Ayberk AKAT, Dilek BALIK TURGUT, Münir AKTAŞ, Nazir DUMANLI

Isolation, cloning and sequence analysis of enolase enzyme encoding gene from theileria annulata for assessment of important residues of this enzyme

Bu yıl ilk kez önemli bir antitheilerial ilaca karşı direnç geliştiği rapor edilmiştir. Yeni antitheilerial ilaçların veya aşıların geliştirilmesi amacıyla Theileria annulata genomik DNAsından enolaz geni izole edilmiş, literatürde ilk kez klonlanmış ve web tabanlı araçlar kullanılarak hem nükleotid hem de amino asit seviyesinde analiz edilmiştir. Bu analizler genin 1365 nükleotidden oluştuğunu ve 40-41 rezidüleri arasında bir intron dizisi içerdiğini göstermiştir. Klonlanan genin restriksiyon enzim haritalama analizinde, TaENO Elazığ soyundakiçifti değişikliklerinin, Ankara soyu ile karşılaştırıldığında, geni kesen ve kesmeyen enzimlerde farklılıklar yarattığı gözlemlenmiştir.farklılıklar, farklı soyların restriksiyon haritalama ile tanımlanmasına yardımcı olabilir ve T. annulata soylarının herhangi bir bölgedeki coğrafik dağılımının belirlenmesini mümkün kılabilir. T. annulata enolaz geni dizisi ile konak Bos taurusun kas enolaz izoformu karşılaştırıldığında, T. annulatada bulunan 4 farklı insersiyonun B. taurus enolazında bulunmadığı belirlenmiş ve bu çalışmanın önemli bir bulgusu olarak rapor edilmiştir. T. annulata enolazının modelleme çalışmaları bu insersiyonların B. taurusta bulunmayan halkalar oluşturduğunu göstermiş ve bu halkaların enzim inhibitörleri için spesifik bağlanma bölgeleri olabileceği önerilmiştir.

Theileria annulatanın Enolaz Enzimini Kodlayan Geninin, Önemli Rezidülerinin Değerlendirilmesi Amacıyla İzolasyonu, Klonlanması ve Dizi Analizinin Yapılması

Drug resistance against one of the important antitheilerial drugs has been reported for the first time in 2010. For the aimdeveloping new antitheilerial drugs or vaccines, enolase gene was isolated from the genomic DNA of Theileria annulata, clonedthe first time in the literature and analyzed at nucleotide and amino acid levels by using different web based tools. These analyses showed that the gene was consisted of 1365 nucleotides including an intron sequence placed between residues 40-41. Restriction enzyme mapping analysis of the cloned gene showed that, base pair changes in TaENO Elazig strain caused differences on cutting and non-cutting restriction enzymes compared to the Ankara strain. These differences may help the identification of different strains by restriction mapping and it may be possible to determine the geological distribution of T. annulata strains in any region. Ascomparison of enolase gene sequences from T. annulata and the muscle enolase isoform of the host Bos taurus was made, four different insertions in T. annulata enolase that do not exist in B. taurus enolase was reported as an important discovery of this study. The modeling studies on T. annulata enolase gene showed that these insertions constituted loops that do not exist in B. taurus enolase, suggesting that these loops could be specific binding sites for enzyme inhibitors.

___

Brown CGD: Control of theileriosis (Theileria annulata infection) ocattle. Parassitologia, 32, 23-31, 1990.
Altay K, Aktas M: Sığır Theileriosisi. FÜ Sağlık Bil Derg, 18 (2): 79-862004.
Mhadhbi M, Naouach A, Boumiza A, Chaabani MF, BenAbderazzak
Darghouth MA: In vivo evidence for the resistance of Theileria annulata o buparvaquone. Vet Parasitol, 169, 241-247, 2010.
Dolan TT, Injairu R, Gisemba F, Maina JN, Mbadi G, Mbwiria SKMulela GHM, Othieno DHA: A clinical trial of buparvaquone in the treatment of EastCoast fever. Vet Rec, 130, 536-538, 1992.
Bishop R, Musokei A, Morzaria S, Gardner M, Nene V: Theileria: Intra- cellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks. Parasitology, 129, 271-283, 2004.
Wilkie GM, Brown CGD, Kirvar E, Thomas M, Williamson SM, Bell- Sakyi LJ, Sparagano O: Chemoprophylaxis of Theileria annulata and Theileria parva infections of calves with buparvaquone. Vet Parasitol, 781-12, 1998.
Dargouth MA: Review on the experience with live attenuated vaccines against tropical theileriosis in Tunisia: Considerations for the present and implications for the future. Vaccine, 26S, G4-G10, 2008.
McHardy N, Wekesa LS, Hudson AT, Randall AW: Antitheilerial activity of BW720C (buparvaquone): A comparison with parvaquone. Res Vet Sci, 39, 29-33, 1985.
Rintelen M, Schein E, Ahmed JS: Buparvaquone but not cyclosporin A prevents Theileria annulata-infected bovine lymphoblastoid cells from stimulating uninfected lymphocytes. Trop Med Parasitol, 41 (2): 203-2071990.
Singh DK, Thakur M, Raghav PRS, Varshney BC: Chemotherapeutic trials with four drugs in crossbred calves experimentally infected with Theileria annulata. Res Vet Sci, 54, 68-71, 1993.
McHardy N, Morgan DW: Field comparison of antitheilerial activity with buparvaquone. Res Vet Sci, 39, 1, 1985.
Pancholi V: Multifunctional a-enolase: Its role in diseases. Cell Mol Life Sci, 58, 902-920, 2001.
Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual I-II-III.3rd ed., CSHL Press, New York, 2001.
Arnold K, Bordoli L, Kopp J, Schwede T: The SWISS-MODEL WorkspaceA web-based environment for protein structure homology modelingBioinformatics, 22, 195-201, 2006.
Dzierszinski F, Mortuaire M, Dendouga N, Popescu O,Tomavo S: Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma Gondii. J Mol Biol, 309, 1017-1027, 2001.
Chaikuad A, Fairweather V, Conners R, Joseph-Horne T, Turgut- Balik D, Brady RL: Structure of lactate dehydrogenase from Plasmodium vivax: Complexes with NADH and APADH. Biochemistry, 44 (49): 16221- 16228, 2005.
Pal-Bhowmick I, Sadagopan K, Vora HK, Sehgal A, Sharma S, Jarori GK: Cloning, over-expression, purification and characterization of Plasmodium Falciparum enolase. Eur J Biochem, 271, 4845-4854, 2004.
Pal-Bhowmick I, Kumar N, Sharma S, Coppens I, Jarori GK: Plasmodium falciparum enolase: Stage-specific expression and suc- cellular localization. Malaria J, 8, 179, 2009.
Dunn CR, Banfield MJ, Barker JJ, Higham CW, Moreton KM, Turgut- Balik D, Brady RL, Holbrook JJ: The structure of lactate dehydrogenase from Plasmodium falciparum reveals a new target for anti-malarial designNat Struct Biol, 3 (11): 912-915, 1996.
Vora HK, Shaik FR, Pal-Bhowmick I, Mout R, Jarori GK: Effect of deletion of a plant like pentapeptide insert on kinetic, structural and immunological properties of enolase from Plasmodium falciparum. Arc Biochem Biophys, 485, 128-138, 2009.
Tindiha HS, Marcottyb T, Naessensa J, Goddeerisc BM, Geysenb D: Demonstration of differences in virulence between two Theileria parva isolates. Vet Parasitol, 168, 223-230, 2010.