Iranian Journal of Biotechnology
Volume:11 Issue: 1, Winter 2013

RNA molecules play many important regulatory, catalytic and structural roles in the cell, and RNA secondary structure prediction with pseudoknots is one the most important problems in biology. An RNA pseudoknot is an element of the RNA secondary structure in which bases of a single-stranded loop pair with complementary bases outside the loop. Modeling these nested structures (pseudoknots) causes numerous computational difficulties and so it has been generally neglected in RNA structure prediction algorithms..

In this study, we present a new heuristic algorithm for the Prediction of RNA Knotted structures using Tree Adjoining Grammars (named PreRKTAG)..

For a given RNA sequence, PreRKTAG uses a genetic algorithm on tree adjoining grammars to propose a structure with minimum thermodynamic energy. The genetic algorithm employs a subclass of tree adjoining grammars as individuals by which the secondary structure of RNAs are modeled. Upon the tree adjoining grammars, new crossover and mutation operations were designed.The fitness function is defined according to the RNA thermodynamic energy function, which causes the algorithm convergence to be a stable structure..

The applicability of our algorithm is demonstrated by comparing its results with three well-known RNA secondary structure prediction algorithms that support crossed structures..

We performed our comparison on a set of RNA sequences from the RNAseP database, where the outcomes show efficiency and practicality of the proposed algorithm..

The witches’ broom disease of lime caused by Candidatus Phytoplasma aurantifolia, is the most devastating disease of acidian lime in the southern parts of Iran.. At present, no efficient method has been developed for controlling the disease, therefore quarantine approaches such as early detection and subsequent eradication of infected trees is very important. Toward this aim, developing a reliable and sensitive detection method would be the first step to prevent transportation of infected plant materials to other places.. In this study, Immunodominant membrane protein (IMP) of the pathogen was selected as a target for detection and preparation of polyclonal antibody. The IMP is the major protein present on the surface of phytoplasma cells. For this purpose, the DNA region encoding IMP gene was isolated and cloned into pET28a bacterial expression vector. The recombinant protein was expressed in a large scale in Escherichia coli. Purification was performed under native conditions and the purity and integrity of produced recombinant protein were confirmed by western immuno blot analysis using anti His-tag and anti-IMP polyclonal antibodies. The purified recombinant IMP was used for immunization of rabbit. Purification of immunoglobulin was performed by affinity chromatography using protein A column. The purified immunoglobulin was conjugated with the alkaline phosphatase enzyme.. The purified antibodies and conjugates were applied for efficient detection of infected plants in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and dot immunosorbent assay (DIBA).. These antibodies were proven to be very powerful tools to detect the Candidatus Phytoplasma aurantifolia in plants..

Protein-protein interactions do not provide any direct information regarding the domains within the proteins that mediate the interactions. The majority of proteins are multi domain proteins and the interaction between them is often defined by the pairs of their domains. Most of the former studies focus only on interacting domain pairs. However they do not consider the interactions that require the presence of a third domain.. In this manuscript, we define the concept of necessary and sufficient triplets of domains and mediator domain.. We approximate these conditions by pragmatic statistical definitions on a set of gold-standard interacting protein pairs and a set of gold-standard non-interacting protein pairs.. In this paper we introduce a new method for the prediction of the interaction between two domains using third domains as a mediator.we show that the mediator domain has an effective role in the interaction between proteins.. By using these concepts, we introduce a method for the prediction of the interaction between two domains. Subsequently by evaluating the performance of our method on the yeast protein interactions data set, we show that the mediator domain has an effective role in the interaction between proteins..

The objectives of this study were isolating rhizosphere-inhabiting indigenous oil-degrading bacteria in wild oat grown in petroleum-polluted areas and in vitro evaluating the efficiency of oil biodegradability by microbial isolates.. Bacteria were isolated from rhizosphere of wild oat grown on contaminated sites in Khuzestan and were identified based on 16S rRNA gene sequencing. The catabolic genes were detected using PCR and hybridization analysis. Hydrocarbon degradation in liquid culture was evaluated by gas chromatography-mass spectrometry (GC-MS).. 23 indigenous oil-degrading bacterial strains were isolated from the wild oat rhizosphere, grown in severely oil contaminated soil in Khuzestan. Based on 16S rRNA gene sequence analyses, isolated strains were classified to Genera Acinetobacter, Pseudomonas, Enterobacter, Stenotrophomonas, Bacillus, Achromobacter, Ochrobactrum, Paenibacillus, Microbacterium, Curtobacterium and Sphingobacterium. Catabolic genes alkM, alkB and xylE, responsible for biodegradation of the alkanes and aromatic petrochemical compounds were detected in bacterial community inhabiting rhizosphere of the wild oat. The GC-MS analysis indicated that consortium of these bacteria was capable of reducing crude petroleum in the liquid culture by 40.5%, after 10 days. The results of the present study revealed the adaptability of microbes to the rhizospheric area and subsequently their great potential to be exploited for cleaning up hydrocarbon contaminated sites.. This study might be an important step towards the development of a phytoremediation strategy in the South of Iran..

