Tomato, which is one of the most important component of human diet, has been genetically modified to develop some properties like delayed ripening and insect resistance. In order to give a choice to the consumer, it is necessary to detect and label GM foods. This study was carried out to detect genetically modified tomato samples purchased from different food markets of Turkey. PCR method was used to detect genetically modified insect resistant tomatoes. The DNAs of collected samples were isolated according to CTAB DNA extraction protocol and the amplification capacity of isolated samples were checked with patatin specific control PCR. Screening tests of tomatoes were done by targeting 35S promoter, Nos terminator and NptII kanamycin resistance gene with four primer sets. It was aimed to detect Cry1A and Cry1Ac genes with three PCR systems, in order to identify insect resistant samples. From twenty-eight samples, twenty-two gave positive amplification signal in NptII specific PCR system and results were confirmed with sequence analysis. Additionally, we observed seventeen and ten DNA fragments with Cry1A-F/Cry1A-R and Cry1Ac-F/Cry1Ac-R primer sets respectively, it is necessary to confirm these results with DNA sequencing.

In recent years, foods produced by genetic engineering technology have been on the world food market. The biosafety aspects, regulations, and labelling of these foods are still contentious issues in most countries. It is necessary to have approval for the use of GMOs in the production of food. Thus, detection and quantification of GMOs play crucial role for developing regulations on GM foods. In this study, raw and processed maize samples were analysed for genetic modification using a DNA based detection method, the Polymerase Chain Reaction. Ten raw food and 18 processed maize food including maize flour, starch, corn flakes, maize chips were collected from different markets located in different places in Turkey. The samples were examined for the presence of genetic elements located in the majority of transgenic crops such as NOS terminator, CaMV 35S promoter, kanamycin resistance (KanR) gene, using conventional PCR with oligonucleotide sets targeting to novel genes. Furthermore screening was conducted via Real-Time PCR assay for NOS terminator and 35S promoter. For confirming the presence of Bt11 maize lines event specific primers were utilised. Quantification of Bt11 maize lines were performed via Real-Time PCR. The result indicates that foreign genetic elements were found in all analysed raw material. In six out of 10 raw material, presence of Bt11 gene were identified. GMO detection was also possible for maize flour and starch, however in processed material as corn starch, corn flakes, corn chips and pop corn, transgenes were not detected.

In highway bridge construction, composite curved girder bridges are becoming more popular recently. Reduced construction time, long span coverage, economics and aesthetics make them more popular than the other structural systems. Although there exist some methods for the analysis of such systems, each have shortcomings. The use of Finite Element Method (FEM) among these methods is limited except in the academic environments. The use of commercial FEM software packages in the analysis of such systems is cumbersome as it takes too much time to form a model. Considering such problems a computational software was developed called UTRAP in 2002 which analyzes bridges for construction loads by taking into account the early age deck concrete. As the topic of this thesis work, this program was restructured and new features were added. In the following thesis work, the program structure, modeling considerations and recommendations are discussed together with the parametric studies.

This thesis is focused on the development of a gene delivery system, especially for the purpose of DNA vaccination. DNA expression vectors have the potential to be useful therapeutics for a wide variety of applications. A carrier system was designed to realize the delivery of genes to cells and the promotion of controlled adequate expression in the target cells. The low gene delivery efficiency observed with systems composed of polyplexes is mainly due to low stability of polycation e.g polyethylenimine-DNA complexes and inability of most of the complexes to the reach nucleus after entering the cells. The encapsulation of polyethylenimine-DNA complexes inside the alginate microspheres was expected to provide protection from nuclease-based attack, thereby, increasing the stability of the complex and also to achieve controlled release of the complex at the target tissue. In this study, controlled release of complexes from alginate microspheres was studied with DNA staining. In Tris-HCl buffer, the release of PEI-DNA complexes were completed in 48 h, however in cell culture medium (DMEM) 18 % of complexes were released in 48 h because of presence of Ca+2 ions in DMEM. Also, in order to provide mucosal gene delivery for mucosal immunization polyethylene glycol (PEG) was introduced into the composition of microspheres and the two systems were compared in terms of release kinetics of the complexes. In the presence of PEG, release of PEI-DNA complexes from alginate microspheres in the cell culture medium (DMEM) were enhanced and 50 % of PEI-DNA were released from the microspheres in 48 h. To understand the effect of the PEG on the surface of microspheres zeta potential analysis and microscopic examination were carried out. By increasing percentage of PEG (0, 15, 30, 50) in microspheres, less negative zeta potential value were measured. Mucoadhesion of alginate and PEG-alginate microspheres were evaluated by using modified microbalance method, and in the presence of PEG enhancement of mucoadhesion was observed. In this way a gene delivery system with a possible route through mucosa of tissues was prepared.

