Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Molecular characterization of Pasteurella multocida isolates obtained from poultry, ruminant, cats and dogs using RAPD and REP-PCR analysis123132370910.22099/mbrc.2016.3709ENHesamaddin Shirzad-AskiDepartment of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.Mohammad TabatabaeiDepartment of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran.0000-0002-3743-6583Journal Article20160223In the present study, Randomly Amplified Polymorphic DNA (RAPD) and Repetitive Extragenic Palindromic sequence-based Polymerase Chain Reaction (REP-PCR) were used to characterize 131 isolates of <em>Pasteurella</em> <em>multocida,</em> originating from different healthy and diseased animal species obtained from several geographical regions of Iran. The RAPD and REP-PCR generated amplified products in the range of 300 to 3400 bp and 200 to 2850 bp, respectively. Among all of the <em>P. multocida</em> isolates, cluster analysis revealed that 63 clusters and nine untypable isolates and 81 clusters and six untypable isolates were produced with RAPD and REP-PCR methods, respectively. The results indicated that the REP-PCR method showed a slightly higher level of discrimination power in differentiating of <em>P. multocida</em> isolates as compared with RAPD. The results showed that a considerable level of genetic diversity exists among <em>P. multocida</em> isolates even in the isolates with the same animal or geographical origins. There was no host- and region-specific pattern. In addition, the isolates obtained from the healthy and diseased animal did not reveal any correlation genotypic profiles, which could be supported by the hypothesis that <em>P. multocida</em> is a strictly opportunistic pathogen. In conclusion, because of a large amount of genetic heterogeneityin the<em> P. multocida</em> isolates, Pasteurellosis may be caused by different clones in the same herd or animal.Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M133142371810.22099/mbrc.2016.3718ENArezoo TavakoliIslamic azad university,Eghlid branch,Fars,Iran.Ainon HamzahSchool of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, MalaysiaAmir RabuSchool of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, MalaysiaJournal Article20151025<span style="font-family: 'Times New Roman','serif'; font-size: 10pt; mso-fareast-font-family: SimSun; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: AR-SA;">In this study, benzoate dioxygenase from <em>Rhodococcus ruber</em> UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (<em>ben</em>A gene) from <em>Rhodococcus ruber</em> UKMP-5M was then expressed, purified, characterized, The <em>ben</em>A gene was amplified (642 bp), and the product was cloned into a pGEM-T vector.The recombinant plasmid pGEMT-benA was digested by double restriction enzymes <em>BamH</em>I and <em>Hind</em>III to construct plasmid pET28b-benA and was then ligated into <em>Escherichia coli</em> BL21 (DE3). The recombinant <em>E. coli</em> was induced with 0.5 mM isopropyl <em>β</em>-<em>D</em>-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (V<sub>max</sub>) and Michaelis constant (K<sub>m</sub>) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).</span>Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production143155374310.22099/mbrc.2016.3743ENFatemeh AzadianDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, IranArastoo Badoei-dalfardDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, IranAbdolhamid Namaki-ShoushtariDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, IranMehdi HassanshahianDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran0000-0001-9899-7168Journal Article20160228A thermophilic strain AMF-07, hydrolyzing carboxymethylcellulose (CMC) was isolated from Kerman hot spring and was identified as Bacillus licheniformis based on 16S rRNA sequence homology. The carboxymethylcellulase (CMCase) enzyme produced by the B. licheniformis was purified by (NH4)2SO4 precipitation, ion exchange and gel filtration chromatography. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 37 kDa. The CMCase enzyme was highly active and stable over broad ranges of temperature (40-80 ºC), pH (6.0-10.0) and NaCl concentration (10-25%) with an optimum at 70 ºC, pH 9.0 and 20% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high activity in the presence of cyclohexane (134%) and chloroform (120%). Saccharification of rice bran and wheat bran by the CMCase enzyme resulted in respective yields of 24 and 32 g L-1 reducing sugars. The enzymatic hydrolysates of rice bran were then used as the substrate for ethanol production by Saccharomyces cerevisiae. Fermentation of cellulosic hydrolysate using S. cerevisiae, reached maximum ethanol production about 0.125 g g-1 dry substrate (pretreated wheat bran). Thus, the purified cellulase from B. licheniformis AMF-07 utilizing lignocellulosic biomass could be greatly useful to develop industrial processes.Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences156166375210.22099/mbrc.2016.3752ENAvijit PatraDepartment of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal, IndiaAnjan MondalDepartment of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal, IndiaSayani BanerjeeDepartment of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal, IndiaHarresh AdikesavaluDepartment of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal, IndiaSiddhartha Narayan JoardarDepartment of Veterinary Microbiology, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata, West Bengal, IndiaThangapalam AbrahamDepartment of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, Chakgaria, Kolkata, West Bengal, IndiaJournal Article20151106The present study characterized<em> Argulus</em> spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among <em>Argulus</em> spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had <em>Argulus</em> infection. The parasitic frequency index (PFI) was observed to be high (20%) in <em>Hypophthalmichthys molitrix</em> and <em>Labeo bata</em>. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five <em>A. bengalensis </em>(KF583878<em>, </em>KF192316,KM016968,KM016969,andKM016970) and one <em>A. siamensis</em> (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of <em>Argulus</em> spp. of NCBI GenBank database. Among the Indian <em>Argulus</em> spp. the sequence homology was 87–100%. Evolutionary pair-wise distances between Indian <em>Argulus</em> spp. and other <em>Argulus </em>spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (<em>Argulus </em>spp.). All <em>Argulus bengalensis</em> clustered together and <em>A. siamensis </em>(KF583879) was closely related to <em>Argulus</em> sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian <em>Argulus</em> species. Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Microsatellite (SSR) amplification by PCR usually led to polymorphic bands: Evidence which shows replication slippage occurs in extend or nascent DNA strands167174378910.22099/mbrc.2016.3789ENAbasalt Hossienzadeh-ColagarDepartment of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, IranMohammad Javad HaghighatniaDepartment of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, IranZahra AmiriDepartment of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, IranMaryam MohadjeraniDepartment of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, IranMajid TafrihiDepartment of Molecular and Cell Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Mazandaran, IranJournal Article20160410Microsatellites or simple sequence repeats (SSRs) are very effective molecular markers in population genetics, genome mapping, taxonomic study and other large-scale studies. Variation in number of tandem repeats within microsatellite refers to simple sequence length polymorphism (SSLP); but there are a few studies that are showed SSRs replication slippage may be occurred during <em>in vitro</em> amplification which are produced ‘stutter products’ differing in length from the main products. The purpose of this study is introducing a reliable method to realize SSRs replication slippage. At first, three unique primers designed to amplify SSRs loci in the great gerbil (<em>Rhombomys opimus</em>) by PCR. Crush and soak method used to isolate interesting DNA bands from polyacrylamide gel. PCR products analyzed using by sequencing methods. Our study has been shown that <em>Taq</em> DNA polymerase slipped during microsatellite <em>in vitro</em> amplification which led to insertion or deletion of repeats in sense or antisense DNA strands. It is produced amplified fragments with various lengths in gel electrophoresis showed as ‘stutter bands’. Thus, in population studies by SSRs markers recommend that replication slippage effects and stutter bands have been considered.Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Structural insights into the effects of charge-reversal substitutions at the surface of horseradish peroxidase175192382310.22099/mbrc.2016.3823ENLeila NavapourBiophysics and Computational Biology Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, IranNavid MogharrabBiophysics and Computational Biology Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran
Institute of Biotechnology, Shiraz University, Shiraz, IranJournal Article20160709Horseradish peroxidase (HRP), has gained significant interests in biotechnology, especially in biosensor field and diagnostic test kits. Hence, its solvent-exposed lysine residues 174, 232, and 241 have been frequently modified with the aim of improving its stability and catalytic efficiency. In this computational study, we investigated the effects of Lys-to-Glu substitutions on HRP structure to model charge-reversal manipulations at the enzyme surface. Simulation results implied that upon these substitutions, the number of stable hydrogen bonds and α-helical content of HRP are increased and the proximal Ca<sup>2+</sup> binding pocket becomes more integrated. The results revealed that although Glu174-heme hydrogen bond is lost after mutation, formation of a new hydrogen bonding network contributes to the stability of heme-protein linkage. Together, it may be concluded that these substitutions enhance the stability of the protein moiety as well as the heme-protein non-covalent interactions. In the enzyme active site, we observed increased accessibility of peroxide binding site and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the bottleneck entry of the peroxide-binding site has become wider and more flexible upon substitutions. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is more extended in mutated enzyme. These observations suggest that the reactivity of the enzyme to its substrates has increased. Together, the results of this simulation study could provide possible structural clues to explain those experimental observations in which the protein stability achieved upon manipulation of charge distribution on protein surface.Shiraz University PressMolecular Biology Research Communications2322-181X5320160901Transcript levels of phytoene desaturase gene in Dunaliella salina Teod. as affected by PbS nanoparticles and light intensity193199382910.22099/mbrc.2016.3829ENAli MoradshahiDepartment of Biology, College of Sciences, Shiraz University, Shiraz, IranHajar ZamaniDepartment of Biology, College of Sciences, Shiraz University, Shiraz, IranJournal Article20160815Phytoene synthase (Psy) and Phytoene desaturase (Pds) are the first two regulatory enzymes in the carotenoids biosynthetic pathway. The genes <em>Psy</em> and <em>Pds</em> are under transcriptional control in many photosynthetic organisms. In the present study, using quantitative real time- PCR (qRT-PCR), the effects of uncoated and gum-Arabic coated PbS nanoparticles (GA-coated PbS NPs) and light intensity on the mRNA levels of <em>Pds </em>were investigated. Relative to mRNA level of <em>Pds</em> at 100 µmol photon m<sup>-2</sup> s<sup>-1</sup> light intensity (control culture), 2.2-fold increase in transcript levels occurred after 12 h of exposure to higher light intensity, which is significantly (P<0.05) different compared to control. After 48 h of exposure, the mRNA level of <em>Pds</em> was reduced to that in control. This indicates that light intensity regulates <em>Pds </em>at the mRNA level. In the presence of uncoated and GA-coated PbS NPs, the transcript levels of <em>Pds </em>were decreased over time, with uncoated PbS NPs having more inhibitory effects on mRNA levels compared to GA-coated PbS NPs. This shows that PbS NPs have adverse effects on transcription or post transcriptional processing and coating nanoparticles with biopolymers reduces their toxicity to organisms. Being under control, it seems that genetic manipulation of <em>Pds </em>may result in increased biotechnological production of carotenoids by<em> D. salina</em>.