National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701The effects of novel mutations in A1 domain of human coagulation factor VIII on its secretion level in cultured mammalian cells1391497115ENGholam Ali KardarNational Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran. Immunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of Medical Sciences, P.O. Box 14185-863, Tehran, I.R. Iran.Alireza ZomorodipourNational Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.0000-0003-0671-4779Mostafa MoinImmunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of
Medical Sciences, P.O. Box 14185-863, Tehran, I.R. Iran.Zahra PourpakImmunology, Asthma and Allergy Research Institute, Children’s Medical Center Hospital, Tehran University of
Medical Sciences, P.O. Box 14185-863, Tehran, I.R. Iran.Mehdi SadeghiNational Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.Fariba AtaeiNational Institute of Genetic Engineering and Biotechnology, P.O. Box 14965/161, Tehran, I.R. Iran.Journal Article20100701Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one of<br />the main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known as<br />BiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of the<br />porcine FVIII and analysis of the hFVIII A110 region, that inhibits its secretion, function of three novel Bdomain deleted hFVIII mutant including; two singlemutants (Leu299Phe and Phe309Thr) and a doublemutant (Tyr323His/Lys325Arg) were examined in three mammalian cell lines (HEK-293T, COS, CHO) for the hFVIII secretion efficiency. The double-mutant construct displayed the highest hFVIII expression level,<br />about seven-fold as much the base-line. The doublemutant hFVIII was biologically active and its inactivation<br />patterns by EDTA and heat was similar to that of the non-mutant hFVIII. Semi-quantitative RT-PCR<br />results showed the highest mRNA level for the doublemutant hFVIII. Both of the mutated residues in the double-mutant are located in a hydrophobic heptamer (320MEAYVKV326) that seems to be involved in Bipbinding activity. None of the L299F and F309T hFVIII mutants exhibited improved secretion. This result has<br />provided convincing evidence for the increasing effect of the double-mutant on the hFVIII secretion and transcription efficiencies.<br /><br />National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701Loss of chloroplast trnLUAA intron in two species of Hedysarum (Fabaceae): evolutionary implications1501557116ENAtefeh AmirahmadiDepartment of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175,
Tehran, I.R. Iran.Shahrokh Kazempour OsalooDepartment of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175,
Tehran, I.R. Iran.Ali Asghar Maassoumi2Department of Botany, Research Institute of Forests and Rangelands, P.O. Box 13185-116, Tehran, I.R. Iran.Journal Article20100701Previous studies have indicated that in all land plants examined to date, the chloroplast gene trnLUAA is<br />interrupted by a single group I intron ranging from 250 to over 1400 bp. The parasitic Epifagus virginiana has<br />lost, however, the entire gene. We report that the intron is missing from the chloroplast genome of two<br />arctic species of the legume genus Hedysarum (H. alpinum, H. boreale). DNA sequencing of the trnL gene<br />and trnL-trnF intergenic spacer (trnL-F), as well as portion of trnF exon in these species confirms the<br />absence of trnL intron and shows that it has been deleted from the gene precisely along established<br />exon/intron splicing sites. Phylogenetic analysis of trnL-F sequence data revealed that they are closely<br />related species. This indicates that the intron was lost from the chloroplast genome before the divergence of<br />the two Hedysarum species. It is concluded that this rare genomic structural mutation may have occurred<br />once during the evolution of land plants.<br /><br />National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701Microsatellite isolation and characterization in pomegranate (Punica granatum L.)1561637117ENSaeideh EbrahimiDepartment of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111,
I.R. Iran.Badraldin Ebrahim Sayed-TabatabaeiDepartment of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111,
I.R. Iran.Bahram SharifnabiDepartment of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111,
