Search results for: gene polymorphism

Authors: Chintanu K. Sarmah, Sandhya Samarasinghe, Don Kulasiri, Daniel Catchpoole

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Gene expression profiling is rapidly evolving into a powerful technique for investigating tumor malignancies. The researchers are overwhelmed with the microarray-based platforms and methods that confer them the freedom to conduct large-scale gene expression profiling measurements. Simultaneously, investigations into cross-platform integration methods have started gaining momentum due to their underlying potential to help comprehend a myriad of broad biological issues in tumor diagnosis, prognosis, and therapy. However, comparing results from different platforms remains to be a challenging task as various inherent technical differences exist between the microarray platforms. In this paper, we explain a simple ratio-transformation method, which can provide some common ground for cDNA and Affymetrix platform towards cross-platform integration. The method is based on the characteristic data attributes of Affymetrix- and cDNA- platform. In the work, we considered seven childhood leukemia patients and their gene expression levels in either platform. With a dataset of 822 differentially expressed genes from both these platforms, we carried out a specific ratio-treatment to Affymetrix data, which subsequently showed an improvement in the relationship with the cDNA data.

Keywords: Gene expression profiling, microarray, cDNA, Affymetrix, childhood leukaemia.

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Authors: M. Maimunah, G.A. Froemming, H. Nawawi, M.I. Nafeeza, O. Effat, M.R. Rohayu Izanwati, M.S. Mohamed Saifulaman

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Attachment of the circulating monocytes to the endothelium is the earliest detectable events during formation of atherosclerosis. The adhesion molecules, chemokines and matrix proteases genes were identified to be expressed in atherogenesis. Expressions of these genes may influence structural integrity of the luminal endothelium. The aim of this study is to relate changes in the ultrastructural morphology of the aortic luminal surface and gene expressions of the endothelial surface, chemokine and MMP-12 in normal and hypercholesterolemic rabbits. Luminal endothelial surface from rabbit aortic tissue was examined by scanning electron microscopy (SEM) using low vacuum mode to ascertain ultrastructural changes in development of atherosclerotic lesion. Gene expression of adhesion molecules, MCP-1 and MMP-12 were studied by Real-time PCR. Ultrastructural observations of the aortic luminal surface exhibited changes from normal regular smooth intact endothelium to irregular luminal surface including marked globular appearance and ruptures of the membrane layer. Real-time PCR demonstrated differentially expressed of studied genes in atherosclerotic tissues. The appearance of ultrastructural changes in aortic tissue of hypercholesterolemic rabbits is suggested to have relation with underlying changes of endothelial surface molecules, chemokine and MMP-12 gene expressions.

Keywords: Ultrastructure of luminal endothelial surface, Macrophage metalloelastase (MMP-12), Real-time PCR.

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Authors: Mehrdad Hashemi, Mitra Behrooz Aghdam, Reza Mahdian, Ahmad Reza Kamyab

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Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value <0.001). These results represent the presence of 3 copies of target gene in DS samples Vs 2 copies in normal controls. The results of quantitative Real-time PCR were in complete agreement with results of cytogenetic analysis. This study confirms previous reports regarding successful implementation of quantitative Real-time PCR for detection of trisomy 21. However, the assay has been improved by using MGB probes and more accurate data analysis. This assay, in particular, when performed in combination with another molecular assay such as QF-PCR or MLPA, can be used as a reliable technique for rapid prenatal diagnosis of trisomy 21.

Keywords: Trisomy 21, Real-time PCR, MGB-TaqMan Probes, Gene Dosage.

