Journal of Cell and Molecular Research

50-55

Authors: Nazila Nouraee, Mohamad Vasei, Shahriar Semnani, Seyed Javad Mowla

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular processes, and their disregulations have been linked to several pathologic conditions, mainly cancers. Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs (miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks, proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection in FFPE samples and NT2 cell line.

62-67

Authors: Balal Sadeghi, Mohammadreza Nassiri, Ali Masoudi-Nejad, Mojtaba Tahmoorespour, Hesam Dehghani and Hamed Ahmadi

MicroRNAs (miRNA) are a class of noncoding and regulatory RNA molecules about 22 nucleotides in length. MicroRNAs regulate gene expression by an RNA interfering pathway through cleavage or inhibition of the translation of target mRNA. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about cattle and sheep miRNAs. The comparative genomics approach due to their conserved nature is a good source for the miRNAs discovery. Cattle and sheep are ideal model organisms for biological and comparative genomics studies. In our study, a computational method based on expressed sequence tag (EST) analysis was used for detection of cattle and sheep miRNAs. In cattle, 25 miRNA candidates found by homology searching frequently clustered at certain chromosomes and 28 miRNAs in sheep had been detected. Our results show that the cattle and sheep miRNA database can be providing useful information for investigating biological functions of miRNAs in cattle and sheep. Furthermore, the bioinformatics approach is a good manner for studying these functions.

76-80

Authors: Eisa Kohan Baghkheirati, Mohammad Bagher Bagherieh-Najjar and Mahnaz Aghdasi

Environmental stresses affect plant growth and cause losses worth hundreds of million dollars to agricultural industry each year. Many genes are induced in response to environmental stresses. The DREB1A gene is a stress-inducible transcription factor which its ectopic over-expression improves plant tolerance to environmental stresses. To produce environmental stress tolerant plants carrying the DREB1A gene, the full length cDNA of the DREB1A gene was amplified from Arabidopsis thaliana Col-0 plants by gene specific primers and cloned into pGEMT-Easy vector, and transformed into E.coli. Presence of the DREB1A gene was confirmed by restriction analysis as well as DNA sequencing. A 668-bp XbaI/BamHI digested fragment of DREB1A gene from the pGEMT::DREB1A construct was sub-cloned into the pBI121 binary vector. The recombinant plasmids were transferred into Agrobacterium tumefaciens cells (strain LBA4404) and screened on LB medium supplied with kanamycin/rifampicin (50 mg/l). Positive bacterial colonies were selected based on colony-PCR analysis and saved for further application in plant materials.

89-96

Authors: Mahnaz Kiani, Homa Zarghami, Farshid Memariani and Ali Tehranifar

Tissue culture methods provide tools to supplement traditional methods for collection, propagation and preservation of endangered plant species. In this study, in vitro propagation of Diaphanoptera khorasanica Rech.f., a rare and threatened plant species with limited distribution range and population was investigated. This species has a potential as an ornamental plant. Single node explants were provided from both adult and seedling sources. Several disinfection treatments were tried to permit selection of a suitable method. Different growth regulators were used for establishment, proliferation and rooting stages. Explants showed the highest establishment percentage after 5 min treatment with 1% sodium hypochlorite (NaOCl), cultured in MS medium containing 2.2 µM 6-benzylaminopurine (BAP) and 2.4 µM indole-3-butyric acid (IBA). The highest proliferation of explants from both adult and seedling source explants was obtained from media supplemented by BA treatment in contrast to TDZ. No significant differences were found between different concentrations of BAP and TDZ. Proliferated shoots in TDZ were longer and had more internode length and less vitrification, in comparison with those in BAP. In vitro rooting of proliferated shoots just induced in liquid half-strength MS medium and rooting was not observed in solid medium. The shoots that originated from adult plants gave rise to the highest rooting rate with 4.8µM α-naphthalene acetic acid (NAA) and 2.4 µM, but NAA rooted plantlets showed higher survival percentage in acclimatization step. This study was aimed towards developing an efficient protocol for in vitro propagation of D. khorasanica and conservation of this vulnerable species.

