Anoxybacillus flavithermus DSM 2641T bakterisinin termofilik GCH-I enziminin klonlanması ve kinetik aktivitesinin belirlenmesi

Phenylketonuria (PKU) is a disease caused by phenylalanine hydroxylase enzyme deficiency in newborn infants and is the most important cause of treatable mental retardation. One of the causes of the disease comes from the defects of the PTPS in the metabolic pathway of phenylalanine metabolisms. Treatment of the disease is not feasible, and life-time tetrahydrobiopterin loading is performed in chronic patients. Today, tetrahydrobiopterin is chemically synthesized. Biological production is a different point of view due to the long duration of chemical synthesis, costs, and exposure to chemical pollutants. NH2TP is a difficult substance to obtain in studies that are difficult to synthesize and need to be used as a substrate. In this study, it was aimed to clone the gch-I gene from the thermophilic A. flavithermus bacterium and to investigate the activity of the enzyme for the enzymatic conversion of NH2TP, which is the substrate of many enzymes and is difficult to synthesize chemically. Fort his purpose, it was aimed to clone the gch-I gene from a thermophilic bacteria, Anoxybacillus flavithermus, and investigate the kinetic activity for enzymatic conversion from GTP as an alternative to the production of NH2TP. For this reason, the gch-I gene from the thermophilic A. flavithermus DSM 2641T was identified by PCR method. We cloned the gchI gene that was 603 bp and its open reading frame has 200 amino acids. The gene was cloned into pET28a(+) expression vector with 6xHis tags and transform in E. coli BL21(DE3)pLys host cells to express with 1 mM IPTG induction. After purification with Ni-NTA resin, we determined that GCH-I is 24 kDa, its optimum pH is 8.0 and temperature is 65C. Under optimal conditions, GCH-I exhibited enzymatic activity with Kmand Vmax- values of 243 ± 23.25 μM and 100.93 ± 3.5 nM/min/mg protein, respectively.: Fenilketonüri (FKU) yenidoğan bebeklerde fenilalanin hidroksilaz enzim eksikliğinin neden olduğu bir hastalıktır ve tedavi edilebilir zihinsel geriliğin en önemli nedenidir. Hastalığın nedenlerinden biri, fenilalanin metabolizmasının metabolik yolundaki PTPS'nin kusurlarından kaynaklanmaktadır. Hastalığın tedavisi mümkün değildir ve kronik hastalarda yaşam boyu tetrahidrobiyopterin yüklemesi yapılır. Bugün, tetrahidrobiyopterin kimyasal olarak sentezlenir. Biyolojik üretim, kimyasal sentezin zaman alıcı olması, maliyetler ve kimyasal kirleticilere maruz kalma nedeniyle farklı bir bakış açısıdır. NH2TP, kimyasal sentezi güç olan ve substrat olarak kullanılması gereken çalışmalarda temini zor bir maddedir. Bu çalışmada, bir çok enzimin substratı olan ve kimyasal yolla sentezlenmesi güç olan NH2TP’nin eldesinde alternatif olarak GTP’den enzimatik dönüşümü için termofilik A. flavithermus bakterisinden gch-I geninin klonlanması ve enzimin aktivitesinin araştırılması amaçlanmıştır. Bu amaç doğrultusunda, termofilik A. flavithermus DSM 2641T 'den gchI geni PCR yöntemi ile tanımlandı. 603 bp olan gchl genini klonlandı ve açık okuma çerçevesi 200 amino aside sahip olduğu tespit edildi. AfgchI geni, 6xHis etiketleri ile pET28a(+) ekspresyon vektörüne klonlandı ve 1 mM IPTG indüksiyonu ile eksprese etmek için E. coli BL21(DE3)pLys konakçı hücrelerine transforme edildi. Ni-NTA afinite kromatografisi ile saflaştırıldıktan sonra GCH-I’in 24 kDa, optimum pH’sının 8,0 ve optimum sıcaklık isteminin 65C olduğu belirlendi. Optimal koşullar altında GCH-I, Km- ve Vmax- değerleri sırasıyla 243 ± 23,25 μM ve 100,93 ± 3,5 nM/dak/mg protein olarak tespit edildi

Analysis of morphological, micromorphological and molecular systematic properties of Rubus l. (Rosaceae) species in Rize province