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Authors: Tom Snir, Eitan Rubin

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Mendelian Disease Genes represent a collection of single points of failure for the various systems they constitute. Such genes have been shown, on average, to encode longer proteins than 'non-disease' proteins. Existing models suggest that this results from the increased likeli-hood of longer genes undergoing mutations. Here, we show that in saturated mutagenesis experiments performed on model organisms, where the likelihood of each gene mutating is one, a similar relationship between length and the probability of a gene being lethal was observed. We thus suggest an extended model demonstrating that the likelihood of a mutated gene to produce a severe phenotype is length-dependent. Using the occurrence of conserved domains, we bring evidence that this dependency results from a correlation between protein length and the number of functions it performs. We propose that protein length thus serves as a proxy for protein cardinality in different networks required for the organism's survival and well-being. We use this example to argue that the collection of Mendelian Disease Genes can, and should, be used to study the rules governing systems vulnerability in living organisms.

Keywords: Systems Biology, Protein Length

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Authors: Ngo Thanh Xuan, Mai Thi Hang, Vu Nguyen Thanh

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A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.

Keywords: Aspergillus niger XP, cloning, over expression, Pichia pastoris, phyA , phytase.

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Authors: Maria E. Manioudaki, Panayiota Poirazi

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Yeast cells live in a constantly changing environment that requires the continuous adaptation of their genomic program in order to sustain their homeostasis, survive and proliferate. Due to the advancement of high throughput technologies, there is currently a large amount of data such as gene expression, gene deletion and protein-protein interactions for S. Cerevisiae under various environmental conditions. Mining these datasets requires efficient computational methods capable of integrating different types of data, identifying inter-relations between different components and inferring functional groups or 'modules' that shape intracellular processes. This study uses computational methods to delineate some of the mechanisms used by yeast cells to respond to environmental changes. The GRAM algorithm is first used to integrate gene expression data and ChIP-chip data in order to find modules of coexpressed and co-regulated genes as well as the transcription factors (TFs) that regulate these modules. Since transcription factors are themselves transcriptionally regulated, a three-layer regulatory cascade consisting of the TF-regulators, the TFs and the regulated modules is subsequently considered. This three-layer cascade is then modeled quantitatively using artificial neural networks (ANNs) where the input layer corresponds to the expression of the up-stream transcription factors (TF-regulators) and the output layer corresponds to the expression of genes within each module. This work shows that (a) the expression of at least 33 genes over time and for different stress conditions is well predicted by the expression of the top layer transcription factors, including cases in which the effect of up-stream regulators is shifted in time and (b) identifies at least 6 novel regulatory interactions that were not previously associated with stress-induced changes in gene expression. These findings suggest that the combination of gene expression and protein-DNA interaction data with artificial neural networks can successfully model biological pathways and capture quantitative dependencies between distant regulators and downstream genes.

Keywords: gene modules, artificial neural networks, yeast, stress

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Authors: D. Vainauska, S. Kozireva, A. Karpovs, M. Chistyakovs, M. Baryshev

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Liposomal magnetofection is a simple, highly efficient technology for cell transfection, demonstrating better outcome than a number of other common gene delivery methods. However, aggregate complexes distribution over the cell surface is non-uniform due to the gradient of the permanent magnetic field. The aim of this study was to estimate the efficiency of liposomal magnetofection for prostate carcinoma PC3 cell line using newly designed device, “DynaFECTOR", ensuring magnetofection in a dynamic gradient magnetic field. Liposomal magnetofection in a dynamic gradient magnetic field demonstrated the highest transfection efficiency for PC3 cells – it increased for 21% in comparison with liposomal magnetofection and for 42% in comparison with lipofection alone. The optimal incubation time under dynamic magnetic field for PC3 cell line was 5 minutes and the optimal rotation frequency of magnets – 5 rpm. The new approach also revealed lower cytotoxic effect to cells than liposomal magnetofection.

