Iranian Journal of Biotechnology
Volume:2 Issue: 2, Spring 2004

Unique benign endophytes from Ascomycets have wide distribution among grass species. The symbiotic fungi enhance plant characters including performance, insect and mammalian deterrence, nematode resistance and tolerance to drought, salt and other biotic and abiotic stresses. Endophytes from genus Neotyphodium (Acremonium) are of the major focus than their ancestors, and Epichloe species, because the formers have lost their sexual reproduction. Therefore they should be genetically stable, and most importantly, they cannot disassociate from host tissues, and are transferred vertically. They are maternally inherited and are therefore attractive for genetic transformation without the concern about gene escape. Some marker genes have been successfully transferred to endophyte Neotyphodium coenophialum and Neotyphodium lolii existing in Festuca arundinacea Schreb. and Lolium perenne L., respectively. Furthermore, gene silencing has been proved to be feasible for eliminating traits, which are economically harmful. Methods of direct DNA uptake using polyethylene glycol (PEG) and electroporation have been found to be useful in transformation of these fungi. Transgenic fungi can be reinserted into the host without need to tissue culture. The endophytic genes responsible for a specific trait can be isolated and transferred to grass species or other microorganisms for direct exploitation of secondary metabolites and endophytic enzymes. Considering advancements in this filed, endophytes can open new horizons faced to scientists and biotechnologist to use them as a surrogate target of transformation.

Due to the restriction of Barley yellow dwarf virus (BYDV)-PAV particles to the phloem tissue and very low virus titers, purification of the virus is difficult. The aim of this study was to prepare antibody against viral coat protein without purifying the virus. To produce recombinant coat protein, the coding sequence was first amplified from a PAV full-length cDNA clone by polymerase chain reaction (PCR), ligated into a vector (pBluescript SK+) to check the sequence, and sub-cloned into an expression vector (pGEX-2T). It was then transformed into Escherichia coli DH5a by electroporation. The open reading frame 3 (ORF3) was linked in-frame to the gene encoding glutathione-S-transferase (GST; 26 kDa) and expression induced by IPTG. The expressed coat protein was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for use as an immunogen. The antisera to BYDV-PAV recombinant coat protein reacted in Western blot analysis with partially purified BYDV-PAV. These antisera were also used to detect BYDV-PAV by immunogold electron microscopy of thin section of barley tissues. The results indicated that BYDV-PAV coat protein can be produced in high yields by E. coli, which provides the ability of simple purification, and because of proper antigencity, can be exploited for diagnostic applications.

One hundred and four isolates of Bradyrhizobium japonicum were isolated from nodules of two different trap plants, Viz. Soya bean cultivars, Maple Glen and Orford which were inoculated with two different soil samples (Ottawa and St-Hugus soils). All isolates were clustered based on PCR/RFLP of 16S-23S rRNA genes. RFLP analysis was performed to characterize all the isolates using six different endonuclease enzymes. The data was analyzed by using Jamp software. Using dendrogram data, all the isolates were grouped into six different clusters. There were four and five clusters of Bradyrhizobium japonicum in Ottawa and St-Hugus soils, respectively. Three clusters were common between two cultivars of Soya bean when inoculated with Ottawa soil and four common clusters were recognized when trap plants inoculated with St-Hugus soil. In Ottawa soil, cluster I was not detected by Orford cultivar, likewise in St-Hugus soil, cluster VI was not detected by Maple Glen cultivar of Soya bean. Isolates of cluster III were dominantly trapped when Maple Glen and Orford cultivars inoculated with Ottawa soil but isolates from clusters I, IV and III were trapped when they were inoculated with St-Hugus soil. Since different cultivars trapped different isolate types it can be concluded that for population studies of rhizobial bacteria different trap plants can provide a better composition of native population of bacteria.

High yield and good quality embryos were obtained from cultures of isolated microspores of Brassica napus L. cv. Global. The donor plants were grown in a growth chamber at 15/10C (day/night) with a 16/8h photoperiod. Microspores were isolated from whole buds of 2.5-3.5 mm in length containing late-uninucleate and early-binucleate microspores. Different heat shock treatments including, 30C for 10, 14 and 18 days, 32C for 2 and 3 days and 35C for 18h followed by 30C for 10 days and various culture densities including 60,000, 40,000 and 20,000 microspores per ml were used. Results showed significant differences among the heat shock treatments, the culture densities and their interaction for embryo induction. A large number of embryos were obtained from the microspores treated at 30C for 18 days, 35C for 18h followed by 30C for 10 days and 30C for 14 days with a density of 60,000 microspores per ml.

