosis, tumor histology, size of tumor, and lymph node involvement between women with breast cancer with or without the BRCA sequence alterations. CONCLUSIONS: Considering the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of BRCA1/2 mutations and reliable genetic counseling for Kazakhstan sporadic breast cancer patients. Evaluation of common polymorphisms and mutations and breast cancer risk in families with genetic predisposition to breast cancer is ongoing in another current investigation.

Direct DNA Lesion Reversal and Excision Repair in Escherichia coli.

Cellular DNA is constantly challenged by various endogenous and exogenous genotoxic factors that inevitably lead to DNA damage: structural and chemical modifications of primary DNA sequence. These DNA lesions are either cytotoxic, because they block DNA replication and transcription, or mutagenic due to the miscoding nature of the DNA modifications, or both, and are believed to contribute to cell lethality and mutagenesis. Studies on DNA repair in Escherichia coli spearheaded formulation of principal strategies to counteract DNA damage and mutagenesis, such as: direct lesion reversal, DNA excision repair, mismatch and recombinational repair and genotoxic stress signalling pathways. These DNA repair pathways are universal among cellular organisms. Mechanistic principles used for each repair strategies are fundamentally different. Direct lesion reversal removes DNA damage without need for excision and de novo DNA synthesis, whereas DNA excision repair that includes pathways such as base excision, nucleotide excision, alternative excision and mismatch repair, proceeds through phosphodiester bond breakage, de novo DNA synthesis and ligation. Cell signalling systems, such as adaptive and oxidative stress responses, although not DNA repair pathways per se, are nevertheless essential to counteract DNA damage and mutagenesis. The present review focuses on the nature of DNA damage, direct lesion reversal, DNA excision repair pathways and adaptive and oxidative stress responses in E. coli.