Pinus nigra subsp. pallasiana is one of the most widespread and economically important forest tree species in Turkey. Primary objective of the present study was to to reveal the effects of forestry practices by determining genetic diversity of natural and managed seed sources by means of RAPD markers. Secondly, two varieties were also investigated to reveal their pattern of genetic variation. Seed stands, seed orchards and plantations were screened against 11 RAPD primers and generated 152 polymorphic DNA loci. Two varieties were compared with a reference seed source and 4 natural seed sources. Seven primers generated 66 polymorphic DNA loci. An overall average for effective number of alleles was 1.68&plusmn / 0.030 / observed heterozygosity was 0.49&plusmn / 0.024 / expected heterozygosity was 0.38&plusmn / 0.014 and proportion of polymorphic loci was 93% for all seed sources considered. Results revealed that there was no considerable variation between seed source categories but some degree of variation was observed within seed orchards and plantations. Mean FST value estimated for the natural populations revealed that 94% of the total genetic variation was within populations. Nei&rsquo / s genetic distance values were also estimated for seed source categories (0.03-0.14). Nevertheless, varieties&rsquo / genetic distance values were considerably higher than other natural seed sources (0.07-0.19). Their dendrogram also claimed that two varieties are genetically different from natural populations. The extent of genetic diversity explored by RAPD markers revealed that forestry practices caused no major changes in the managed populations with respect to natural populations. Moreover, further study is needed to illustrate genetic divergence of varieties.

Yellow rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriksson is one of the most severe leaf diseases of wheat. Aim of this study is to illuminate the downstream signaling pathways upon incompetible infection of rust pathogen in wheat, thus to understand the genes involved in resistance mechanism. The strategy used is the dual bait yeast two-hybrid analysis which is the most powerful method for in vivo detection of protein-protein interactions. The bait proteins used are / the domains of Yr10 yellow rust resistance gene, Rad6 gene which is considered to have a critical role in R gene mediated signaling pathway, and WR5 gene fragment which is an unknown protein having homology to the WD40 repeat containing protein with apoptosis related activity. Screening of a yeast prey library with these baits revealed proteins having mostly apoptosis related functions (SRP72, POR1, CSE1), translation initiation control in response to stress conditions (Gcn2p, Eap1p), phosphorylation (SKY1) and dephosphorylation activities (GAC1), cell cycle control (FAR1), oxidative stress control (OXR1), protein degradation control (TOM1), protein folding control (CPR7) and ion homeostasis in the cell (POR1, GAC1). The significance of the study can be summarized as i) being the first yeast two hybrid analysis of a wheat R gene, ii) being able to detect interacting partners with anticipated functions, iii) most importantly, initiating further detailed analysis of the key interactors.

Kangal dogs are the most popular dogs of Turkey due to their strength, intelligence, loyalty, endurance to extreme temperatures and their lack of predatory behavior towards livestock. In this study to provide genetic information about the distinctness of Kangal and Akbash dogs and hence to provide a basis to conserve them separately, 585 base pair of the mitochondrial DNA (mtDNA) control region sequence was analysed in 105 Kangal and 9 Akbash dog samples. All the results indicated that Kangal and Akbash dogs were different from each other. Comparison of the Turkish data with those from other dogs revealed that Kangal dogs harbour a rare haplogroup which is only seen in Scandinavian (36%), Portuguese (20%), Turkish (20%) dogs and only one Spanish dog, but not in Akbash, Middle Eastern, other European, Eastern Asian and Indian dogs. Furthermore, comparison of the Kangal and Akbash dogs with the dogs from different geographic regions indicated that Kangal dogs are genetically closer to Scandinavian and South West Asian dogs whereas Akbash dogs are more similar to European and East Asian dogs, based on the mtDNA control region sequences Today, the sizes of Kangal and especially Akbash populations are decreasing. An urgent program for their conservation is needed. In order to conserve them separately, it must be understood that these two dogs are genetically distinct. That is why, the main purpose of the present study is to provide genetic information about the distinctness of Kangal and Akbash dogs.

Facultative phototrophic bacterium Rhodobacter capsulatus has two c-type electron carrier cytochromes (cyt) / the soluble cyt c2 and the membrane-attached cyt cy, that act as electron carriers during respiratory and photosynthetic growth of this species. Previously, a soluble form of cyt cy was constructed by fusing genetically the signal sequence of cyt c2 to the cyt c domain of cyt cy. The obtained novel soluble cyt cy (cyt S-cy) was unable to support photosynthetic growth of R. capsulatus but yielded photosynthetically functional (Ps+) revertants frequently. In the first part of this study, photosynthetic electron transfer properties of some of Ps+ revertants of cyt S-cy were analyzed by biochemical and biophysical methods and compared with the cyt cy and cyt c2. Reduction-oxidation titration of membrane supernatants showed that the redox midpoint potential of cyt S-cy was +338 mV which is similar to midpoint potentials of cyt cy or the cyt c2. However, light-activated, time resolved spectroscopy revealed that reaction center mediated oxidation kinetics of cyt S-cy exhibited only a slow phase, unlike cyt c2 which has both fast and slow phases. It therefore appeared that during electron transfer cyt S-cy does not interact with the reaction centre as tightly as cyt c2. These findings imply that attaching electron carrier cyts to the membrane allowed them to weaken their interactions with their partners, while restricting their spatial diffusion, so that they accomplish rapid multiple turnovers. In the second part of this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes, cyt cbb3 deleted and Qox deleted strains were compared with the wild type. Under photoheterotrophic growth conditions with limiting nitrogen source, the excess reducing equivalents generated by organic acid oxidation are consumed to reduce protons into hydrogen by the activity of nitrogenase in R. capsulatus. The results indicated that the hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3-5 folds, and the hydrogen production rate of the cyt cbb3- mutant increased significantly. Moreover in this study, the hydrogen production efficiency of different R. capsulatus strains was increased by the chromosomal inactivation of uptake hydrogenase genes and enzymatic activity of uptake hydrogenase of R. capsulatus strains were determined.

