Search results for: mecA genes

Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Oya Bulut, Oguzhan Avci, Zafer Bulut, Atilla Simsek

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Livestock breeders have focused on the improvement of production traits with little or no attention for improvement of disease resistance traits. In order to determine the association between the genetic structure of the individual gene loci with possibility of the occurrence and the development of diseases, MHC (major histocompatibility complex) are frequently used. Because of their importance in the immune system, MHC locus is considered as candidate genes for resistance/susceptibility against to different diseases. Major histocompatibility complex (MHC) molecules play a critical role in both innate and adaptive immunity and have been considered candidate molecular markers of an association between polymorphisms and resistance/susceptibility to diseases. The purpose of this study is to give some information about MHC genes become an important area of study in recent years in terms of animal husbandry and determine the relation between MHC genes and resistance/susceptibility to disease.

Keywords: MHC, polymorphism, disease, resistance

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Authors: Suwattana Visetnan, Premruethai Supungul, Sureerat Tang, Ikuo Hirono, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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In animals, infection by Gram-negative bacteria and certain viruses activates the Imd signaling pathway wherein the a NF-κB transcription factor, Relish, is a key regulatory protein for the synthesis of antimicrobial proteins. Infection by yellow head virus (YHV) activates the Imd pathway. To investigate the expression of genes involved in YHV infection and under the influence of PmRelish regulation, RNA interference and suppression subtractive hybridization (SSH) are employed. The genes in forward library expressed in shrimp after YHV infection and under the activity of PmRelish were obtained by subtracting the cDNAs from YHV-infected and PmRelish-knockdown shrimp with cDNAs from YHV-infected shrimp. Opposite subtraction gave a reverse library whereby an alternative set of genes under YHV infection and no PmRelish expression was obtained. Sequencing of 252 and 99 cDNA clones from the respective forward and reverse libraries were done and annotated through blast search against the GenBank sequences. Genes involved in defense and homeostasis were abundant in both libraries, 31% and 23% in the forward and reverse libraries, respectively. They were predominantly antimicrobial proteins, proteinases and proteinase inhibitors. The expression of antimicrobial protein genes, ALFPm3, crustinPm1, penaeidin3 and penaeidin5 were tested under PmRelish silencing and Gram-negative bacterium V. harveyi infection. Together with the results previously reported, the expression of penaeidin5 and also penaeidin3 but not ALFPm3 and crustinPm1 were under the regulation of PmRelish in the Imd pathway.

Keywords: relish, yellow head virus, penaeus monodon, antimicrobial proteins

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Authors: Shahram Nanekarania, Majid Goodarzia

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Calpastatin and callipyge have been known as one of the candidate genes in meat quality and quantity. Calpastatin gene has been located to chromosome 5 of sheep and callipyge gene has been localized in the telomeric region on ovine chromosome 18. The objective of this study was identification of calpastatin and callipyge genes polymorphism and analysis of genotype structure in population of Lori sheep kept in Iran. Blood samples were taken from 120 Lori sheep breed and genomic DNA was extracted by salting out method. Polymorphism was identified using the PCR-RFLP technique. The PCR products were digested with MspI and FaqI restriction enzymes for calpastatin gene and callipyge gene, respectively. In this population, three patterns were observed and AA, AB, BB genotype have been identified with the 0.32, 0.63, 0.05 frequencies for calpastatin gene. The results obtained for the callipyge gene revealed that only the wild-type allele A was observed, indicating that only genotype AA was present in the population under consideration.

Keywords: polymorphism, calpastatin, callipyge, PCR-RFLP, Lori sheep

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Authors: Zhang Lei, Zhao Qingrong, Wang Chen, Zhang Sufang, Zhang Hanguo

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Larch is the main afforestation and timber tree species in Northeast China, and drought is one of the main factors limiting the growth of Larch and other organisms in Northeast China. In order to further explore the mechanism of Larch drought resistance, PEG was used to simulate drought stress. The full-length sequencing of Larch embryogenic callus under PEG simulated drought stress was carried out by combining Illumina-Hiseq and SMRT-seq. A total of 20.3Gb clean reads and 786492 CCS reads were obtained from the second and third generation sequencing. The de-redundant transcript sequences were predicted by lncRNA, 2083 lncRNAs were obtained, and the target genes were predicted, and a total of 2712 target genes were obtained. The de-redundant transcripts were further screened, and 1654 differentially expressed genes (DEGs )were obtained. Among them, different DEGs respond to drought stress in different ways, such as oxidation-reduction process, starch and sucrose metabolism, plant hormone pathway, carbon metabolism, lignin catabolic/biosynthetic process and so on. This study provides basic full-length sequencing data for the study of Larch drought resistance, and excavates a large number of DEGs in response to drought stress, which helps us to further understand the function of Larch drought resistance genes and provides a reference for in-depth analysis of the molecular mechanism of Larch drought resistance.

Keywords: larch, drought stress, full-length transcriptome sequencing, differentially expressed genes

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Authors: Moses Ikechukwu Benjamin

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The continued increase in methicillin-resistant Staphylococcuspseudintermedius (MRSP) among dogs and the zoonotic transmission event of MRSP from dogs to humans threaten veterinary medicine and public health. The cardinal objective of this study was to determine the antibiogram and frequency of toxingenes in MRSP obtained from shelter dogs with skin infections and dog owners in Abakaliki, Eastern Nigeria. Skinswabs from 61 shelter dogs with skin infections and 33 nasal swabs from dog owners were processed and analyzed using standard microbiological techniques. Susceptibility to antibiotics was determined by Kirby Bauer disc diffusion technique. The screening for Seccanine, lukD, siet, and exitoxin genes was carried out by PCR. A total of 23 (37.7 %) and 1 (3 %) MRSP strains were obtained from shelter dogs and dog owners, respectively. Generally, isolates exhibited high resistance to amoxicillin-clavulanic acid, ceftazidime, and cefepime (100 % - 66.7 %) but were very susceptible (100 % - 70.7 %) to chloramphenicol and doripenem. The only isolate from dog owners harbouredseccanine, lukD, and siet toxin genes while solatesfrom shelter dogs harbouredseccanine16 (69.6 %), lukD 17 (73.9 %), siet 20 (87 %), and exi1 (4.4 %) toxin genes. Isolates were generally observed to be more resistant than other reports from the literature. Interesting, there was a similarity in the resistance antibiotypes and frequency of toxin genes harboured by MRSP isolates between shelter dogs with skin infections and their owner in a sampled household, thus suggesting a likely zoonotic transmission event. This report of the occurrence of MRSP and high frequency of toxin genes (Seccanine,lukD, and siet) in shelter dogs and dog owners represent a major challenge, especially in terms of antibiotic therapy, and is a serious concern for both animal and public health.