Keywords: Dynamic gradient magnetic field, gene delivery, liposomal magnetofection, prostate cancer cell line

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Authors: M. Omer Faruk, A. M. A. M. Zonaed Siddiki, M. Fazal Karim, Md. Masuduzzaman, S. Chowdhury, Md. Shafiqul Islam, M. Alamgir Hossain

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The dog tapeworms Echinococcus granulosus develop hydatid cysts in various organs in human and domestic animals worldwide including Bangladesh. The aim of this study was to identify and characterize the genotype of E. granulosus isolated from cattle using 12S rRNA and Cytochrome oxidase 1 (COX 1) genes. A total of 43 hydatid cyst samples were collected from 390 examined cattle samples derived from slaughterhouses. Among them, three cysts were fertile. Genomic DNA was extracted from germinal membrane and/or protoscoleces followed by PCR amplification of mitochondrial 12S rRNA and Cytochrome oxidase 1 gene fragments. The sequence data revealed existence of G1 (64.28%) and possible G3 (21.43%) genotypes for the first time in Bangladesh. The study indicates that common sheep strain G1 is the dominant subtype of E. granulosus in Chittagong region of Bangladesh. This will increase our understanding of the epidemiology of hydatidosis in the southern part of the country and will be useful to plan suitable control measures in the long run.

Keywords: Echinococcus granulosus, molecular characterization, cattle, Bangladesh.

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Authors: Ines Hamdi, Mohamed Ben Ahmed

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The understanding of the system level of biological behavior and phenomenon variously needs some elements such as gene sequence, protein structure, gene functions and metabolic pathways. Challenging problems are representing, learning and reasoning about these biochemical reactions, gene and protein structure, genotype and relation between the phenotype, and expression system on those interactions. The goal of our work is to understand the behaviors of the interactions networks and to model their evolution in time and in space. We propose in this study an ontological meta-model for the knowledge representation of the genetic regulatory networks. Ontology in artificial intelligence means the fundamental categories and relations that provide a framework for knowledge models. Domain ontology's are now commonly used to enable heterogeneous information resources, such as knowledge-based systems, to communicate with each other. The interest of our model is to represent the spatial, temporal and spatio-temporal knowledge. We validated our propositions in the genetic regulatory network of the Aarbidosis thaliana flower

Keywords: Ontological model, spatio-temporal modeling, Genetic Regulatory Networks (GRNs), knowledge representation.

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Authors: Alexander Kaplunovsky, Vladimir Khailenko, Alexander Bolshoy, Shara Atambayeva, AnatoliyIvashchenko

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Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from the RNA transcripts before translation into a protein. The exon-intron structures of different eukaryotic species are quite different from each other, and the evolution of such structures raises many questions. We try to address some of these questions using statistical analysis of whole genomes. We go through all the protein-coding genes in a genome and study correlations between the net length of all the exons in a gene, the number of the exons, and the average length of an exon. We also take average values of these features for each chromosome and study correlations between those averages on the chromosomal level. Our data show universal features of exon-intron structures common to animals, plants, and protists (specifically, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Cryptococcus neoformans, Homo sapiens, Mus musculus, Oryza sativa, and Plasmodium falciparum). We have verified linear correlation between the number of exons in a gene and the length of a protein coded by the gene, while the protein length increases in proportion to the number of exons. On the other hand, the average length of an exon always decreases with the number of exons. Finally, chromosome clustering based on average chromosome properties and parameters of linear regression between the number of exons in a gene and the net length of those exons demonstrates that these average chromosome properties are genome-specific features.

Keywords: Comparative genomics, exon-intron structure, eukaryotic clustering, linear regression.

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Authors: Sang-Ho Baik

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D-erythro-cyclohexylserine (D chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on genomic sequence of Shinorhizobium meliloti Sequence analysis of the cloned DNA fragment revealed one open-reading frame of 1059 bp and 386 amino acids. This putative D-TA gene was cloned into NdeI and EcoRI (pEnsi His-tag sequence or BamHI (pEnsi-DTA[2]) sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA[1] compared to E. coli BL21(DE3) transformed with pEnsi-DTA[2]. When the cells expressing the wild used for D-TA enzyme activity, 12 mM glycine was successfully detected in HPLC analysis. Moreover, the whole cells harbouring the recombinant D-TA was able to synthesize D-erythro of 0.6 mg/ml in a batch reaction.