Acid proteases, commonly known as aspartic proteases are degredative enzymes which catalyze the cleavage reaction of peptide bonds in proteins with a pH optimum in the acidic range (pH 3-4). Acid proteases have crucial roles in metabolism. Moreover, they are used in different fields of industry. Thermophilic microorganisms, especially archaea, gain special interest because of their thermal stability for both fundamental and industrial researches. Thermopsin is an extracellular acid protease and a member of A5 family of proteases. This thermophilic enzyme has no characteristic active aspartyl residue, is insensitive to pepstatin and no apparent sequence homology to other acid proteases and therefore represents a new class of acid proteases. Thermophilic archaeal strain Thermoplasma volcanium GSS1 (optimum temperature 550C and pH 2.7) in the genome has a putative thermopsin gene encoding 998 amino acid enzyme. In this study thermopsin gene from Thermoplasma volcanium was expressed in E. coli as fusion with 6xHis tag under the control of T5 transcription/translation system. Putative thermopsin gene from Thermoplasma volcanium was amplified by PCR method using two primer sets and cloned. A 3080 bp and a 3070bp PCR products were obtained by using TP1/TP2 primer set (thermopsin gene with the start codon) and TP1&rsquo / /TP2 primer set (thermopsin gene missing start codon) respectively. PCR amplified thermopsin genes pDrive and pUC18 vectors in E. coli TG1 were cloned using and then cloned genes were sub-cloned directionally into pQE triple vector set for expression. In these expression vectors, cloned genes are placed downstream of a 6XHis tag to produce an expression fusion. E.coli strains (M15[pREP4], SG13009[pREP4], and TG1) used as hosts. Recombinant colonies screened by colony blot/hybridization method based on immunological detection of the expressed 6XHis tag fusion by Anti-His HRP conjugates which are specific for 6xHis tag, and DAB chromogenic substrate was used for colony blot procedure. PCR amplified thermopsin gene containing 3080bp could not expressed in pQE30 and 31 vectors in TG1 strains. It is thought that pQE32 open reading frame can be true for thermopsin gene (3080bp). Three expression constructs, pQE31-1, pQE31-4 and pQE31-6 plasmids containing PCR amplified 3070bp thermopsin gene were confirmed as true recombinant plasmids according to both colony blot hybridization result and restriction digestion profile the agarose gel.

In this study, we have characterized some biochemical and electrophoretic features of recombinant 20S Proteasome from a thermoacidophilic archaeon Thermoplasma volcanium. As revealed by SDS-PAGE the 20S Proteasome was composed of two subunits, &amp / #945 / - and &amp / #946 / - subunits with estimated molecular masses of 24 kDa and 23 kDa, respectively. The highest chymotryptic activity was observed over an alkaline pH range (pH 8.0 &ndash / pH 9.0) and the optimum temperature for the activity was determined as 85oC. The heat stability of proteasome was quite high after treatment at 98oC for 30 minutes, 64 % of the activity has still been retained. The highest activity associated with the Thermoplasma volcanium proteasome was found to be peptidylglutamyl peptidase activity. Within the scope of this project, also, we have cloned a 26S Proteasome related Regulatory Subunit gene of Thermoplasma volcanium. For cloning we have followed a PCR based approach. Amplification of 26S Proteasome Regulatory Subunit gene from chromosomal DNA of Tp. volcanium yielded a product of 1419 bp containing an open reading frame of 1128 bp comprising the structural gene. The PCR amplicon was cloned using pDrive vector in E.coli TG-1 cells. Out of ten putative recombinants, three plasmids, E.coli pD1-6, E.coli pD2-3, E.coli pD3-1, were proved to be true recombinants and selected for further characterization by restriction mapping and expression studies. ATPase activities of cell free extracts from both recombinant and non-recombinant E.coli strains were measured and found that ATPase activities in cell free extracts of recombinant strains were 10 times higher than non-recombinants. This result indicates sucessful expression of the cloned regulatory subunit gene with ATPase activity in E.coli.