Keywords: methicillin-resistant S. pseudintermedius, zoonotic transmission, antibiotic resistance, companion dogs, toxin genes

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Authors: Paul Shize Li, Frank Alber

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A fundamental task of clinical genomics is to unravel the functions of genes and their associations with disorders. Although experimental biology has made efforts to discover and elucidate the molecular mechanisms of individual genes in the past decades, still about 40% of human genes have unknown functions, not to mention the diseases they may be related to. For those biologists who are interested in a particular gene with unknown functions, a powerful computational method tailored for inferring the functions and disease relevance of uncharacterized genes is strongly needed. Studies have shown that genes strongly linked to each other in multiple biological networks are more likely to have similar functions. This indicates that the densely connected subgraphs in multiple biological networks are useful in the functional and phenotypic annotation of uncharacterized genes. Therefore, in this work, we have developed an integrative network approach to identify the frequent local clusters, which are defined as those densely connected subgraphs that frequently occur in multiple biological networks and consist of the query gene that has few or no disease or function annotations. This is a local clustering algorithm that models multiple biological networks sharing the same gene set as a three-dimensional matrix, the so-called tensor, and employs the tensor-based optimization method to efficiently find the frequent local clusters. Specifically, massive public gene expression data sets that comprehensively cover dynamic, physiological, and environmental conditions are used to generate hundreds of gene co-expression networks. By integrating these gene co-expression networks, for a given uncharacterized gene that is of biologist’s interest, the proposed method can be applied to identify the frequent local clusters that consist of this uncharacterized gene. Finally, those frequent local clusters are used for function and disease annotation of this uncharacterized gene. This local tensor clustering algorithm outperformed the competing tensor-based algorithm in both module discovery and running time. We also demonstrated the use of the proposed method on real data of hundreds of gene co-expression data and showed that it can comprehensively characterize the query gene. Therefore, this study provides a new tool for annotating the uncharacterized genes and has great potential to assist clinical genomic diagnostics.

Keywords: local tensor clustering, query gene, gene co-expression network, gene annotation

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Authors: Chul Lee, Seoae Cho, Erich D. Jarvis, Heebal Kim

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Vocal learning, the ability to imitate vocalizations based on auditory experience, is a homoplastic character state observed in different independent lineages of animals such as songbirds, parrots, hummingbirds and human. It has now become possible to perform genome-wide molecular analyses across vocal learners and vocal non-learners with the recent expansion of avian genome data. It was analyzed the whole genomes of human and 48 avian species including those belonging to the three avian vocal learning lineages, to determine if behavior and neural convergence are associated with molecular convergence in divergent species of vocal learners. Analyses of 8295 orthologous genes across bird species revealed 141 genes with amino acid substitutions specific to vocal learners. Out of these, 25 genes have vocal learner specific genetic homoplasies, and their functions were enriched for learning. Several sites in these genes are estimated under convergent evolution and positive selection. A potential role for a subset of these genes in vocal learning was supported by associations with gene expression profiles in vocal learning brain regions of songbirds and human disease that cause language dysfunctions. The key candidate gene with multiple independent lines of the evidences specific to vocal learners was DRD5. Our findings suggest cAMP-based learning pathway in avian vocal learners, indicating molecular homoplastic changes associated with a complex behavioral trait, vocal learning.

Keywords: amino acid substitutions, convergent evolution, positive selection, vocal learning

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Authors: Muhammad Imran, Sarfraz Shafiq, Xiangru Tang

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Alternative splicing (AS) is an important post-transcriptional regulatory mechanism to generate transcripts variability and proteome diversity in plants. Fragrant rice (Oryza sativa L.) has a high economic and nutritional value, and the application of micronutrients regulate 2-acetyl-1-pyrroline (2-AP) production, which is responsible for aroma in fragrant rice. However, no systematic investigation of AS events in response to micronutrients (Zn) has been performed in fragrant rice. Furthermore, the post-transcriptional regulation of genes involved in 2-AP biosynthesis is also not known. In this study, a comprehensive analysis of AS events under two gradients of Zn treatment in two different fragrant rice cultivars (Meixiangzhan-2 and Xiangyaxiangzhan) was performed. A total of 386 and 598 significant AS events were found in Meixiangzhan-2 treated with low and high doses of Zn, respectively. In Xiangyaxiangzhan, a total of 449 and 598 significant AS events were found in low and high doses of Zn, respectively. Go analysis indicated that these genes were highly enriched in physiological processes, metabolism, and cellular process in both cultivars. However, genotype and dose-dependent AS events were also detected in both cultivars. By comparing differential AS (DAS) events with differentially expressed genes (DEGs), we found a weak overlap among DAS and DEGs in both fragrant rice cultivars, indicating that only a few genes are post-transcriptionally regulated in response to Zn treatment. We further report that Zn differentially regulates the expression of 2-AP biosynthesis-related genes in both cultivars, and Zn treatment altered the editing frequency of SNPs in the genes involved in 2-AP biosynthesis. Finally, we showed that epigenetic modifications associated with active gene transcription are generally enriched over 2-AP biosynthesis-related genes. Taken together, our results provide evidence of the post-transcriptional gene regulation in fragrant rice in response to Zn treatment and highlight that the 2-AP biosynthesis pathway may also be post-transcriptionally regulated through epigenetic modifications. These findings will serve as a cornerstone for further investigation to understand the molecular mechanisms of 2-AP biosynthesis in fragrant rice.