Keywords: About four key words or phrases in alphabetical order, separated by commas.

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Authors: Huihai Wu, Xiaohui Liu

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Using Dynamic Bayesian Networks (DBN) to model genetic regulatory networks from gene expression data is one of the major paradigms for inferring the interactions among genes. Averaging a collection of models for predicting network is desired, rather than relying on a single high scoring model. In this paper, two kinds of model searching approaches are compared, which are Greedy hill-climbing Search with Restarts (GSR) and Markov Chain Monte Carlo (MCMC) methods. The GSR is preferred in many papers, but there is no such comparison study about which one is better for DBN models. Different types of experiments have been carried out to try to give a benchmark test to these approaches. Our experimental results demonstrated that on average the MCMC methods outperform the GSR in accuracy of predicted network, and having the comparable performance in time efficiency. By proposing the different variations of MCMC and employing simulated annealing strategy, the MCMC methods become more efficient and stable. Apart from comparisons between these approaches, another objective of this study is to investigate the feasibility of using DBN modeling approaches for inferring gene networks from few snapshots of high dimensional gene profiles. Through synthetic data experiments as well as systematic data experiments, the experimental results revealed how the performances of these approaches can be influenced as the target gene network varies in the network size, data size, as well as system complexity.

Keywords: Genetic regulatory network, Dynamic Bayesian network, GSR, MCMC.

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Authors: A. Molee, N. Duanghaklang, P. Mernkrathoke

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The objective was to determine the single gene and interaction effect of composite genotype of beta-kappa casein and DGAT1 gene on milk yield (MY) and milk composition, content of milk fat (%FAT), milk protein (%PRO), solid not fat (%SNF), and total solid (%TS) in crossbred Holstein cows. Two hundred and thirty- one cows were genotyped with PCR-RFLP for DGAT1 and composite genotype data of beta-kappa casein from previous work were used. Two model, (1), and (2), was used to estimate single gene effect, and interaction effect on the traits, respectively. The significance of interaction effects on all traits were detected. Most traits have consistent pattern of significant when model (1), and (2) were compared, except the effect of composite genotype of betakappa casein on %FAT, and the effect of DGAT1 on MY, which the significant difference was detected in only model (1).The results suggested that when the optimum of all traits was necessary, interaction effect should be concerned.

Keywords: composite genotype of beta-kappa casein, DGAT1gene, Milk composition, Milk yield

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Authors: Ekaterina Y. Lukianova-Hleb, Dmitri O. Lapotko

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Cell processing techniques for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in heterogeneous cell systems. Using our novel on-demand nonstationary intracellular events instead of permanent materials, plasmonic nanobubbles, generated with a short laser pulse only in target cells, we achieved simultaneous multifunctional cell-specific processing with the rate up to 50 million cells per minute.

Keywords: Delivery, cell separation, graft, laser, plasmonic nanobubble, cell therapy, gold nanoparticle.

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Authors: Atnaf Tiruneh Mulugeta, Mohammed Ali Hussein, Zelleke Habtamu

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Thirty six genotypes (8 parents and 28 F1 diallel crosses) were grown in randomized complete block design during 2006 at Mandura, North western Ethiopia. The experiment was executed to study the inheritance of two primary yield component traits: number of seeds per pod and 1000 seed weight. Statistical significant difference was observed between genotypes, parents, and crosses for these traits. The mean square due to GCA was significant for the two traits. However, SCA mean square was significant only for number of seeds per pod. Thus both additive and non-additive types of gene actions were important in the inheritance of number of seeds per pod. Significant b1 component was obtained for this trait. The b2 and b3 components, however, were not significant, suggesting the absence of gene asymmetry. From Wr/Vr graph, inheritance of seeds per pod was governed by partial dominance with additive gene action.

Keywords: Diallel crosses, General combining ability, Phaseolus vulgaris L., Specific combining ability

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcus lactis, recombinant, phytase, poultry.

