Iranian Journal of Biotechnology
Volume:2 Issue: 3, Summer 2004

Preimplantation genetic diagnosis (PGD) is a novel approach for the prevention of genetic disorders in couples at risk of having offspring with genetic disease. Although the original idea dates back to early 1960s when sexing of rabbit blastocysts was attempted, the first clinical application of PGD was reported about three decades later, describing the use of PCR for sexing embryos from couples at risk of X-linked disease. The development of PCR-based tests led to PGD for screening well known monogenic diseases such as thalassaemia and cystic fibrosis. The introduction of fluorescence in situ hybridization (FISH) quickly replaced PCR-based methods which had led to misdiagnoses for sexing embryos. FISH can be used for aneuploidy screening of up to seven clinically significant chromosomes and translocation detection. The advent of molecular genetic techniques has brought forth new procedures for in situ chromosomal analysis. One of these techniques is the primed in situ labeling (PRINS) procedure which constitutes a fast and efficient alternative to conventional fluorescence in situ hybridization for nucleic acid detection. This technique has the potential to become a powerful tool for cytogenetic investigations. The recent achievements reported show that PRINS can constitute an efficient complement to PCR and FISH. Adaptation of this technique to preimplantation embryo screening both at chromosomal level and gene localization opens a promising perspective for being used in the field of PGD.

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. A gene construct was generated, consisting of 10.7 kbp of the ovine beta-lactoglobulin (oBLG) gene including its promoter and 3 flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. The gene construct was purified using CsCl gradient, released from vector, and gel-purified. It was microinjected into fertilized mouse oocytes. These oocytes were then transferred to pseudo-pregnant foster mice. The pups born from foster mice were genotyped using PCR, slot blot, and Southern blot techniques. Among 9 mice which showed positive PCR results, only 6 mice transmitted the transgene to the next generation. Therefore, 6 transgenic lines were established which stably transmitted their transgene to their progeny.

We have developed an allotyping assay for detection of four HLA DRB1*01 group alleles based on polymerase chain reaction and solution hybridization in a microtiter plate. Using group specific primers a region within exon 2 of HLA DRB1 gene was amplified by PCR. Labeling of PCR product was achieved by adding small amount of Dig-dUTP in place of dTTP. Labeled PCR product was hybridized to allele (HLA DRB1*0101, *0102, *0103 and *0104) specific and a group (HLA DRB1*01) specific oligonucleotide probes in separate wells of the plate. Hybridized amplicones were detected by an enzymatic procedure. Ninety DNA samples were tested in parallel with PCR-SSP typing. The results were found to be well correlated by two methods. These results further suggest that, PCR-ELISA would be a rapid, specific and simple method that can be used for high resolution HLA typing before bone marrow and stem cell transplantation.

Toxoplasmosis is a worldwide infection which is commonly asymptomatic but can cause serious health problems in immunocompromised individuals and fetus. GRA2 is a dense granule protein of Toxoplasma gondii, which induces strong antibody and T-cell response in human and mice. The purpose of this study was to prepare recombinant GRA2 and evaluate its antigenic properties using infected mice sera. To reach this point, GRA2 gene was highly expressed as a fusion protein with Thioredoxin (TRX) in Escherichia coli BL21pLysS strain. The protein was purified in a single step on Ni-NTA affinity column. TRX-GRA2 was confirmed by Western blot technique using a monoclonal antibody specific for GRA2. Sera from mice infected with Toxoplasma gondii showed high reactivity toward GRA2 and the level of IgG2a isotype was predominant and significantly higher than IgG1.Taken together, TRX-GRA2 might be considered as an ideal antigen to be used as a vaccine target as well as diagnostic tool for detection of toxoplasmosis.

GAL gene family is a set of structural and regulatory genes that enables cells to utilize galactose as a carbon source in Saccharomyces cerevisiae. Phospho-rylation of intracellular galactose can be catalyze by galactokinase (encoded by GAL1 gene). In this study role of GAL1 gene on ethanol production by S. cerevisiae together with physiological characterization of GAL1 mutant strain was studied. Aerobic cultivation was carried out with wild-type strain and the GAL1 mutant. The GAL1 mutant strain displayed fermentative growth in early exponential phase. Deletion of the GAL1 gene was shown to have a major impact on biomass and ethanol formation. The GAL1 mutant exhibited a decrease in growth rate and increased ethanol production. Furthermore the results showed that glucose consumption by GAL1 mutants did not favor biomass formation, rather cause excessive respiro-fermentative metabolism, with whereas could linear increase in ethanol production.

Botulinum neurotoxins constitute a family of bacterial toxins for botulism syndrome in human. The toxins bind with high affinity to nerve cells where they cause a complete inhibition and release of neurotransmitters and thereby produce flaccid paralysis. In this study the binding domain of type E neurotoxin was isolated by PCR and expressed in a proper expression vector. The results of this investigation can be used as a tool to study the mechanism of binding of holotoxins. This study is also implicated in antibody production against botulism syndrome.

A simple preparative method was developed for purification of Tyrosinase from edible mushroom (Agaricus bispora). Homogenized extract of mushroom was first saturated by ammonium sulfate. The desired precipitate was mixed thoroughly with DEAE-Cellulose (DE-52) and washed out to produce melanin free precipitate. The obtained protein solution was dialyzed against running water for 4 hrs, then, concentrated and chromatographed on a DE-52 column. On the basis of the activities assay, the eluted fractions by 150 mM salt solution were selected for further purification. The collected fractions were pooled and chromatographed on a Sephadex G-200 column. Polyacrylamide gel electrophoresis (PAGE) of the purified tyrosinase produced a single band right beside the commercial sample obtained from Sigma Company at 128 kDa. The lyophilized form of the purified Tyrosinase had a purification degree of 104 and showed strong cresolase and catecholase activities when compared to a commerically available tyrosinase.

Two different DNA-based techniques viz, randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to estimate genetic diversity among bread wheat. A total of 188 clear and repeatable bands were amplified from 17 selected RAPD primers, and 101 fragments were detected from 35 SSR primer pairs. The level of polymorphism was 88% with RAPDs compared to 100% with SSRs. Mean genetic similarity was estimated to be 0.88 based on RAPDs and 0.85 using SSRs. The wide range of genetic similarity was obtained by SSR than RAPD, reflecting the hypervariability of SSR markers and their high resolution power. Matrix correlation analyses suggested that a good representation of the relationships among the bread wheat cultivars/lines can be obtained by using RAPDs alone or in combination with SSRs, but SSRs alone cannot be used for this purpose. Both techniques discriminated the genotypes very effectively. On the hand, RAPDs were able to discriminate the cultivars Alvand and Ghods, whereas the cultivars Sardari and Ghods were discriminated only by SSRs. The use of PCR-based assays having advantage of being quick, easy to use and refractory to many environmental influences can complement traditional methods of germplasm characterization.

NAD(P)H: quinone oxidoreductase (NQO1) plays an important role in detoxification of numerous endogenous and foreign compounds. This gene has a single nucleotide polymorphism at site of codon 187 (CCT? TCT). Recently, it has been demonstrated that individuals with T allele may exhibit resistance to quinone based anticancer drugs such as mitomycin C. In the present study, a simple and feasible method was developed for detection of NQO1 genotype. In this modified procedure, dimethylsulfoxide (DMSO) and Triton X-100 were eliminated, also, PCR cycling conditions were modified to improve the PCR products from blood and formalin-fixed, paraffin-embedded tissues. PCR-RFLP and DNA sequencing analysis carried out on a limited number of blood and archival samples. It is suggested that this procedure convenient for NQO1 genotyping.