Genetics in the Third Millennium
Volume:10 Issue: 3, 2013

Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking acetylcholine release in motor neurons. Each BoNT is composed of a 50 kDa light chain of that acts as a metalloprotease، and of a 100 kDa heavy chain. The carboxy terminal domain of heavy chain (HC) that mediates the binding to specific cell surface receptors. The HC domain has considerable immunogenicity without any significant toxicity. In this study the BoNT/A& B-HC were joined together for producing a recombinant chimeric protein as a vaccine candidate. For this، the relative genes were synthesized in pET28a (+) expression vector separately. Then appropriate primers were designed to amplify the BoNT/B-HC gene. The restriction sites for NcoI and NdeI endonuclease were placed at 5'' end of forward and reverse primers respectively. The PCR products then were digested whit NcoI and NdeI enzymes. At the same time the construct of pET28a (+) –BoNT/A-HC gene was digested by the same enzymes. Then these digested fragments were joined together by T4 DNA ligase. The recombinant construct then introduced into E. coli DH5α strain. Our enzymatic digestion، PCR amplification and sequencing of recombinant construct confirmed correct fusion of BoNT/B-HC and BoNT/A-HC genes

Transgenic plastids offer unique advantages in plant biotechnology، including high-level foreign protein expression and posing lower environmental ricks. As regard to sequencing of lettuce plastid genome، many Homologous segments can be used for vector designing to production recombinant proteins in this plant. We have described the development of a plastid transformation system for lettuce، (Lactuca sativa L. TN-96-39-B). The transforming DNA carries a betaine aldehyde -resistance gene BADH cassette (lettuce rrn promoter-BADH- lettuce psbA terminator) under control of lettuce chloroplast regulatory expression elements، flanked by two adjacent lettuce plastid genome sequences allowing its targeted insertion between the rbcL and accD genes. This system will open up new possibilities for the efficient production of plasma proteins، enzymes، growth factors، recombinant edible vaccines and antibodies in lettuce.

Lipid transfer proteins (LTPs) are a family group of proteins with low average molecular weight in plants that have numerous potential applications especially in drug delivery systems. The aim of this research was cloning of LTP coding sequence in a prokaryotic expression vector to produce a fusion protein with GST in E. coli (TOP10). As there is no intron segment in rice LTP2 gene، specific primers were implemented to introduce BamHI and XhoI restriction sites at the head and tail of amplified LTP2 coding sequence respectively، using extracted rice genome as a template. After treatment by respected restriction endonucleases، the amplified fragment was inserted into the BamHI-XhoI sites in pGEX-6p-2 vector downstream of GST to make a chimeric DNA for further production of GST-LTP fusion protein. At the next step recombinant vector was transformed into TOP10 E. coli cells for amplifying purposes. To ensure the accuracy of gene cloning، extracted recombinant vector were sent for sequencing the inserted fragment. Data revealed that cloned fragment was bona fide sequence of rice LTP2 as was reported to NCBI. Results showed that PCR amplification of GC-rich DNA sequences could be improved by Betaine، DMSO and AMS. Finally expression of LTP2 in E. coli was confirmed by SDS-PAGE technique.

Securigera securidaca Degen&Dörfl (Fabaceae) is an annual herb occurring naturally in Europe، Asia and Australia. The seeds of this plant have been applied to a variety of ailments like diabetes and hyperlipidemia in Iranian folk medicine. Herpes simplex virus type 2 (HSV-2) is an enveloped virus which causes genital herpes infection and some other important complications such asencephalitis and eye infections. In present study antiherpetic activity of the seed crude methanol extract of S. securidaca was investigated for the first time. Methanol seed extracts were prepared; Therefore، extracts were added directly to the Vero cell suspension containing virus. To determine the inhibitory effects of methanol extract on herpes simplex virus type 2 Plaque forming assay was used. According to the results، methanol seed extract (20 µg/ml) exerted an inhibitory effect of 98±5. 1%. In conclusion، methanol seed extract of Securigera securidaca can be a good candidate as a natural source for antiviral drug development against HSV-2.

Glucose-6-phosphate dehydrogenase enzyme deficiency is one of the most frequent human genetic disorders so that more than 400 million people are affected worldwide. G6PD enzyme has a critical role in detoxification and neutralization of free radicals and other oxidative agents in cells، because it catalyzes the first step in pentose phosphate pathway that provides cells with NADPH required for detoxification mechanisms such as glutathione redux cycle. This gene is expressed in all body tissues but the major clinical complications are just seen in erythrocytes because these cells do not have supplies for production of new enzyme and their only source of NADPH is the pentose phosphate pathway. As a result erythrocytes are highly sensitive to oxidative stress. The main clinical manifestations of G6PD deficiency include hemolytic non-spherocytic chronic anemia، neonatal jaundice and acute hemolytic anemia induced by infection or ingestion of fava bean and certain oxidative drugs and chemical agents. This gene is located on distal long arm of X chromosome with a length of about 18Kb that consists of 13 exons and 12 introns. This gene is highly polymorphic so that about 400 biochemical variants and 170 mutations have been reported so far. According to previous studies the most prevalent mutations in Iran are Mediterranean، Chatham and Cosenza. In this paper the role and importance of g6pd and a review of studies on this enzyme in Iran will be described.

The hypothesis that the sex chromosomes evolved from a pair of autosomes has been accepted by many evolutionary geneticists. The latest findings revealed that، mammalian sex chromosomes evolved from a pair of autosome، 166 million years ago. While the X is highly conserved in size، gene content and gene order among different species of placental mammals، Y chromosome has been undergone many degeneration and lost more than 1000 genes during the evolution. It is predicted that، at the observed rate of attrition، the human Y chromosome will be gone within 5-10 million years. Stepwise cessation of recombination initiated with an inversion in the Y، is the main cause of Y chromosome attrition. Higher mutation rate due to special structure of Y، accumulation of transposable elements and inefficiency of selection in non-recombinant parts of Y for elimination of deleterious mutations، are the major causes of Y chromosome degeneration. It is predicted that after complete extinction of Y، a new sex determination should be evolved. In two groups of rodents، the mole vole (Ellobius) and the Japanese spiny rat (Tokudaya)، Y chromosome has been removed completely and a new sex determination system evolved. However، it is yet to be detected.