I.R. Iran.Journal Article20100701Development of microsatellite markers has been an increasing trend in crop genetic studies because of<br />their applicability in breeding programs. Here we report the development of inter simple sequence<br />repeat (SSRs) in pomegranate (Punica granatum L.) using an enrichment method that makes use of magnetic<br />beads. Enriched genomic libraries with AG and ATG microsatellite motifs were constructed, and 60 positive clones were detected by a colony PCR technique, of which 43 clones showed high-quality sequences. Out of these, 32 (74.4%) contained microsatellite sequences and 25 primer pairs were designed, of which 11 (44%) revealed polymorphisms, 12 (48%) showed monomorphic patterns and 2 (8%) generated poor amplification on a set of 20 pomegranate genotypes. Eleven microsatellite primers (two of them amplified two loci) were selected to assess polymorphism in the set of genotypes. There were 44 alleles amplified over 13 loci, with an average of 3.38 alleles per locus. The mean polymorphism information content (PIC) value was 0.433 over 13 loci, which shows that the majority of the microsatellite loci are highly informative. Cluster analysis was able to separate genotypes based on their geographical distribution and type (i.e., wild or domestic). This study shows the isolation efficiency of the magnetic beads technique, the abundance of microsatellites in pomegranate, and their potential application in pomegranate genome mapping and genotyping.<br /><br />National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701A new changeable bioreactor for detection of organophosphate in a flow-through system1641717118ENBahman EbrahimiBiotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University,
P.O. Box 14115-143, Tehran, I.R. Iran.Seyed Abbas ShojaosadatiBiotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University,
P.O. Box 14115-143, Tehran, I.R. Iran.Seyyed Mohammad MousaviBiotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University,
P.O. Box 14115-143, Tehran, I.R. Iran. 2Department of Chemical Technology, Lappeenranta University of Technology, Lappeenranta, Finland.Journal Article20100701A flow-through biosensor consisting of a fixed bed bioreactor was employed to detect the insecticide<br />paraoxon. Based on the inhibition of organophosphorous insecticide to the enzymatic activity of acetylcholinesterase (AChE), using paraoxon as a model compound, the condition for detection of the insecticide were optimized. The influence of enzyme loading on the packing surface was studied and AChE loading was set at 0.36 U/cm2 for subsequent studies.<br />Maximum value of absorbance response occurred at the residence time in bioreactor of 5 min, that was chosen as the optimal residence time. This flow-through system gave a linear response (R2 = 0.9869) to<br />acetylthiocholine iodide (ATChI) at concentrations of 0.050 to 1 mM. Under appropriate conditions, the inhibition of paraoxon was proportional to its concentration in two ranges, from 0.25 to 25 mg/l and 25 to 60 mg/l and the detection limit for paraoxon was 7.3×10-8 M.<br />The incubation time was 14 min. These results demonstrate that silicate-multiwall carbon nanotube<br />(MWCNT) sol film is very efficient for retaining the activity of AChE with a good long-term stability.<br /><br />National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701Polymorphism in prolactin and PEPCK-C genes and its association with economic traits in native fowl of Yazd province1721777114ENHakimeh Emamgholi BegliDepartment of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
386, Gorgan, I.R. Iran.Saeed ZerehdaranDepartment of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
386, Gorgan, I.R. Iran.Saeed HassaniDepartment of Animal Science, Gorgan University of Agricultural Sciences and Natural Resources, P.O. Box
386, Gorgan, I.R. Iran.Mokhtar Ali AbbasiAnimal Science Research Institute of Iran, P.O. Box 3146618361, Karaj, I.R. Iran
3Gonbad Higher Education Center, P.O. Box 163, Gonbad, I.R. Iran.Alireza Khan AhmadiAnimal Science Research Institute of Iran, P.O. Box 3146618361, Karaj, I.R. Iran
3Gonbad Higher Education Center, P.O. Box 163, Gonbad, I.R. Iran.Journal Article20100701The objective of the present study was to investigate the polymorphism of prolactin promoter and cytosolic<br />phosphoenol pyruvate carboxykinase (PEPCK-C) intron 3 to exon 3 regions, and its association with economic<br />traits in native fowl of Yazd province. These traits consisted of body weight at 8 (BW8) and 12 (BW12) weeks of age, age at sexual maturity (ASM), weight at sexual maturity (WSM), mean egg weight at<br />28, 30, and 32 weeks (MEW), and the number of eggs during the first 12 weeks of laying period (EN). Blood<br />samples were collected from 159 pedigreed fowl at native fowl breeding center of Yazd province, and DNA<br />was extracted from the samples according to saltingout protocol. PCR amplification together with restriction<br />fragment length polymorphism was used to identify different genotypes of prolactin and PEPCK-C<br />genes. The effect of prolactin genotypes on economic traits was analyzed using general linear model. A 24-<br />bp indel (insertion or deletion) at nucleotide position (np) 358 was identified, but no polymorphism was<br />found for PEPCK-C. Based on our results, the