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Authors: Jafar Ahmadi, Zhohreh Asiaban, Sedigheh Fabriki Ourang

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Brassinosteroids (BRs) regulate cell elongation, vascular differentiation, senescence, and stress responses. BRs signal through the BES1/BZR1 family of transcription factors, which regulate hundreds of target genes involved in this pathway. In this research a comprehensive genome-wide analysis was carried out in BES1/BZR1 gene family in Arabidopsis thaliana, Cucumis sativus, Vitis vinifera, Glycin max and Brachypodium distachyon. Specifications of the desired sequences, dot plot and hydropathy plot were analyzed in the protein and genome sequences of five plant species. The maximum amino acid length was attributed to protein sequence Brdic3g with 374aa and the minimum amino acid length was attributed to protein sequence Gm7g with 163aa. The maximum Instability index was attributed to protein sequence AT1G19350 equal with 79.99 and the minimum Instability index was attributed to protein sequence Gm5g equal with 33.22. Aliphatic index of these protein sequences ranged from 47.82 to 78.79 in Arabidopsis thaliana, 49.91 to 57.50 in Vitis vinifera, 55.09 to 82.43 in Glycin max, 54.09 to 54.28 in Brachypodium distachyon 55.36 to 56.83 in Cucumis sativus. Overall, data obtained from our investigation contributes a better understanding of the complexity of the BES1/BZR1 gene family and provides the first step towards directing future experimental designs to perform systematic analysis of the functions of the BES1/BZR1 gene family.

Keywords: BES1/BZR1, Brassinosteroids, Phylogenetic analysis, Transcription factor.

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Authors: M. F. Miah, K. M. A. Zinnah, M. J. Raihan, H. Ali, M. N. Naser

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As genetic diversity is most important for existing, breeding and production of any fish; this study was undertaken for investigating genetic diversity of freshwater mud eel, Monopterus cuchia at population level where three ecological populations such as flooded area of Sylhet (P1), open water of Moulvibazar (P2) and open water of Sunamganj (P3) districts of Bangladesh were considered. Four arbitrary RAPD primers (OPB-12, C0-4, B-03 and OPB-08) were screened and RAPD banding patterns were analyzed among the populations considering 15 individuals of each population. In total 174, 138 and 149 bands were detected in the populations of P1, P2 and P3 respectively; however, each primer revealed less number of bands in each population. 100% polymorphic loci were recorded in P2 and P3 whereas only one monomorphic locus was observed in P1, recorded 97.5% polymorphism. Different genetic parameters such as inter-individual pairwise similarity, genetic distance, Nei genetic similarity, linkage distances, cluster analysis and allelic information, etc. were considered for measuring genetic diversity. The average inter-individual pairwise similarity was recorded 2.98, 1.47 and 1.35 in P1, P2 and P3 respectively. Considering genetic distance analysis, the highest distance 1 was recorded in P2 and P3 and the lowest genetic distance 0.444 was found in P2. The average Nei genetic similarity was observed 0.19, 0.16 and 0.13 in P1, P2 and P3, respectively; however, the average linkage distance was recorded 24.92, 17.14 and 15.28 in P1, P3 and P2 respectively. Based on linkage distance, genetic clusters were generated in three populations where 6 clades and 7 clusters were found in P1, 3 clades and 5 clusters were observed in P2 and 4 clades and 7 clusters were detected in P3. In addition, allelic information was observed where the frequency of p and q alleles were observed 0.093 and 0.907 in P1, 0.076 and 0.924 in P2, 0.074 and 0.926 in P3 respectively. The average gene diversity was observed highest in P2 (0.132) followed by P3 (0.131) and P1 (0.121) respectively.

Keywords: Genetic diversity, Monopterus cuchia, population, RAPD, Bangladesh.