Keywords: fragrant rice, 2-acetyl-1-pyrroline, gene expression, zinc, alternative splicing, SNPs

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Authors: T. O. Ajewole, K. S. Olorunmiaye, D. A. Animasaun, M. Okpeku

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Drought impact on the production of food crops for the benefit of mankind cannot be overemphasized. This study shows the different kind of genes expressed at various level of drought regimes on Basella alba and rubra using a real-time PCR machine. The planting was done in the screen house while the gene expression study was carried out in the laboratory. Sandy-loamy soil was collected and four levels of drought regime was used as treatment and a control experiment was set up for the two vegetables. Drought interval of 5, 10, 15 and 20 days were used as treatments while a control experiment which was not starved of water at any point was also set up, five replicates were set up for each treatment. Stress was introduced at 12 Weeks after planting (WAP). From the result of this study, Basella alba shows the highest amplicon size of 34.6 and 52.32 for GmPCS5 and HVA1 respectively which by implication means these genes were expressed the more as the stress period interval increases.

Keywords: water stress, basella alba, basella rubra, HVA1

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Authors: Aktar-Uz-Zaman, M. Tuhina-Khatun, Mohamed Hanafi Musa

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Three rust diseases: leaf (brown) rust caused by Puccinia triticina Eriks, stripe (yellow) rust caused by Puccinia striiformis West, and stem (black) rust caused by Puccinia graminis f. sp. tritici are economically important diseases of wheat in world wide. Yield loss due to leaf rust is 40% in susceptible cultivars. Yield losses caused by the stem rust pathogens in the mid of 20 century reached 20-30% in Eastern and Central Europe and the most virulent stem rust race Ug99 emerged first in Uganda and after that in Kenya, Ethiopia, Yemen, in the Middle East and South Asia. Yield losses were estimated up to 100%, whereas, up to 80% have been reported in Kenya during 1999. In case of stripe rust, severity level has been recorded 60% - 70% as compared to 100% severity of susceptible check in disease screening nurseries in Kenya. Improvement of resistant varieties or cultivars is the sustainable, economical and environmentally friendly approaches for increasing the global wheat production to suppress the rust diseases. More than 68 leaf rust, 49 stripe rust and 53 stem rust resistance genes have been identified in the global wheat cultivars or varieties using different molecular breeding approaches. Among these, Lr1, Lr9, Lr10, Lr19, Lr21, Lr24, Lr25, Lr28, Lr29, Lr34, Lr35, Lr37, Lr39, Lr47, Lr51, Lr3bg, Lr18, Lr40, Lr46, and Lr50 leaf rust resistance genes have been identified by using molecular, enzymatic and microsatellite markers from African, Asian, European cultivars of hexaploid wheat (Triticum aestivum), durum wheat and diploid wheat species. These genes are located on 20, of the 21 chromosomes of hexaploid wheat. Similarly, Sr1, Sr2, Sr24, and Sr3, Sr31 stem rust resistance genes have been recognized from wheat cultivars of Pakistan, India, Kenya, and Uganda etc. A race of P. striiformis (stripe rust) Yr9, Yr18, and Yr29 was first observed in East Africa, Italy, Pakistan and India wheat cultivars. These stripe rust resistance genes are located on chromosomes 1BL, 4BL, 6AL, 3BS and 6BL in bread wheat cultivars. All these identified resistant genes could be used for notable improvement of susceptible wheat cultivars in the future.

Keywords: hexaploid wheat, resistance genes, rust disease, triticum aestivum

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Authors: Mayada Mazher, Ahmed Moustafa, Ahmed Abdellatif

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Background: Autophagy is a eukaryotic, highly conserved catabolic process implicated in many pathophysiologies such as wound healing. Autophagy-associated genes serve as a scaffolding platform for signal transduction of macrophage polarization during the inflammatory phase of wound healing and tissue repair process. In the current study, we report a model for the interplay between autophagy-associated genes and macrophages polarization associated genes. Methods: In silico analysis was performed on 249 autophagy-related genes retrieved from the public autophagy database and gene expression data retrieved from Gene Expression Omnibus (GEO); GSE81922 and GSE69607 microarray data macrophages polarization 199 DEGS. An integrated protein-protein interaction network was constructed for autophagy and macrophage gene sets. The gene sets were then used for GO terms pathway enrichment analysis. Common transcription factors for autophagy and macrophages' polarization were identified. Finally, microRNAs enriched in both autophagy and macrophages were predicated. Results: In silico prediction of common transcription factors in DEGs macrophages and autophagy gene sets revealed a new role for the transcription factors, HOMEZ, GABPA, ELK1 and REL, that commonly regulate macrophages associated genes: IL6,IL1M, IL1B, NOS1, SOC3 and autophagy-related genes: Atg12, Rictor, Rb1cc1, Gaparab1, Atg16l1. Conclusions: Autophagy and macrophages' polarization are interdependent cellular processes, and both autophagy-related proteins and macrophages' polarization related proteins coordinate in tissue remodelling via transcription factors and microRNAs regulatory network. The current work highlights a potential new role for transcription factors HOMEZ, GABPA, ELK1 and REL in wound healing.

Keywords: autophagy related proteins, integrated network analysis, macrophages polarization M1 and M2, tissue remodelling

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Authors: Mona T. Kashef, Omneya M. Helmy

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Multidrug resistant (MDR) bacteria have become a significant public health threat. The prevalence rates, of Gram negative MDR bacteria, are in continuous increase. However, few data are available about these resistant strains. Since, third generation cephalosporins are one of the most commonly used antimicrobials, we set out to investigate the prevalence, different mechanisms and clonal relatedness of multidrug resistance among third generation resistant Gram negative clinical isolates. A total of 114 Gram negative clinical isolates, previously characterized as being resistant to at least one of 3rd generation cephalosporins, were included in this study. Each isolate was tested, using Kirby Bauer disk diffusion method, against its assigned categories of antimicrobials. The role of efflux pump in resistance development was tested by the efflux pump inhibitor-based microplate assay using chloropromazine as an inhibitor. Detecting different aminoglycosides, β-lactams and quinolones resistance genes was done using polymerase chain reaction. The genetic diversity of MDR isolates was investigated using Random Amplification of Polymorphic DNA technique. MDR phenotype was detected in 101 isolates (89%). Efflux pump mediated resistance was detected in 49/101 isolates. Aminoglycosides resistance genes; armA and aac(6)-Ib were detected in one and 53 isolates, respectively. The aac(6)-Ib-cr allele, that also confers resistance to floroquinolones, was detected in 28/53 isolates. β-lactam resistance genes; blaTEM, blaSHV, blaCTX-M group 1 and group 9 were detected in 52, 29, 61 and 35 isolates, respectively. Quinolone resistance genes; qnrA, qnrB and qnrS were detectable in 2, 14, 8 isolates respectively, while qepA was not detectable at all. High diversity was observed among tested MDR isolates. MDR is common among 3rd generation cephalosporins resistant Gram negative bacteria, in Egypt. In most cases, resistance was caused by different mechanisms. Therefore, new treatment strategies should be implemented.

