Iranian Journal of Biotechnology
Volume:8 Issue: 3, Summer 2010

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 B-domain deleted hFVIII mutant including; two single-mutants (Leu299Phe and Phe309Thr) and a double-mutant (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 double-mutant 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 double-mutant hFVIII. Both of the mutated residues in the double-mutant are located in a hydrophobic heptamer (320MEAYVKV326) which seems to be involved in Bip-binding 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 circumboreal species of the legume genus Hedysarum (H. alpinum, H. boreale). DNA sequencing of the trnL gene, trnL-trnF intergenic spacer, and portion of trnF exon in these taxa confirms the absence of trnL intron and shows that it has been deleted from the gene precisely along established exon/intron splicing sites. On the basis of distribution of the intron loss, we conclude that this rare genomic structural mutation has been occurred once in the common ancestor of H. alpinum and H. boreale during the evolution of the flowering 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 SSRs in pomegranate 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. Out of these, 32 (74.4%) were found to contain microsatellite sequences and 25 primer pairs were designed, of which 44% revealed polymorphisms, 48% showed monomorphic patterns and 8% generated weak amplification on a set of 20 pomegranate genotypes. Eleven microsatellite primers 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 procedure, the abundance of microsatellites in pomegranate, and their potential usefulness in pomegranate genome mapping and for evaluating pomegranate genotypes.

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 conditions for detection of the insecticide were explored. The influence of enzyme loading on the packing surface was studied and AChE loading was set at 0.36 U.cm-2 for subsequent studies. Maximum value of absorbance response occurred at the residence time in bioreactor of 5 min, and 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 optimal conditions, the inhibition of paraoxon was proportional to its concentration in two ranges, from 0.25 to 25 mg.L-1 and 25 to 60 mg.L-1 and the detection limit for paraoxon was equal to 7.3×10-12 mol paraoxon. 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 cetylcholinesterase with a good long-term stability.

The objective of present study was to determine the polymorphism of prolactin promoter region and its association with age at sexual maturity, weight at sexual maturity, average egg weight at 28th, 30th and 32nd weeks and the number of eggs during the first 12 weeks of laying period (EN) in Iranian native fowls. Blood samples were collected from 159 pedigreed native fowls of Yazd breeding center and the DNA of blood samples were extracted according to salting out protocol. Polymerase chain reaction and restriction fragment length polymorphism were used to identify different genotypes of prolactin gene. The effect of prolactin genotypes on economic traits was analyzed using general linear model. A 24-bp indel (insertion or deletion) at the site of -358 was identified. Based on present 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, 0.390 and 0.044, respectively. Genotypes II and ID were significantly associated with incresased EN (P<0.01). Nevertheless, genotypes of the 24-bp indel were not significantly associated with BW8, BW12, ASM, WSM and MEW (P> 0.05).

Fermentation characteristics of four strains of brewer''s Saccharomyces (Saccharomyces cerevisiae 70424, Saccharomyces rouxii 2535, Saccharomyces rouxii 2531 and Saccharomyces ludwigii 3447) in Yeast-Mold-broth medium containing four different fermentable sugars (glucose or fructose or maltose or sucrose) were studied with the aim of considering the suitability of treatments (strains/sugars) for production of non-alcoholic beer as well as realizing the best treatments resulting in the greatest growth rate of yeast cells. Experimental parameters were yeast cells growth trend, ethanol production trend, pH drop trend and changes in fermenting media attenuation (°Pl) during 48-hour fermentation period. Fermentation was performed at 24°C using periodic aeration practice. For S. cerevisiae, the greatest growth rate throughout the fermentation period achieved in presence of sucrose. The maximum and minimum ethanol contents at the end of fermentation were related to the sucrose-containing treatment (0.94% V/V) and glucose-containing one (0.4% V/V), respectively. In the case of S. ludwigii, fructose caused the highest growth rate and the maximum and minimum ethanol contents at the end of fermentation were observed in sucrose-containing treatment (0.49%) and maltose-containing one (0.04%), respectively. For S. rouxii 2535, the greatest growth rate was observed in the presence of fructose/glucose. The maximum and minimum ethanol contents belonged to the fructose/glucose-containing treatments (about 0.40) and maltose/sucrose-containing ones (about 0.01%), 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 was found in glucose-containing treatment (0.01%) and maltose/sucrose-containing ones (0.00%), respectively.

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. Keywords: Peroxisome; FnIII domain; Hydrophobic domain; SKI tripeptide; Protein targeting

AbstractGene replacement protease deficient mutants of A. hydrophila indicated that mutant activities were lower until 90% than wild type. Fish injected with the A. hydrophila parental strain die more rapidly than those are injected with isogenic and Tn5-induced protease mutants. In addition, proteases are also major antigenic components of a vaccine against hemorrhagic septicaemia in fish. Besides, bacterial-based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as a vaccination strategy. Developments in genetic engineering have given these Gram-positive lactic acid bacteria (LAB) the advantage to be used as a host expression system for antigen delivery to induce immune response.A fragment containing the full length of protease gene was amplified by PCR with the forward and reverse primers; using Aeromonas hydrophila strains genomic DNA as template. The amplified 1038-bps fragment was digested by PstI and HindIII, followed by ligation into the corresponding sites on pCR®-Blunt II-TOPO and pNZ8048 plasmids. The ligated DNA was transformed into Escherichia coli TOP10 and Lactococuss lactis NZ9000 cells by the heat-shock and electroporation methods respectively. Verification of cloning was done using of RE digestion and DNA sequencing. SDS-PAGE analysis also detected the expression of the recombinant protease protein. In the present work, cloning of eprA1 gene, a temperature-stable metalloprotease, of fish isolated Aeromonas hydrophila into E. coli and expression in Lactococcus lactis system were successfully done. This can develop a useful and safe system to control microbial infections in different living things.