Bu çalışma ile Rize ilinde yayılış gösteren 8 Rubus türü morfolojik, mikromorfolojik ve moleküler sistematik yönden araştırıldı. Bitki materyalleri 2019 yılında yapılan arazi çalışmaları ile toplandı. Araştırma bölgesinden toplanan örnekler teşhis edildi ve herbaryum örneği haline getirildi. Bu türlerin gövde, yaprak ve çiçek gibi kısımları stereomikroskopta morfolojik, SEM’de ise mikromorfolojik olarak incelendi. Sonrasında örneklere ait ITS bölgeleri PZR ile çoğaltılarak dizi analizi yapıldı. Analiz sonucu 8 tür arasındaki benzerlik ilişkisini ortaya koyan filogenetik ağaç oluşturuldu. Mikromorfolojik görüntülere bakıldığında 8 tür arasında gövde, yaprak tüylenmesi, tüy tipleri ve diken yapısının ayırıcı özellikte olduğu tespit edildi. Morfolojik bulgular sonucunda 8 Rubus türünün yaprak, meyve, diken, petal, yaprak ve yaprakçık ölçüsü, meyve tanelerinin yapısı, sayısı ve şekli, yaprak alt ve üst yüzey, petal yüzeyi tüylenme durumu, meyve ve sepallerin rengi, yaprak kenarı yapısı bakımından birbirinden ayrıldığı, bu özelliklerin karakteristik olduğu belirlendi. Rubus türlerinin dizi analizi sonucunda elde edilen moleküler bulgularda ortalama baz uzunluğunun 709 ile 731 bp arasında değiştiği belirlendi. Oluşturulan filogenetik ağaç neticesinde Rosacea familyasına en yakın türlerin Rubus hirtus ve Rubus tereticaulis, en uzak türün ise Rubus sanctus olduğu belirlendi.Rubus species in Rize province were compared morphologically and micromorphologically along with molecular systematics of plants in this work. Analysed plant materials were sampled during field studies conducted in 2019. The collected samples were identified according to known methods and were transferred into herbarium plants. A total of 8 taxa belonging to Rubus species were found. Plant parts such as stem, leaves and flowers were examined morphologically and micromorphologically under stereo microscope and SEM. Then, plant genomic DNA was isolated from fresh leaf samples by suitable methods to determine ITS profiles. Then the ITS regions were amplified by PCR and sequenced. A phylogenetic tree showing the similarity among this 8 species was then created based on the sequence analysis. The stem, leaf trichomes, trichome types and thorn structure were found to be distinctive among these 8 species when micromorphological images were examined. As a result of morphological findings, Rubus plants constitute 8 distinct species in terms of leaf, fruit, thorn, shape, petal, leaf and leaflet size, structure, number and shape of fruit grains, leaf adaxial and abaxial surface, trichome amount of petal surface, color of fruit and sepals and leaf edge structure. These features were characteristic for all the species examined. Molecular data showed that the average base length of Rubus species was found to be between 709 and 731 bps. Created phylogenetic tree revealed that the species closest to the Rosacea family were Rubus hirtus and Rubus tereticaulis while the most distant species were Rubus sanctus

Evaluation of genetic diversity of cultivated tea clones (Camellia sinensis (L.) Kuntze) in the eastern black sea coast by inter-simple sequence repeats (ISSRS)

SANDALLI, Cemal/0000-0002-1298-3687; PEHLIVAN, NECLA/0000-0002-2045-8380WOS: 000376744800008Tea is the most globally consumed drink after spring water and an important breeding plant with high economical value in Turkey. in half a century, various kinds of tea cultivars have been bred in Turkey to improve the quality and yield of tea plants. Since tea reproduces sexually, tea fields vary in quality. Thus, determining the genetic diversity and relationship of the plants to support breeding and cultivation is important. in this study we aimed to determine the genetic diversity of tea cultivars breeding in the Eastern Black Sea coast of Turkey and the genetic relationship between them, to verify whether the qualitative morphological designations of the clones are genetically true by the ISSR markers. Herein, the genetic diversity and relationships of 18 Turkish tea cultivars were determined using 15 ISSR markers with sizes ranging from 250 to 3000 base pairs. the similarity indices among these cultivars were between 0.456 and 0.743. Based on cluster analysis using UPGMA, some of tea cultivars originating from the same geographical position were found to be clustered closely. Our data provide valuable information and a useful basis to assist selection and cloning experiments of tea cultivars and also help farmers to find elite parental clones for tea breeding in the Eastern Black Sea coast of Turkey.Recep Tayyip Erdogan UniversityRecep Tayyip Erdogan University [BAP_2013.102.03.4]This work was supported by Recep Tayyip Erdogan University Research Fund Grants BAP_2013.102.03.4