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Authors: Dana Gabriková, Martin Mistrík, Jarmila Bernasovská, Iveta Tóthová, Jana Kisková

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The Roma (Gypsies) is a transnational minority with a high degree of consanguineous marriages. Similar to other genetically isolated founder populations, the Roma harbor a number of unique or rare genetic disorders. This paper discusses about a rare form of Charcot-Marie-Tooth disease – type 4G (CMT4G), also called Hereditary Motor and Sensory Neuropathy type Russe, an autosomal recessive disease caused by mutation private to Roma characterized by abnormally increased density of non-myelinated axons. CMT4G was originally found in Bulgarian Roma and in 2009 two putative causative mutations in the HK1 gene were identified. Since then, several cases were reported in Roma families mainly from Bulgaria and Spain. Here we present a Slovak Roma family in which CMT4G was diagnosed on the basis of clinical examination and genetic testing. This case is a further proof of the role of the HK1 gene in pathogenesis of the disease. It confirms that mutation in the HK1 gene is a common cause of autosomal recessive CMT disease in Roma and should be considered as a common part of a diagnostic procedure.

Keywords: Gypsies, HK1, HSMN-Russe, rare disease.

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Authors: Wiriya Loongyai, Kiettisak Promphet, Nilubol Kangsukul, Ratchawat Noppha

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Polymerase chain reaction (PCR) assay and conventional microbiological methods were used to detect bacterial contamination of egg shells and egg content in different commercial housing systems, open house system and evaporative cooling system. A PCR assay was developed for direct detection using a set of primers specific for the invasion by A gene (invA) of Salmonella spp. PCR detected the presence of Salmonella in 2 samples of shell egg from the evaporative cooling system, while conventional cultural methods detected no Salmonella from the same samples.

Keywords: egg content, egg shell, invA gene, PCR, Salmonellaspp.

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Authors: Archana Panchapakesan Iyer, Taha Abdullah Kumosani

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Many high-risk pathogens that cause disease in humans are transmitted through various food items. Food-borne disease constitutes a major public health problem. Assessment of the quality and safety of foods is important in human health. Rapid and easy detection of pathogenic organisms will facilitate precautionary measures to maintain healthy food. The Polymerase Chain Reaction (PCR) is a handy tool for rapid detection of low numbers of bacteria. We have designed gene specific primers for most common food borne pathogens such as Staphylococci, Salmonella and E.coli. Bacteria were isolated from food samples of various food outlets and identified using gene specific PCRs. We identified Staphylococci, Salmonella and E.coli O157 using gene specific primers by rapid and direct PCR technique in various food samples. This study helps us in getting a complete picture of the various pathogens that threaten to cause and spread food borne diseases and it would also enable establishment of a routine procedure and methodology for rapid identification of food borne bacteria using the rapid technique of direct PCR. This study will also enable us to judge the efficiency of present food safety steps taken by food manufacturers and exporters.

Keywords: food borne pathogens, PCR, food safety, rapiddetection.

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Authors: Fabian Horn, Reinhard Guthke

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In molecular biology, microarray technology is widely and successfully utilized to efficiently measure gene activity. If working with less studied organisms, methods to design custom-made microarray probes are available. One design criterion is to select probes with minimal melting temperature variances thus ensuring similar hybridization properties. If the microarray application focuses on the investigation of metabolic pathways, it is not necessary to cover the whole genome. It is more efficient to cover each metabolic pathway with a limited number of genes. Firstly, an approach is presented which minimizes the overall melting temperature variance of selected probes for all genes of interest. Secondly, the approach is extended to include the additional constraints of covering all pathways with a limited number of genes while minimizing the overall variance. The new optimization problem is solved by a bottom-up programming approach which reduces the complexity to make it computationally feasible. The new method is exemplary applied for the selection of microarray probes in order to cover all fungal secondary metabolite gene clusters for Aspergillus terreus.