Magnetite (Fe3O4) nanoparticles are currently one of the important and acceptable magnetic nanoparticles for biomedical applications. To use magnetite nanoparticles for bacteria cell separation, the surface of nanoparticles would be modified for immobilizing of nanoparticles on the surface of bacteria. Functionalization of magnetite nanoparticles is performed by different surfactants such as glycine or oleic acid to attach on the bacteria cell surface simultaneously. The magnetic nanoparticles have very low toxicity on the living cells. There are some studies on evaluating the toxicity of magnetite nanoparticles on eukaryote cells, which their results showed negligible toxicity in eukaryote cells of the modified magnetite nanoparticles with different surfactants. But the toxicity of magnetite nanoparticles on bacteria cells is not reported.. in this study, the effect of the magnetic nanoparticles iron oxide (Fe3O4) on the growth rate of the genetically engineered Pseudomonas aeruginosa (PTSOX4) cells in different media with different magnetic nanoparticles concentration have been investigated.. In this study, the genetically manipulated bacterial cells, Pseudomonas aeruginosa (PTSOX4), were coated with magnetic Fe3O4 nanoparticles to evaluate the toxicity effect of these nanoparticles on the growth rate of this strain in Laurial Bertany (LB) and Basal Salt media (BSM) separately. In addition the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) tests of these nanoparticles were examined.. A low concentration of nanoparticles has little toxicity effect on the cell growth in this bacterium. Maximal level of the growth obtained in the late stationary phase, using a concentration of 500 ppm or more of Fe3O4 nanoparticles, but a high concentration of these nanoparticles, more than 1000 PPM, resulted in reducing the cell growth rate. However, there was not a considerable lethal effect on the cell viability. Moreover, using a high nanoparticle concentration leads to a high level of bacterial cell coating due to more contact of the nanoparticles to bacterial cell surface.. It is concluded that magnetite nanoparticles have negligible toxicity on the living bacteria cells and they are so applicable in different parts of biotechnology fields..

Leukemia inhibitor factor (LIF) is a very important pleiotropic cytokine which belongs to interleukin-6 (IL-6) family. LIF exerts multiple effects on different types of cells and tissues with numerous regulatory effects in vivo and in vitro. It is a lymphoid factor, which performs a number of activities including cholinergic neuron differentiation, control of stem cell pluripotency, bone and fat metabolism, and is important for embryo implantation and promoting megakaryocytes production in vivo. Human LIF is a potential therapeutic candidate for some diseases such as multiplesclerosis (MS).. Because of aforementioned applications of the LIF protein in biological systems, the LIF gene has been cloned in various species. In this study a useful novel method was used to clone an optimized LIF sequence.. In this study, the optimized cDNA form of human leukemia inhibitory factorwas cloned into the pET-28a (+) expression vector under control of T7lacpromoter using BamHI and XbaI restriction enzymes. The recombinant vector was transformed into Escherichia coli strain BL21 (DE3). The cloned hLIF cDNA was expressed as a fusion protein with His-tag. Cloning of hLIF cDNA was confirmed by digestion and DNA sequencing. Appropriate expression of recombinant hLIF was examined by SDS-PAGE after induction with isopropylthio-β-galactoside (IPTG).. The results confirmed the expression of the 19.7 kDa rhLIF protein in the bacterial expression system.. We could show that codon optimization might increase the production of recombinant hLIF in the E. coli. This result is useful in similar cases, in which the level of expressed gene is critical..

PhiC31 integrase is a DNA site-specific recombinase integrates DNA into the chromosomes between the two sites of attB and attP. Several pseudo attPs have been identified in mammalian genomes with critical features for long-term expression of transgene. In this manuscript, we report a novel intrinsic pseudo attP site named CHOL1 in the Chinese hamster genome implementing an inverse Polymerase Chain Reaction (IPCR).. Identification of pseudo attP site(s) of Tenecteplase cDNA integration in the genome of stable transformed CHO cell line.. First, genome was extracted from a stable transformed CHO cell line expressing Tenecteplase. By creating of minicircle DNA in the last step, sequencing was performed.. We obtained one band. BLAST analysis of the respective sequence of inverse PCR band identified a pseudo attP site.. Data demonstrated that the phiC31 integrase provides a suitable insertion site in the genome to express the gene of interest..

Aqueous extract of dried fourth stomach of unweaned bovine contains prochymosin, that subsequently is converted to active chymosin, a milk-coagulating enzyme in the cheese industry. Recombinant chymosin has been found to be a suitable replacement for natural bovine chymosin. Meanwhile it possesses several advantages to other plant, fungal and bacterial milk-clotting enzymes.. In present research we evaluate the expression of this critical enzyme in a eukaryotic system for future use in cheese industry.. We have cloned bovine prochymosin gene in methylotrophic yeast, P. pastoris, using pPIC9K as an expression vector. The recombinant plasmid was transformed into the host by electroporation, and it was expressed in optimum conditions (temperature 29oC, 200 rpm, 2% methanol for induction, and 5 days of incubation). Transcription and expression of the recombinant prochymosin was evaluated by the reverse transcription polymerase chain reaction (RT-PCR), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis as well as western blotting and enzyme-linked immunosorbent assay (ELISA).. In optimum conditions, only a low level of this heterologous protein was detected using ELISA method and subsequently confirmed by RT-PCR.. Since it has been reported that P. pastoris is an appropriate host for the expression of recombinant proteins, a low level of expression of prochymosin in this host should be explored in our future research..