Iranian Journal of Biotechnology
Year:2010 | Volume:8 | Issue:3|Papers Count:8

Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one of the main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known as BiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of the 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, about seven-fold as much the base-line. The doublemutant hFVIII was biologically active and its inactivation patterns by EDTA and heat was similar to that of the non-mutant hFVIII. Semi-quantitative RT-PCR 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 provided convincing evidence for the increasing effect of the double-mutant on the hFVIII secretion and transcription efficiencies.

Previous studies have indicated that in all land plants examined to date, the chloroplast gene trnLUAA is interrupted by a single group I intron ranging from 250 to over 1400 bp. The parasitic Epifagus virginiana has lost, however, the entire gene. We report that the intron is missing from the chloroplast genome of two arctic species of the legume genus Hedysarum (H. alpinum, H. boreale). DNA sequencing of the trnL gene and trnL-trnF intergenic spacer (trnL-F), as well as portion of trnF exon in these species confirms the absence of trnL intron and shows that it has been deleted from the gene precisely along established exon/intron splicing sites. Phylogenetic analysis of trnL-F sequence data revealed that they are closely related species. This indicates that the intron was lost from the chloroplast genome before the divergence of the two Hedysarum species. It is concluded that this rare genomic structural mutation may have occurred once during the evolution of land plants.

Development of microsatellite markers has been an increasing trend in crop genetic studies because of their applicability in breeding programs. Here we report the development of inter simple sequence repeat (SSRs) in pomegranate (Punica granatum L.) using an enrichment method that makes use of magnetic 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.

A flow-through biosensor consisting of a fixed bed bioreactor was employed to detect the insecticide 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. 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 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. The incubation time was 14 min. These results demonstrate that silicate-multiwall carbon nanotube (MWCNT) sol film is very efficient for retaining the activity of AChE with a good long-term stability.

The objective of the present study was to investigate the polymorphism of prolactin promoter and cytosolic phosphoenol pyruvate carboxykinase (PEPCK-C) intron 3 to exon 3 regions, and its association with economic 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 28, 30, and 32 weeks (MEW), and the number of eggs during the first 12 weeks of laying period (EN). Blood samples were collected from 159 pedigreed fowl at native fowl breeding center of Yazd province, and DNA was extracted from the samples according to saltingout protocol. PCR amplification together with restriction fragment length polymorphism was used to identify different genotypes of prolactin and PEPCK-C genes. The effect of prolactin genotypes on economic traits was analyzed using general linear model. A 24- bp indel (insertion or deletion) at nucleotide position (np) 358 was identified, but no polymorphism was found for PEPCK-C. Based on our results, the frequency of I and D alleles were 0.761 and 0.239, respectively. Frequencies of II, ID and DD genotypes were 0.566, 0.389 and 0.044, respectively. Genotypes II and ID were significantly associated with incresased EN (P<0.01). Meanwhile, the genotypes of the 24-bp indel site were not significantly associated with BW8, BW12, ASM, WSM and MEW (P> 0.05). The results of current study showed that using information of genes related to egg production could be used to improve the performance of native fowl of Yazd province.

Fermentation characteristics of four strains of brewer's yeast (Saccharomyces cerevisiae strain 70424, S. rouxii strain 2535, S. rouxii strain 2531 and Saccharomyces ludwigii strain 3447) in Yeast Moldbroth containing four different fermentable sugars (glucose, fructose, maltose, or sucrose) were studied. The aim was to consider the suitability of different strain/sugar treatments for the production of non-alcoholic beer as well as to devise treatments resulting in greatest growth rate of yeast cells. Experimental parameters were yeast cell growth, ethanol production, pH drop, and changes in fermenting media attenuation (°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.

The recently cloned peroxisomal matrix protein (PeP), contains two hydrophobic domains at 12-31 and 152- 169 amino acid residues and a fibronectin type III (FnIII) domain between residues 31-114. To understand the importance of the above mentioned domains in peroxisome sorting of PeP, site-directed mutagenesis was performed to delete these domains separately. Amplified mutants of PEP cDNA were constructed downstream of enhanced green fluorescent protein (EGFP) cDNA for transfection into the chinese hamster ovary (CHO) K1 and P19 cell lines. Upon their transfections, numerous green fluorescent punctuate structures, superimposable on those punctuates stained with the anti-catalase antibody, appeared in the cells. Thus, the aforementioned domains do not exert the targeting signal activity for the peroxisomal sorting of PeP.

Bacterial-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 acid bacteria (LAB) the advantage of being used as a host expression system for antigen delivery to induce 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. 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 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.