frequency of I and D alleles were 0.761 and 0.239,<br />respectively. Frequencies of II, ID and DD genotypes were 0.566, 0.389 and 0.044, respectively. Genotypes<br />II and ID were significantly associated with incresased EN (P<0.01). Meanwhile, the genotypes of the 24-bp<br />indel site were not significantly associated with BW8, BW12, ASM, WSM and MEW (P> 0.05). The results of<br />current study showed that using information of genes related to egg production could be used to improve the<br />performance of native fowl of Yazd province.National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701Characteristics of different brewer’s yeast strains used for non-alcoholic beverage fermentation in media containing different fermentable sugars1781857119ENSarah SohrabvandiDepartment of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology,
University of Tehran, Postal code 31587-77871, Karaj, Iran.Seyyed Hadi RazaviDepartment of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology,
University of Tehran, Postal code 31587-77871, Karaj, Iran.Seyyed Mohammad MousaviDepartment of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology,
University of Tehran, Postal code 31587-77871, Karaj, Iran.Amir Mohammad MortazavianDepartment of Food Science and Technology, Faculty of Nutrition Sciences, Food Science Technology/National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, P.O. Box 19395-4741, Tehran, Iran.Journal Article20100701Fermentation characteristics of four strains of brewer's yeast (Saccharomyces cerevisiae strain 70424, S.<br />rouxii strain 2535, S. rouxii strain 2531 and Saccharomyces ludwigii strain 3447) in Yeast Moldbroth<br />containing four different fermentable sugars (glucose, fructose, maltose, or sucrose) were studied. The<br />aim was to consider the suitability of different strain/sugar treatments for the production of non-alcoholic<br />beer as well as to devise treatments resulting in greatest growth rate of yeast cells. Experimental<br />parameters were yeast cell growth, ethanol production, pH drop, and changes in fermenting media attenuation<br />(°Pl), during a 48 h fermentation period. Fermentation was performed at 24°C using periodic aeration practice. For S. cerevisiae, the greatest growth rate was achieved in presence of sucrose. The maximum and minimum ethanol contents at the end of fermentation were related to sucrose- (0.94% V/V) and glucose-containing (0.4% V/V) treatments, respectively. In the case of S. ludwigii, fructose stimulated the highest growth rate and the maximum and minimum ethanol contents at the end of fermentation were observed in sucrose- (0.49%), and maltose-containing (0.04%) treatments, respectively. For S. rouxii 2535, highest growth rate was observed in the presence of fructose/glucose. The maximum and minimum ethanol contents belonged to the fructose/glucose- (~ 0.40) and maltose/sucrose-containg (~ 0.01%) treatments, respectively. In the case of S. rouxii 2531, glucose and to lesser extent, fructose led to the highest growth rate and the maximum and minimum ethanol contents were observed in glucose (0.01%) and maltose/sucrose (0.00%) treatments, respectively. Applying different strains of Saccharomyces in presence of different types of sugars caused various fermentation characteristics especially with regard to growth rate and ethanol production.<br /><br />National Institute of Genetic Engineering and Biotechnology of IranIranian Journal of Biotechnology1728-30438320100701Cloning of EprA1 gene of Aeromonas hydrophila in Lactococcus lactis1931987120ENHosseinali SasanDepartment of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, P.O. Box: 76169141111, Kerman, I.R. Iran.Journal Article20100701Bacterial-based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. Developments in genetic engineering have given Gram-positive lactic<br />acid bacteria (LAB) the advantage of being used as a host expression system for antigen delivery to induce<br />the immune response. A fragment containing the full length of the “eprA1” gene, encoding a temperaturestable metalloprotease of Aeromonas hydrophila isolated from fish was amplified by polymerase chain reaction (PCR) using the genomic DNA of this bacterium as template. The amplified 1038 bp fragment was digested by PstI and HindIII, followed by ligation into the corresponding site on the pNZ8048 plasmid. The ligated DNA was then transformed into Lactococcus lactis NZ9000 cells by electroporation method.<br />Verification of cloning was carried out using restriction enzyme digestion and DNA sequencing. Gel electrpophoresis technique also detected the expression of the recombinant protease protein. The successful<br />cloning and expression of the eprA1 gene into L. lactis can be developed as a useful and safe system to control A. hydrophila infections in fish.<br /><br />