1-10

Authors: Mahnaz Aghdasi, Fariba Fazli and Mohammad Bagher Bagherieh

TRR14 is a novel protein important in trehalose (α-D-glucosyl-[1,1]-α-D-glucopyranoside) signaling in Arabidopsis. In this research, we provided evidences to demonstrate that TRR14 plays role in Arabidopsis responses to salt and drought stress. Transgenic Arabidopsis plants over-expressing TRR14 under the control of CaMV 35S promoter were generated. The transformed lines showed higher transcript levels of TRR14 than that of the Wild Type (WT) Arabidopsis plants. The RT-PCR results showed that TRR14 transcript level increased markedly by salt and drought stress both in WT and transformed lines. Further experiments indicated that the TRR14 transformed lines had unchanged seed germination, root length and chlorophyll content under stress conditions. In addition, the activity of oxidative enzymes like peroxidase and catalase were significantly induced in transformed lines under salt and drought treatments. Thus, the present data indicate that a novel protein, TRR14, is involved in plant salt and drought tolerance.

18-27

Authors: Hassan Monhemi, Mohammad Reza Housaindokht, Mohammad Reza Bozorgmehr and Ahmad Reza Bahrami

Signaling pathways are not isolated from their surroundings. They are also intervened by other signaling pathways known as “crosstalk mechanism”. One of the most important crosstalk mechanisms is the insulin- EGF network. Although insulin and epidermal growth factor (EGF) networks have some complexity in their isolated forms, their complexities will grow in the crosstalk network. In this study, we used the analytical tools of the systems biology workbench for elucidating some ambiguities of the insulin-EGF crosstalk. Based on sensitivity analysis, we reconstructed an elucidated model with 51 chemical reactions in comparison with the previous model with 111 chemical reactions. Interestingly, this reduced model reproduces the results of the original model in synergy conditions. We noticed two controlling pathways with direct participation of phosphorylated insulin and EGF receptors that involve Insulin Receptor Substrate (IRS) and Src kinase modules. Also, insulin pathway by producing phosphatidylinositol-3, 4, 5-triphosphate (PIP3), and EGF pathway by activation of GAB1, control the downstream events and lead to potentialities in the mitogenic signal. Surprisingly, Shc and phosphatase SHP2-dependent reactions have no significant roles in the synergy conditions and are not involved in the reduced model. Regarding sensitivity analysis, all Ras/ERK cascade reactions are crucial for signal transduction and were kept in the reduced model.

34-42

Authors: Somayeh Panahi Moghadam, Zohreh Hojati and Majid Motovali-Bashi

StrR is a putative pathway specific regulator of streptomycin production in Streptomyces griseus. Because of finding new spo0j domain in strR by bioinformatics methods, the purpose of this study was to suggest another role for strR gene. This domain can be seen in proteins that are involved in initiation of sporulation and normal chromosome partitioning. So, 51 bps of strR in accordance to spo0j domain was deleted to investigate effects of deletion mutation in StrR functions. A unique specific procedure, including three consecutive PCRs, known as SOEing PCR were used here for site directed mutagenesis. Application and feasibility of this PCR was studied here. Bioinformatic studies were carried out for comparison of the sequence similarities between StrR and Spo0J proteins. An exclusive procedure, including three consecutive PCRs, was designed here in order to delete a 51 bp from the native strR. Other PCRs such as Semi-Nested PCR and RFLP PCR were used for strR isolation and structural confirmation of the isolated strR and deleted strR genes. Routine genetic engineering procedures were conducted in order to clone the native and deleted strR genes into E. coli. Obtained sequence information, from Conserved Domains Database (CDD) and Clustal W program, revealed that the StrR is similar to members of ParB family. Here, the strR was initially isolated from Iranian strain of Streptomyces griseus (PTCC1127). It was then confirmed as StrR by Semi-Nested PCR and RFLP-PCR. A 51 base pair region of the strR gene was deleted by specifically designed overlapped primers. A ten nucleotide overlap region was considered for a set of these primers. The recombinant cassette pSPMstrRΔ17 was constructed and cloned in E. coli. The sequencing results showed that a specific deletion is produced in the desired site and region in the strR gene. Therefore the designed three steps PCRs (known as SOing PCR) is a very rapid, cheap, and precise method for introducing such a deletion in any preferred gene.