Effect of microwave irradiation on the synthesis of 1,2,4-triazol-3-one derivatives and their antimicrobial activities

mentese, emre/0000-0003-4105-8666WOS: 000308541900014An easy efficient method for the synthesis of N-(3,5-dichlorophenyl)-5-alkyl/aryl-3H-1,2,4-triazol-3-one derivatives under microwave irradiation has been developed using the reaction of 3,5-dichloroaniline and ethoxycarbonylhydrazones. This reaction occurs more efficiently and faster than the conventional heating method. We obtained 5-mercapto-1,3,4-oxadiazol derivatives by this method. the newly synthesised compounds have been tested for their antimicrobial activity against Enterobacter cloaceae, Escherichia coli, Klebsiella pneumonie, Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhimurium, Yersinia pseudotuberculosis, Bacillus subtilis and Staphylococcus aereus

Microwave-assisted synthesis of some new benzimidazole derivatives with their antimicrobial activity

WOS: 0002185277000062-(2-Chlorobenzyl)-1H-benzimidazole (2) was prepared from the reaction of o-phenylenediamine and ethylimido-o-chlorophenylacetate hydrochloride (1) under microwave irradiation. the acetate and hydrazide derivatives of 2(2- chlorobenzyl)-1H-benzimidazole (2) were synthesized respectively. Then, 2-[2-(2-chlorobenzyl)-1H-benzimidazol-1yl]-N-(2,5-dioxopyrrolidin-1-yl) acetamide (5), 5-{[2-(2-chlorobenzyl)-1H-benzimidazol-1-yl]methyl}-1,3,4-oxadiazole2-thiol (6), acetohydrazide (7a-i) and N-phenylhydrazinecarbo thioamide derivatives (8a-c) were synthesized from hydrazide derivatives. Finally, the intramolecular cyclization of N-phenylhydrazinecarbo thioamide derivatives (8a-c) gave the corresponding 1,2,4-triazole-3-thione derivatives (9a-c). the newly synthesized compounds have been tested for their antimicrobial activity against three Gram-positive and three Gram-negative bacterias

Distribution and molecular differentiation of Culex pipiens complex species in the Middle and Eastern Black Sea Regions of Turkey

Culex pipiens species complex is commonly found in tropical and subtropical areas. Identification of species complexes and their spatial distribution patterns are fundamental steps needed to improve our understanding of arboviral disease circulation and their control. The specimens were differentiated using the Ace-2 gene, and the ecological forms of Culex pipiens were identified by analysis of the CQ11 microsatellite locus. Results showed that all sample points comprised of Culex pipiens s.s., and seven points contained Culex torrentium. Further, we determined that 22 haplotypes from five haplogroups were identified within the Ace-2 marker, and three haplotypes were contained within the CQ11 microsatellite locus. We determined that there was a high degree of the genetic flow of each marker between populations. In conclusion, Culex pipiens complex and sibling species were determined to be distributed throughout the coastal and inland parts of the Black Sea region in Turkey. The occurrence of two distinct ecological forms has the potential to create hybridization zones and a high risk of WNV transmission

Monitoring and distribution of kdr and ace-1 mutation variations in Culex pipiens L., 1758 (Diptera: Culicidae) in artificial sites and agricultural fields in the central and eastern Black Sea Region of Türkiye

Culex pipiens L., 1758 (Diptera: Culicidae) is one of the most important pests and disease vectors in the world. It is of major importance to monitor the development of insecticide resistance in order to effectively control. This study investigated the presence of mutations in specific loci of the Vgsc (kdr L1014F/C) and ace-1 (G119S, F290V) gene, associated with insecticide resistance in Culex pipiens collected from nine provinces in central and eastern Black Sea Region of Turkiye in the 2020 active season. For kdr, L1014F mutation was determined for each region with three different silent mutations for wild and resistant type alleles, while L1014C was not recorded in any of the analyzed populations. For ace-1, substitution F290V was detected at a low frequency in heterozygosity, while G119S was more widespread, in the analyzed populations. For ace-1, G119I (6 populations) and G119A (5 populations) substitution was firstly described. Types of mutations differences related to the resistance between artificial sites and agricultural fields were not significantly different