Keywords: bottom-up approach, gene clusters, melting temperature, metabolic pathway, microarray probe design, probe selection

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Authors: Rosarin Rujananon, Poonsuk Prasertsan, Amornrat Phongdara, Tanate Panrat, Jibin Sun, Sugima Rappert, An-Ping Zeng

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In this study, a synthetic pathway was created by assembling genes from Clostridium butyricum and Escherichia coli in different combinations. Among the genes were dhaB1 and dhaB2 from C. butyricum VPI1718 coding for glycerol dehydratase (GDHt) and its activator (GDHtAc), respectively, involved in the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). The yqhD gene from E.coli BL21 was also included which codes for an NADPHdependent 1,3-propanediol oxidoreductase isoenzyme (PDORI) reducing 3-HPA to 1,3-propanediol (1,3-PD). Molecular modeling analysis indicated that the conformation of fusion protein of YQHD and DHAB1 was favorable for direct molecular channeling of the intermediate 3-HPA. According to the simulation results, the yqhD and dhaB1 gene were assembled in the upstream of dhaB2 to express a fusion protein, yielding the recombinant strain E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP41Y3). Strain BP41Y3 gave 10-fold higher 1,3-PD concentration than E. coliBL21 (DE3)//pET22b+::yqhD-dhaB1_dhaB2 (strain BP31Y2) expressing the recombinant enzymes simultaneously but in a non-fusion mode. This is the first report using a gene fusion approach to enhance the biological conversion of glycerol to the value added compound 1,3- PD.

Keywords: Recombinant E.coli, 1, 3-propanediol, glycerol, fusion protein.

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Authors: T. Holden, G. Tremberger, Jr, E. Cheung, R. Subramaniam, R. Sullivan, N. Gadura, P. Schneider, P. Marchese, A. Flamholz, T. Cheung, D. Lieberman

Abstract:

A nucleotide sequence can be expressed as a numerical sequence when each nucleotide is assigned its proton number. A resulting gene numerical sequence can be investigated for its fractal dimension in terms of evolution and chemical properties for comparative studies. We have investigated such nucleotide fluctuation in the 16S rRNA gene of archaea thermophiles. The studied archaea thermophiles were archaeoglobus fulgidus, methanothermobacter thermautotrophicus, methanocaldococcus jannaschii, pyrococcus horikoshii, and thermoplasma acidophilum. The studied five archaea-euryarchaeota thermophiles have fractal dimension values ranging from 1.93 to 1.97. Computer simulation shows that random sequences would have an average of about 2 with a standard deviation about 0.015. The fractal dimension was found to correlate (negative correlation) with the thermophile-s optimal growth temperature with R2 value of 0.90 (N =5). The inclusion of two aracheae-crenarchaeota thermophiles reduces the R2 value to 0.66 (N = 7). Further inclusion of two bacterial thermophiles reduces the R2 value to 0.50 (N =9). The fractal dimension is correlated (positive) to the sequence GC content with an R2 value of 0.89 for the five archaea-euryarchaeota thermophiles (and 0.74 for the entire set of N = 9), although computer simulation shows little correlation. The highest correlation (positive) was found to be between the fractal dimension and di-nucleotide Shannon entropy. However Shannon entropy and sequence GC content were observed to correlate with optimal growth temperature having an R2 of 0.8 (negative), and 0.88 (positive), respectively, for the entire set of 9 thermophiles; thus the correlation lacks species specificity. Together with another correlation study of bacterial radiation dosage with RecA repair gene sequence fractal dimension, it is postulated that fractal dimension analysis is a sensitive tool for studying the relationship between genotype and phenotype among closely related sequences.

Keywords: Fractal dimension, archaea thermophiles, Shannon entropy, GC content

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Authors: Alexandra Oliveros, Miguel Sotaquirá

Abstract:

DNA microarray technology is widely used by geneticists to diagnose or treat diseases through gene expression. This technology is based on the hybridization of a tissue-s DNA sequence into a substrate and the further analysis of the image formed by the thousands of genes in the DNA as green, red or yellow spots. The process of DNA microarray image analysis involves finding the location of the spots and the quantification of the expression level of these. In this paper, a tool to perform DNA microarray image analysis is presented, including a spot addressing method based on the image projections, the spot segmentation through contour based segmentation and the extraction of relevant information due to gene expression.