Keywords: gram negative, multidrug resistance, RAPD typing, resistance genes

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Authors: Atena Sadat Hosseini, Mohammadhossein Habibi

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Acute myeloid leukemia (AML) is the most typically diagnosed leukemia. In older adults, AML imposes a dismal outcome. AML originates with a dominant mutation, then adds collaborative, transformative mutations leading to myeloid transformation and clinical/biological heterogeneity. Several chemotherapeutic drugs are used for this cancer. These drugs are naturally associated with several side effects, and finding a more accurate molecular mechanism of these drugs can have a significant impact on the selection and better candidate of drugs for treatment. In this study, we evaluated bortezomibin myeloma cells using bioinformatics analysis and evaluation of RNA-Seq data. Then investigated the molecular pathways proteins- proteins interactions associated with this chemotherapy drug. A total of 658upregulated genes and 548 downregulated genes were sorted.AUF1 (hnRNP D0) binds and destabilizes mRNA, degradation of GLI2 by the proteasome, the role of GTSE1 in G2/M progression after G2 checkpoint, TCF dependent signaling in response to WNT demonstrated in upregulated genes. Besides insulin resistance, AKT phosphorylates targets in the nucleus, cytosine methylation, Longevity regulating pathway, and Signal Transduction of S1P Receptor were related to low expression genes. With respect to this results, HIST2H2AA3, RP11-96O20.4, ZED9, PRDX1, and DOK2, according to node degrees and betweenness elements candidates from upregulated genes. in the opposite side, PYURF, NRSN1, FGF23, UPK3BL, and STAG3 were a prominent role in downregulated genes. Sum up, Using in silico analysis in the present study, we conducted a precise study ofbortezomib molecular mechanisms in myeloma cells. so that we could take further evaluation to discovermolecular cancer therapy. Naturally, more additional experimental and clinical procedures are needed in this survey.

Keywords: myeloma cells, acute myeloid leukemia, bioinformatics analysis, bortezomib

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Authors: Jie Lin, Yiwen Pan, Li Zhang, Zhangyong Xia

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Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids, immune cells, and extracellular matrix in the arterial walls. This pathological process can lead to the formation of plaques that can obstruct blood flow and trigger various cardiovascular diseases such as heart attack and stroke. The underlying molecular mechanisms still remain unclear, although many studies revealed the dysfunction of endothelial cells, recruitment and activation of monocytes and macrophages, and the production of pro-inflammatory cytokines and chemokines in atherosclerosis. This study aimed to identify hub genes involved in the progression of atherosclerosis and to analyze their biological function in silico, thereby enhancing our understanding of the disease’s molecular mechanisms. Through the analysis of microarray data, we examined the gene expression in media and neo-intima from plaques, as well as distant macroscopically intact tissue, across a cohort of 32 hypertensive patients. Initially, 112 differentially expressed genes (DEGs) were identified. Subsequent immune infiltration analysis indicated a predominant presence of 27 immune cell types in the atherosclerosis group, particularly noting an increase in monocytes and macrophages. In the Weighted gene co-expression network analysis (WGCNA), 10 modules with a minimum of 30 genes were defined as key modules, with blue, dark, Oliver green and sky-blue modules being the most significant. These modules corresponded respectively to monocyte, activated B cell, and activated CD4 T cell gene patterns, revealing a strong morphological-genetic correlation. From these three gene patterns (modules morphology), a total of 2509 key genes (Gene Significance >0.2, module membership>0.8) were extracted. Six hub genes (CD36, DPP4, HMOX1, PLA2G7, PLN2, and ACADL) were then identified by intersecting 2509 key genes, 102 DEGs with lipid-related genes from the Genecard database. The bio-functional analysis of six hub genes was estimated by a robust classifier with an area under the curve (AUC) of 0.873 in the ROC plot, indicating excellent efficacy in differentiating between the disease and control group. Moreover, PCA visualization demonstrated clear separation between the groups based on these six hub genes, suggesting their potential utility as classification features in predictive models. Protein-protein interaction (PPI) analysis highlighted DPP4 as the most interconnected gene. Within the constructed key gene-drug network, 462 drugs were predicted, with ursodeoxycholic acid (UDCA) being identified as a potential therapeutic agent for modulating DPP4 expression. In summary, our study identified critical hub genes implicated in the progression of atherosclerosis through comprehensive bioinformatic analyses. These findings not only advance our understanding of the disease but also pave the way for applying similar analytical frameworks and predictive models to other diseases, thereby broadening the potential for clinical applications and therapeutic discoveries.

Keywords: atherosclerosis, hub genes, drug prediction, bioinformatics

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Authors: Sabin Aslam, Sultan Habibullah Khan, James G. Thomson, Abhaya M. Dandekar

