Journal of Cell and Molecular Research
Volume:4 Issue: 2, Winter and Spring 2012

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamentalcellular 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.

Agrobacterium tumefaciens is capable to transfer genes across kingdoms. It can genetically transform not only plant cells, but also many other bacterial, algal, fungal, animal and human cells. This depends on the interactions among a variety of both Agrobacterium and host genes. Inside the host cell, RAD52 which is involved in DNA repair is a key gene determining integration of T-DNA by homologous recombination. Here, using Saccharomyces cerevisiae haploid strains BY4741 and BY4742, a rad52 diploid deletion strain is constructed in yeast BY4743 background.This model organism was employed to show that RAD52 deletion severely decreases frequencies of Agrobacterium genetic transformation mediated by either an integrative T-DNA or a circular non-integrative T-DNA. Indeed, the frequencies of such Agrobacterium-mediated transformation (AMT) decreased by ca. 25-fold. Hence, host RAD52 deletion might affect AMT by a mechanism which differs from its only involvement in DNA repair in yeast.

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.

Micro-organisms such as bacteria, yeasts, molds and algae that accumulate lipid more than 20% of their biomass are called oleaginous. Microbial lipid has a lot of similarity to the oil obtained from plants and animals. Microbial lipids are renewable sources that can use for different purposes such as biodiesel production. Production of oil from yeasts has more advantages than plant’s oil. According to this, isolation of oleaginous yeast with high ability of lipid production is very valuable. In this investigation 176 yeasts were isolated. Between them 68 lipid producing yeasts were found that they were isolated from 34 soil samples. The strains were screened by an enrichment technique in glycerol and then Sudan black staining. After extraction of lipid by Bligh & Dyer method, best strain was selected which has lipid quantity, dry biomass and lipid productivity of 10.97g/l, 18.84g/l and 58.2% in optimized condition. Extracted lipid was analyzed by thin layer chromatography and FTIR spectroscopy.

Environmental stresses affect plant growth and cause losses worth hundreds of million dollars of agricultural industry each year. Many genes induced in response to environmental stresses. The DREB1A gene is a stress-inducible transcription factor that its overexpression increased tolerance to environmental stress in transgenic plants. DNA was extracted from Arabidopsis thaliana var. Col.0 plants and DREB1A gene amplified by particular primers. After purification, PCR products were cloned into pGEMT-EASY vector and transferred into E. coli strain DH5α competent cells. Blue/white colonies were analyzed and present of the DREB1A gene revealed by restriction analysis and sequencing test. A 668-bp XbaI/BamHI digested fragment of DREB1A gene from pGEMT::DREB1A construct cloned in pBI121 plasmid. The recombinant plasmids were transferred into Agrobacterium tumefaciens cells strain of LBA4404 and transformed cells were cultured on selective medium that supplied with kanamycin and rifampycin. As a result of gel electrophoresis of Agrobacterium colonies-PCR product, band existing on expected size show that the DREB1A gene was cloned in to pBI121 binary vector. At time, Agrobacterium cells containing of pBI121::DREB1A construct are using for product of environmental stress tolerant plants.

Plant regeneration was achieved in Verbascum speciosum Schard. via organogenesis and somatic embryogenesis by culture of mature embryo explants. Two types of calli, embryogenic and non embryogenic, were induced from mature embryo explants on Murashige and Skoog (MS) medium supplemented with different concentrations of benzyl adenine (BA) and α-naphthalene acetic acid (NAA). In order to further proliferate the somatic embryoids, the yellow and friable embryogenic calli were transferred on MS medium containing 0.5 mg-1 charchol and 0.1 or 1 mg-1 2,4-dichlorophenoxy acetic acid (2,4-D) or into MS medium containing 60 g-1 sucrose, 50 mgl-1 casein hydrolysate (CH), 0.5 mg-1 kinetin (Kin), 5 mg-1 2,4 D and 0.5 mg-1 charchol. Among the 3 tested media, MS medium containing 0.1 mg-1 2,4-D and 0.5 mg-1 charchol was more effective for proliferation of embryonic calli. Somatic embryos were transferred to hormone free MS medium for maturation and shoot regeneration. In addition, shoots and roots regenerated from non embryogenic calli in hormone free MS medium or containing NAA and BA. Shoot buds were obtained from non embryogenic calli and they were transferred to MS medium supplemented with 1 mg-1 BA or Kin for further growth and multiplication. Regenerated plants then were potted and maintained in the greenhouse.

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.