Keywords: Contour segmentation, DNA microarrays, edge detection, image processing, segmentation, spot addressing.

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

Abstract:

Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA microarray. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 NanoLabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring softwares analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with downregulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38-MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: CARD16, CASP10, cDNA microarray, PTPRR, Thymoquinone.

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Authors: C. Gunavathi, K. Premalatha

Abstract:

Tumor classification is a key area of research in the field of bioinformatics. Microarray technology is commonly used in the study of disease diagnosis using gene expression levels. The main drawback of gene expression data is that it contains thousands of genes and a very few samples. Feature selection methods are used to select the informative genes from the microarray. These methods considerably improve the classification accuracy. In the proposed method, Genetic Algorithm (GA) is used for effective feature selection. Informative genes are identified based on the T-Statistics, Signal-to-Noise Ratio (SNR) and F-Test values. The initial candidate solutions of GA are obtained from top-m informative genes. The classification accuracy of k-Nearest Neighbor (kNN) method is used as the fitness function for GA. In this work, kNN and Support Vector Machine (SVM) are used as the classifiers. The experimental results show that the proposed work is suitable for effective feature selection. With the help of the selected genes, GA-kNN method achieves 100% accuracy in 4 datasets and GA-SVM method achieves in 5 out of 10 datasets. The GA with kNN and SVM methods are demonstrated to be an accurate method for microarray based tumor classification.

Keywords: F-Test, Gene Expression, Genetic Algorithm, k- Nearest-Neighbor, Microarray, Signal-to-Noise Ratio, Support Vector Machine, T-statistics, Tumor Classification.

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Authors: Dalia. G. Aseel

Abstract:

Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.

Keywords: Begomovirus, AV1 gene, sequence, cloning, whitefly, okra, cotton, tomato, RAPD, phylogenetic tree and SDS-PAGE.

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Authors: Bezuidt O., Lima-Mendez G., Reva O. N.

Abstract:

SeqWord Gene Island Sniffer, a new program for the identification of mobile genetic elements in sequences of bacterial chromosomes is presented. This program is based on the analysis of oligonucleotide usage variations in DNA sequences. 3,518 mobile genetic elements were identified in 637 bacterial genomes and further analyzed by sequence similarity and the functionality of encoded proteins. The results of this study are stored in an open database http://anjie.bi.up.ac.za/geidb/geidbhome. php). The developed computer program and the database provide the information valuable for further investigation of the distribution of mobile genetic elements and virulence factors among bacteria. The program is available for download at www.bi.up.ac.za/SeqWord/sniffer/index.html.

Keywords: mobile genetic elements, virulence, bacterial genomes

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Authors: Sawangjit Sopid

Abstract:

Atrazine, a herbicide widely used in sugarcane and corn production, is a frequently detected groundwater contaminant. An atrazine-degrading bacterium, strain KB02, was obtained from long-term atrazine-treated sugarcane field soils in Kanchanaburi province of Thailand. Strain KB02 had a rod-to-coccus morphological cycle during growth. Sequence analysis of the PCR product indicated that the 16S rRNA gene in strain KB02 was ranging from 97-98% identical to the same region in Klebsiella sp. Based on biochemical, physiological analysis and 16S rDNA sequence analysis of one representative isolate, strain KB02, the isolates belong to the genus Klebsiella in the family Enterobacteriaceae. Interestingly that the various primers for atzA, B and C failed to amplify genomic DNA of strain KB02. Whereas the expected PCR product of atzA, B and C were obtained from the reference strain, Arthrobacter sp. strain KU001.

Keywords: Atrazine, atz gene, Biodegradation, bioremediation, Klebsiella

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