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Losses due to viral diseases are posing a serious threat to crop production. A quick breakdown of resistance to viruses like Cotton Leaf Curl Virus (CLCuV) demands the application of a proficient technology to engineer durable resistance. Gene stacking has recently emerged as a potential approach for integrating multiple genes in crop plants. In the present study, recombinase technology has been used for site-specific gene stacking. A target vector (pG-Rec) was designed for engineering a predetermined specific site in the plant genome whereby genes can be stacked repeatedly. Using Agrobacterium-mediated transformation, the pG-Rec was transformed into Coker-312 along with Nicotiana tabacum L. cv. Xanthi and Nicotiana benthamiana. The transgene analysis of target lines was conducted through junction PCR. The transgene positive target lines were used for further transformations to site-specifically stack two genes of interest using Bxb1 and PhiC31 recombinases. In the first instance, Cas9 driven by multiplex gRNAs (for Rep gene of CLCuV) was site-specifically integrated into the target lines and determined by the junction PCR and real-time PCR. The resulting plants were subsequently used to stack the second gene of interest (AVP3 gene from Arabidopsis for enhancing cotton plant growth). The addition of the genes is simultaneously achieved with the removal of marker genes for recycling with the next round of gene stacking. Consequently, transgenic marker-free plants were produced with two genes stacked at the specific site. These transgenic plants can be potential germplasm to introduce resistance against various strains of cotton leaf curl virus (CLCuV) and abiotic stresses. The results of the research demonstrate gene stacking in crop plants, a technology that can be used to introduce multiple genes sequentially at predefined genomic sites. The current climate change scenario highlights the use of such technologies so that gigantic environmental issues can be tackled by several traits in a single step. After evaluating virus resistance in the resulting plants, the lines can be a primer to initiate stacking of further genes in Cotton for other traits as well as molecular breeding with elite cotton lines.

Keywords: cotton, CRISPR/Cas9, gene stacking, genome editing, recombinases

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Authors: Aileen Editha

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The legal recognition of property rights over human genes is a divisive topic to which there is no resolution. As a frequently discussed topic, scholars and practitioners often highlight the inadequacies of a proprietary regime. However, little has been said in regard to the nature of human genetic materials (HGMs). This paper proposes approaching the issue of property over HGMs from an alternative perspective that looks at the personal and social value and valuation of HGMs. This paper will highlight how the unique and unresolved status of HGMs is incompatible with the main tenets of property and, consequently, contributes to legal ambiguity and uncertainty in the regulation of property rights over human genes. HGMs are perceived as part of nature and a free-for-all while also being within an individual’s private sphere. Additionally, it is also considered to occupy a unique “not-private-nor-public” status. This limbo-like position clashes with property’s fundamental characteristic that relies heavily on a clear public/private dichotomy. Moreover, as property is intrinsically linked to the legal recognition of one’s personhood, this irresolution benefits some while disadvantages others. In particular, it demands the publicization of once-private genes for the “common good” but subsequently encourages privatization (through labor) of these now-public genes. This results in the gain of some (already privileged) individuals while enabling the disenfranchisement of members of minority groups, such as Indigenous communities. This paper will discuss real and intellectual property rights over human genes, such as the right to income or patent rights, in Canada and the US. This paper advocates for a sui generis approach to governing rights and interests over human genes that would not rely on having a strict public/private dichotomy. Not only would this improve legal certainty and clarity, but it would also alleviate—or, at the very least, minimize—the role that the current law plays in further entrenching existing systemic inequalities. Despite the specificity of this topic, this paper argues that there are broader lessons to be learned. This issue is an insightful case study on the interconnection of various principles in law, society, and property, and what must be done when discordance between one or more of those principles has detrimental societal outcomes. Ultimately, it must be remembered that property is an adaptable and malleable instrument that can be developed to ensure it contributes to equity and flourishing.

Keywords: property rights, human genetic materials, critical legal scholarship, systemic inequalities

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Authors: Sontana Mimapan, Phattarawadee Wattanasuntorn, Phanom Saijit

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Aflatoxin contamination in maize, one of several agriculture crops grown for livestock feeding, is still a problem throughout the world mainly under hot and humid weather conditions like Thailand. In this study Aspergillus flavus (A. Flavus), the key fungus for aflatoxin production especially aflatoxin B1 (AFB1), isolated from naturally infected maize were identified and characterized according to colony morphology and PCR using ITS, Beta-tubulin and calmodulin genes. The strains were analysed for the presence of four aflatoxigenic biosynthesis genes in relation to their capability to produce AFB1, Ver1, Omt1, Nor1, and aflR. Aflatoxin production was then confirmed using immunoaffinity column technique. A loop-mediated isothermal amplification (LAMP) was applied as an innovative technique for rapid detection of target nucleic acid. The reaction condition was optimized at 65C for 60 min. and calcein flurescent reagent was added before amplification. The LAMP results showed clear differences between positive and negative reactions in end point analysis under daylight and UV light by the naked eye. In daylight, the samples with AFB1 producing A. Flavus genes developed a yellow to green color, but those without the genes retained the orange color. When excited with UV light, the positive samples become visible by bright green fluorescence. LAMP reactions were positive after addition of purified target DNA until dilutions of 10⁻⁶. The reaction products were then confirmed and visualized with 1% agarose gel electrophoresis. In this regards, 50 maize samples were collected from dairy farms and tested for the presence of four aflatoxigenic biosynthesis genes using LAMP technique. The results were positive in 18 samples (36%) but negative in 32 samples (64%). All of the samples were rechecked by PCR and the results were the same as LAMP, indicating 100% specificity. Additionally, when compared with the immunoaffinity column-based aflatoxin analysis, there was a significant correlation between LAMP results and aflatoxin analysis (r= 0.83, P < 0.05) which suggested that positive maize samples were likely to be a high- risk feed. In conclusion, the LAMP developed in this study can provide a simple and rapid approach for detecting AFB1 producing A. Flavus genes from maize and appeared to be a promising tool for the prediction of potential aflatoxigenic risk in livestock feedings.

Keywords: Aflatoxin B1, Aspergillus flavus genes, maize, loop-mediated isothermal amplification

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Authors: Taru Singh, Shukla Das, V. G. Ramachandran

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Objectives: To develop SYBR green based real-time PCR assay to detect carbapenemases (NDM, IMP) genes in E. coli. Methods: A total of 40 E. coli from stool samples were tested. Six were previously characterized as resistant to carbapenems and documented by PCR. The remaining 34 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial RNA was extracted using manual method. The real-time PCR was performed using the Light Cycler III 480 instrument (Roche) and specific primers for each carbapenemase target were used. Results: Each one of the two carbapenemase gene tested presented a different melting curve after PCR amplification. The melting temperature (Tm) analysis of the amplicons identified was as follows: blaIMP type (Tm 82.18°C), blaNDM-1 (Tm 78.8°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. Conclusions: The new assay was able to detect the presence of two different carbapenemase gene type by real-time PCR.