The second exon of the bovine Major Histocompati- bility Complex (MHC) class II DRB3 gene was amplified by polymerase chain reaction (PCR) from 50 DNA samples of Iranian Sarabi cattle. Bovine DNA was isolated from aliquots of whole blood. A two-step polymerase chain reaction followed by digestion with restriction endonucleases RsaI, BstyI, and HaeIII was conducted on DNA from samples. Fifteen Bovine Lymphocyte Antigen (BoLA)-DRB3 alleles were assigned, including some that were only recently described for zebu cattle. Allelic frequencies ranged from 0.02 to 0.23. The most frequent alleles were *52 (frequency = 0.23), *11 (0.18) and *23 (0.15). Results of this study demonstrate that the BoLA-DRB3.2 locus is highly polymorphic in Sarabi cattles.Keywords: BoLA-DRB3.2, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), Iranian Sarabi cows.Keywords

The reverse micelles have been used for extraction and purification of proteins and enzymes in downstream processing. In this study a simple complexation model was developed for protein extraction using reverse micelles. We assumed that the size of protein-reverse micelle complex is a function of net charge of protein and salt concentration. The model has been applied to correlate the experimental data for reverse micellar extraction of bovine serum albumin (BSA) and lysozyme. The solutions of reverse micelles for extraction of BSA and lysozyme were composed of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, and sodium bis(2-ethylhexyl) phosphate (NaDEHP), an anionic surfactant, respectively. Moreover, the effects of surfactant concentration, pH of aqueous phase, and salt concentration were investigated. In comparison with experiment the results of the model for both systems are in very good agreement.

A simple fed-batch process with pre-determined exponential feeding strategy for high-cell-density cultivation of recombinant E. coli BL21 (DE3) in defined medium was developed. In this feeding method glucose and glycerol were used as the sole sources of carbon and energy to increase the cell density exponentially at controlled specific growth rates, which do not cause the accumulation of acetate. Thus, sophisticated feedback control or extra equipment to prevent the accumulation of toxic level of acetate is not necessary. The final cell densities of 100 and 118 gl-1 of dry cell mass for recombinant E. coli producing human interferon-g (hIFN-g) were obtained by using glucose and glycerol, respectively. The concentration of acetate was always maintained below toxic level. The specific yield of hIFN-g with glucose and glycerol was 93 and 92 mgg-1 of dry cell mass, and the overall productivity of hIFN-g was 0.16 and 0.14 gl-1h-1 for these two carbon sources, respectively.

Several reports indicate that the nonconserved genes of Helicobacter pylori (H. pylori) in particular its cytotoxin are widely heterogeneous among various geographic locations and this is manifested at the protein level ranging from 5-15% which demands access to locally deduced protein antigens for inclusion into diagnostic kits and/or inclusion as a vaccine component for the target population. We have previously demonstrated such variations via PCR-RFLP analysis between Iranian and western H. pylori strains. vacA gene from a selected strain of the most prevalent RFLP category among Iranian strains, was partially sequenced which revealed 8.3% dissimilarity with reference strains at protein level. This drastic difference prompted us to subclone the vacA coding region into an expression vector to produce the recombinant protein. Full sequencing of the coding region demonstrated 8-9% amino acid difference with American and German reference strains. Recombinant protein expression yielded 4% of the total E. coli proteins. Histidine tag allowed for purification of the recombinant VacA using immobilized metal affinity chromatography (IMAC). Identity of the recombinant protein was repeatedly confirmed by Western blot analysis using patient serum, rabbit hyper immune serum as well as anti-His monoclonal antibody.

Hemophilia B is an X-linked recessive bleeding disorder caused by heterogeneous mutations in factor IX gene. In about one-third of cases it arises by a new mutation in germ-line cells. In this study carrier testing was performed for females of a family with only one affected individual by single strand conformation polymorphism (SSCP). Results indicated that the SSCP band shift in the propositus was de novo and his mother and also sisters were not carrier. This finding was also confirmed by sequencing.