فهرست مطالب

Molecular Biology Research Communications - Volume:5 Issue:3, 2016
  • Volume:5 Issue:3, 2016
  • تاریخ انتشار: 1395/07/05
  • تعداد عناوین: 7
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  • Hesamaddin Shirzad, Aski, Mohammad Tabatabaei* Pages 123-132
    In 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 Pasteurella multocida, 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 P. multocida 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 P. multocida isolates as compared with RAPD. The results showed that a considerable level of genetic diversity exists among P. multocida 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 P. multocida is a strictly opportunistic pathogen. In conclusion, because of a large amount of genetic heterogeneityin the P. multocida isolates, Pasteurellosis may be caused by different clones in the same herd or animal.
    Keywords: Pasteurella multocida, Molecular analysis, RAPD, REP, PCR
  • Arezoo Tavakoli *, Ainon Hamzah, Amir Rabu Pages 133-142
    In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA 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 BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-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 (Vmax) and Michaelis constant (Km) 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).
    Keywords: Benzoate dioxygenase, Rhodococcus ruber, Purification, GC, MS analysis
  • Fatemeh Azadian, Arastoo Badoei, Dalfard*, Abdolhamid Namaki, Shoushtari, Mehdi Hassanshahian Pages 143-155
    A 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.
    Keywords: Cellulosic hydrolysate, Bioethanol, Purification, Characterization
  • Avijit Patra, Anjan Mondal, Sayani Banerjee, Harresh Adikesavalu, Siddhartha Narayan Joardar, Thangapalam Abraham* Pages 156-166
    The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316,KM016968,KM016969,andKM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87–100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus 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 (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species.
    Keywords: Aquaculture, Argulus spp, 18S rRNA, Phylogenetic relationship
  • Abasalt Hossienzadeh, Colagar*, Mohammad Javad Haghighatnia, Zahra Amiri, Maryam Mohadjerani, Majid Tafrihi Pages 167-174
    Microsatellites 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 in vitro 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 (Rhombomys opimus) 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 Taq DNA polymerase slipped during microsatellite in vitro 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.
    Keywords: Microsatellites, Taq polymerase slippage, Replication slippage
  • Leila Navapour, Navid Mogharrab* Pages 175-192
    Horseradish 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 Ca2 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.
    Keywords: Horseradish peroxidase, Charge, reversal substitutions, Molecular dynamics simulation, Stability
  • Ali Moradshahi *, Hajar Zamani Pages 193-199
    Phytoene synthase (Psy) and Phytoene desaturase (Pds) are the first two regulatory enzymes in the carotenoids biosynthetic pathway. The genes Psy and Pds 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 Pds were investigated. Relative to mRNA level of Pds at 100 µmol photon m-2 s-1 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
    Keywords: Phytoene desaturase, qRT, PCR, Carotenoids, Lead sulfide