Keywords: resistance, b-lactamases, E. coli, real-time PCR

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Authors: Amal Saafan, Kareem Al Sofy, Sameh AbdelGhani, Magdy Amin

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Background: Acinetobacter spp. resistance towards β-lactam antibiotics is mediated mainly by different classes of β-lactamases production; detection of some genes responsible for production of β-lactamases is the objective of the study. Methods: One hundred fifty bacterial isolates were recovered from blood, sputum, and urine specimens from different hospitals in Egypt. Sixty-nine isolate were identified as Acinetobacter baumannii using traditional biochemical tests, CHROM agar, MicroScan and PCR amplification of blaoxa-51like gene. Acinetobacterbaumannii isolates were grouped into carbapenem resistant group (GP1), cefotaxime, ceftazidime and cefoxitin resistant group (GP2) and carbapenem and cephalosporin non-resistant group (GP3). Carbapenemase activity was screened using modified Hodge test (MHT) for GP1.Metallo-β-lactamases screening was performed for MHT positive isolates using double disk synergy test (DDST) and combined disk test (CDT). Amp C activity was screened using Amp C disk test with Tris-EDTA, DDST, and CDT for GP2. Finally, PCR amplification of blaoxa-51like, blaoxa-23like, blaIMP-like, blaVIM-like, and blaADC-like genes was performed for isolates that showed, at least, two positive results of three for both AmpC and carbapenemases phenotypic screening tests (obvious activity), in addition to GP3 (for comparison). Detection of blaoxa-51like and blaADC-like genes preceded by ISAba1 was also performed. Results: Antibiogram of 69 pure Acinetobacter baumannii isolates resulted in 57, 64, and 2 isolates enrolled into GP1, GP2, and GP3, respectively. Carbapenemase activity was shown by 49(85.9%) isolate using MHT. Metallo-β-lactamases screening revealed 32(65.3%) and 35(71.4%) using DDST and CDT, respectively.AmpC activity was shown by 43(67.2%) and 50 (78.1%) isolates using AmpC disk test with Tris-EDTA, and both DDST and CDT, respectively. Twenty-seven isolates showed obvious activity, all of them (100%) were harboring blaoxa-51like and blaADC-like genes, while blaoxa-23like, blaIMP-like andblaVIM-like genes were harbored by 23(85.2%), 9 (33.%) and no isolate respectively. Only 12 (44.4%) isolates harbored blaoxa-51like and blaADC-like genes preceded by ISAba1. GP3 isolates showed only positive blaoxa-51like and blaADC-like genes. Conclusion: It is not possible to correlate resistance with presence of blaoxa-51like and blaADC-like genes and presence of ISAba1 was immediate as transcriptional promoter. A blaoxa-23like gene played an important role in carbapenem resistance when compared with blaIMP-like and blaVIM-like gene.

Keywords: acinetobacter, beta-lactams, resistance, antimicrobial agents

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Authors: Vijay Kumar Singh, Pooja Kushwaha, Prabhat Ranjan, Krishna Kumar Ojha, Jitendra Kumar

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Present day high-throughput genomic technologies, NGS/microarrays, are producing large volume of data that require improved analysis methods to make sense of the data. The correlation between genes and samples has been regularly used to gain insight into many biological phenomena including, but not limited to, co-expression/co-regulation, gene regulatory networks, clustering and pattern identification. However, presence of outliers and violation of assumptions underlying Pearson correlation is frequent and may distort the actual correlation between the genes and lead to spurious conclusions. Here, we report a method to measure the strength of association between genes. The method assumes that the expression values of a gene are Bernoulli random variables whose outcome depends on the sample being probed. The method considers the two genes as uncorrelated if the number of sample with same outcome for both the genes (Ns) is equal to certainly expected number (Es). The extent of correlation depends on how far Ns can deviate from the Es. The method does not assume normality for the parent population, fairly unaffected by the presence of outliers, can be applied to qualitative data and it uses the binomial distribution to assess the significance of association. At this stage, we would not claim about the superiority of the method over other existing correlation methods, but our method could be another way of calculating correlation in addition to existing methods. The method uses binomial distribution, which has not been used until yet, to assess the significance of association between two variables. We are evaluating the performance of our method on NGS/microarray data, which is noisy and pierce by the outliers, to see if our method can differentiate between spurious and actual correlation. While working with the method, it has not escaped our notice that the method could also be generalized to measure the association of more than two variables which has been proven difficult with the existing methods.

Keywords: binomial distribution, correlation, microarray, outliers, transcriptome

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Authors: M. Sunitha, B. Nithyavani, Mathew Yohannan, S. Thiruvieni Balajji, M. A. Rathi, C. Arul Raj, P. Ragavendran, V. K. Gopalkrishnan

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Establishment of an early and reliable biomarker for ovarian carcinogenesis whose expression can be monitored through noninvasive techniques will enable early diagnosis of cancer. Carbonic anhydrases (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. In von Hippel-Lindau (VHL)-defective tumors, the cell surface transmembrane carbonic anhydrase (CA) CA XI and CA XII genes are overexpressed because of the absence of pVHL. These enzymes are involved in causing a hypoxia condition, thereby providing an environment for metastasis. Aberrant expression of the facilitative glucose transporter GLUT I is found in a wide spectrum of epithelial malignancies. Studying the mRNA expression of CA IX, CA XII and Glut I isozymes in ovarian cancer cell lines (OAW-42 and PA-1) revealed the expression of these hypoxia genes. Immunohistochemical staining of carbonic anhydrases was also performed in 40 ovarian cancer tissues. CA IX and CA XII were expressed at 540 bp and 520 bp in OAW42, PA1 in ovarian cancer cell lines. GLUT-1 was expressed at 325bp in OAW 42, PA1 genes in ovarian cancer cell lines. Immunohistochemistry revealed high to moderate levels of expression of these enzymes. The immuostaining was seen predominantly on the cell surface membrane. The study concluded that these genes CA IX, CA XII and Glut I are expressed under hypoxic condition in tumor cells. From the present results expression of CA IX, XII and Glut I may represent potential targets in ovarian cancer therapy.

Keywords: ovarian cancer, carbonic anhydrase IX, XII, Glut I, tumor markers

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Authors: Rozhgar A. Khailany, Mehri Igci, Emine Bayraktar, Sakip Erturhan, Metin Karakok, Ahmet Arslan

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Kidney cancer is the most lethal urological cancer accounting for 3% of adult malignancies. VHL, a tumor-suppressor gene, is best known to be associated with renal cell carcinoma (RCC). The VHL functions as negative regulator of hypoxia inducible factors. Recent sequencing efforts have identified several novel frequent mutations of histone modifying and chromatin remodeling genes in ccRCC (clear cell RCC) including PBRM1 and SETD2. The PBRM1 gene encodes the BAF180 protein, which involved in transcriptional activation and repression of selected genes. SETD2 encodes a histone methyltransferase, which may play a role in suppressing tumor development. In this study, RNAs of 30 paired tumor and normal samples that were grouped according to the types of kidney cancer and clinical characteristics of patients, including gender and average age were examined by RT-PCR, SSCP and sequencing techniques. VHL, PBRM1 and SETD2 expressions were relatively down-regulated. However, statistically no significance was found (Wilcoxon signed rank test, p > 0.05). Interestingly, no mutation was observed on the contrary of previous studies. Understanding the molecular mechanisms involved in the pathogenesis of RCC has aided the development of molecular-targeted drugs for kidney cancer. Further analysis is required to identify the responsible genes rather than VHL, PBRM1 and SETD2 in kidney cancer.

Keywords: kidney cancer, molecular biomarker, expression analysis, mutation screening

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Authors: Maricarmen Herrera, Pierre Chymkowitch, Joe Robertson, Jens Eriksson, Jorrit Enserink

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tRNA genes are transcribed by RNA polymerase III. During recent years, it has become clear that tDNA transcription fluctuates during the cell cycle. However, the mechanism by which the cell cycle controls the amplitude of tDNA transcription remains unknown. We found that the cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to sharply increase tRNA synthesis during a brief interval in the cell cycle. We show that Cdk1 promotes the interaction of TFIIIB with TFIIIC, that it stimulates the recruitment of TFIIIC to tRNA genes, that it prevents the formation of an overly stable TFIIIB-tDNA complex and that it augments the dynamics of RNA polymerase III. Furthermore, we identify Bdp1 as a novel Cdk1 substrate, and phosphorylation of Bdp1 is required for the cell cycle-dependent increase in tDNA transcription. In addition, we show that phosphorylation of the Cdk1 substrate Nup60 mediates formation of a Nup60-Nup2 complex at tRNA genes, which is also required for cell cycle-dependent tDNA transcription. Together, our findings indicate that Cdk1 activity gates tRNA synthesis by regulating the dynamics of the TFIIIB-TFIIIC-RNAPIII complex, and that it may promote the formation of a nuclear pore microenvironment conducive to efficient tDNA transcription.

Keywords: Cdk1, cell cycle, RNAPIII machinery, tRNA

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Authors: Othman Elmahdy Othman

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The objectives of this study were to identify polymorphisms in the STAT5A using Restriction Fragment Length Polymorphism and DGAT1 using Single-Strand Conformation Polymorphism genes among three Egyptian goat breeds (Barki, Zaraibi, and Damascus) as well as investigate the effect of their genotypes on milk composition traits of Zaraibi goats. One hundred and fifty blood samples were collected for DNA extraction, 60 from Zaraibi, 40 from Damascus and 50 from Barki breeds. Fat, protein and lactose percentages were determined in Zaraibi goat milk using an automatic milk analyzer. Two genotypes, CC and CT (for STAT5A) and C-C- and C-C+ (for DGAT1), were identified in the three Egyptian goat breeds with different frequencies. The associations between these genotypes and milk fat, protein and lactose were determined in Zaraibi breed. The results showed that the STAT5A genotypes had significant effects on milk yield, protein, fat and lactose with the superiority of CT genotype over CC. Regarding DGAT1 polymorphism, the result showed the only association between it with milk fat where the animals with C-C+ genotype had greater milk fat than animals possess C-C- genotype. The association of combined genotypes with milk trait declared that the does with heterozygous genotypes for both genes are preferred than does with homozygous genotypes where the animals with CTC-C+ have more milk yield, fat and protein than those with CCC-C- genotype. In conclusion, the result showed that C/T and C-/C+ SNPs of STAT5A and DGAT1 genes respectively may be useful markers for assisted selection programs to improve goat milk composition

Keywords: DGAT1, genetic polymorphism, milk trait, STAT5A

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Authors: M. Moaeen-ud-Din, G. Bilal, M. Yaqoob

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Identification of genes of importance regarding production traits in buffalo is impaired by a paucity of genomic resources. Choice to fill this gap is to exploit data available for cow. The cross-species application of comparative genomics tools is potential gear to investigate the buffalo genome. However, this is dependent on nucleotide sequences similarity. In this study gene diversity between buffalo and cattle was determined by using 86 gene orthologues. There was about 3% difference in all genes in term of nucleotide diversity; and 0.267±0.134 in amino acids indicating the possibility for successfully using cross-species strategies for genomic studies. There were significantly higher non synonymous substitutions both in cattle and buffalo however, there was similar difference in term of dN – dS (4.414 vs 4.745) in buffalo and cattle respectively. Higher rate of non-synonymous substitutions at similar level in buffalo and cattle indicated a similar positive selection pressure. Results for relative rate test were assessed with the chi-squared test. There was no significance difference on unique mutations between cattle and buffalo lineages at synonymous sites. However, there was a significance difference on unique mutations for non synonymous sites indicating ongoing mutagenic process that generates substitutional mutation at approximately the same rate at silent sites. Moreover, despite of common ancestry, our results indicate a different divergent time among genes of cattle and buffalo. This is the first demonstration that variable rates of molecular evolution may be present within the family Bovidae.

Keywords: buffalo, cattle, gene diversity, molecular evolution

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Authors: Masoud Soltanialvar, Ali Bagherpour

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Documented cases of human infection with H9N2 avian influenza viruses, first detected in 1999 in Hong Kong and China, indicate that these viruses can be directly transmitted from birds to humans. In this study, we characterized the mutation in the Hemagglutinin (HA) genes and proteins that correlates with a shift in affinity of the Hemagglutinin (HA) protein from the “avian” type sialic receptors to the “human” type in 10 Iranian isolates. We delineated the genomes and receptor binding profile of HA gene of some field isolates and established their phylogenetic relationship to the other Asian H9N2 sub lineages. A total of 1200 tissue samples collected from 40 farms located in various states of Iran during 2008 – 2010 as part of a program to monitor Avian Influenza Viruses (AIV) infection. To determine the genetic relationship of Iranian viruses, the Hemagglutinin (HA) genes from ten isolates were amplified and sequenced (by RT-PCR method). Nucleotide sequences (orf) of the (HA) genes were used for phylogenetic tree construction. Deduced amino acid sequences showed the presence of L226 (234 in H9 numbering) in all ten Iranian isolates which indicates a preference to binding of α (2–6) sialic acid receptors, so these Iranian H9N2 viruses have the potential to infect human beings. These isolates showed high degree of homology with 2 human H9N2 isolates A/HK/1073/99, A/HK/1074/99. Phylogenetic analysis of showed that all the HA genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage which had contributed as donor of six internal genes to H5N1 highly pathogenic avian influenza. The results of this study indicated that all Iranian viruses have the potential to emerge as highly pathogenic influenza virus, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.

Keywords: influenza virus, hemagglutinin, neuraminidase, Iran

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Authors: Mehdi Nasr-Esfahani, Leila Mohammad Bagheri, Ava Nasr-Esfahani

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Root and collar rot disease caused by Phytophthora capsici (Leonian) is one of the most serious diseases in pepper, Capsicum annuum L. In this study, a diverse collection of 37 commercial edible and ornamental pepper genotypes infected with P. capsici were investigated for biomass parameters and enzymatic activity of peroxidase or peroxide reductases (EC), superoxide dismutase (SOD), polyphenol oxidase (PPOs), catalase (CAT) and phenylalanine ammonia-lyase (PAL). Seven candidate DEG genes were also evaluated on resistant and susceptible pepper cultivars, through measuring product formation, using spectrophotometry and real-time polymerase chain reaction. All the five enzymes and seven defense-gene candidates were up-regulated in all inoculated pepper accessions to P. capsici. But, the enzymes and DEG genes were highly expressed in resistant cv. 19OrnP-PBI, 37ChillP-Paleo, and “23CherryP-Orsh". The expression level of enzymes were 1.5 to 5.6-fold higher in the resistant peppers, than the control non-inoculated genotypes. Also, the transcriptional levels of related candidate DEG genes were 3.16 to 5.90-fold higher in the resistant genotypes. There was a direct and high correlation coefficient between resistance, bio-mass parameters, enzymatic activity, and resistance gene expression. The related enzymes and candidate genes expressed herein will provide a basis for further gene cloning and functional verification studies, and also will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

Keywords: AP2/ERF, cDNA, enzymes, MIP gene, q-RTPCR, XLOC

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Authors: Andrey V. Khromov, Antonida V. Makhotenko, Ekaterina A. Snigir, Svetlana S. Makarova, Natalia O. Kalinina, Valentin V. Makarov, Mikhail E. Taliansky

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Due to its simplicity, CRISPR/Cas9 has become widely used and capable of inducing mutations in the genes of organisms of various kingdoms. The aim of this work was to develop applications for the efficient modification of DNA coding sequences of phytoene desaturase (PDS), coilin and vacuolar invertase (Solanum tuberosum) genes, and to develop a new nanoparticles carrier efficient technology to deliver the CRISPR/Cas9 system for editing the plant genome. For each of the genes - coilin, PDS and vacuolar invertase, five single RNA guide (sgRNAs) were synthesized. To determine the most suitable nanoplatform, two types of NP platforms were used: magnetic NPs (MNPS) and gold NPs (AuNPs). To test the penetration efficiency, they were functionalized with fluorescent agents - BSA * FITS and GFP, as well as labeled Cy3 small-sized RNA. To measure the efficiency, a fluorescence and confocal microscopy were used. It was shown that the best of these options were AuNP - both in the case of proteins and in the case of RNA. The next step was to check the possibility of delivering components of the CRISPR/Cas9 system to plant cells for editing target genes. AuNPs were functionalized with a ribonucleoprotein complex consisting of Cas9 and corresponding to target genes sgRNAs, and they were biolistically bombarded to axillary buds and apical meristems of potato plants. After the treatment by the best NP carrier, potato meristems were grown to adult plants. DNA isolated from this plants was sent to a preliminary fragment of the analysis to screen out the non-transformed samples, and then to the NGS. The present work was carried out with the financial support from the Russian Science Foundation (grant No. 16-16-04019).

Keywords: biobombardment, coilin, CRISPR/Cas9, nanoparticles, NPs, PDS, sgRNA, vacuolar invertase

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Authors: Rudzani Manafe, Ezekiel Green

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Brucella, has many different virulence factors that act as a causative agent of brucellosis, depending on the environment and other factors, some factors may play a role more than others during infection and as a result, play a role in becoming a causative agent for pathogenesis. Brucella melitensis and Brucella abortus are considered to be pathogenic to humans. The genetic regularity of nine potential causes of virulence of two Brucella species in Eastern Cape livestock have been examined. A hundred and twenty isolates obtained from Molecular Pathogenesis and Molecular Epidemiology Research Group (MPMERG) were used for this study. All isolates were grown on Brucella agar medium. Nine primer pairs were used for the detection of virB2, virB5, vceC, btpA, btpB, prpA, betB, bpe275, and bspB virulence factors using Polymerase chain reaction (PCR). Approximately 100% was observed for genes BecC and BetB from B. arbotus. While the lowest gene observed was PrpA at 4.6% from B. arbotus. BetB was detected in 34.7%, while virB2 and prpA (0%) were not detected in B. melitensis. The results from this research suggest that most isolates of Brucella have virulence-related genes associated with disease pathogenesis. Finally, our findings showed that Brucella strains in the Eastern Cape Province are extremely virulent as virulence characteristics exist in most strains investigated.

Keywords: putative virulence genes, brucella, polymerase chain reaction, milk

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