Identifying cis-acting DNA elements within the control region of glycogen phosphorylase 2 by DNaseI footprinting in Dictyostelium discoideum

Dictyostelium discoideum organizmasında, gp2 geni tarafından kodlanan Glikojen fosforilaz 2 enzimi bu organizmanın biyolojik gelişim evrelerinde kilit bir rol oynar. Gp2 enzimi glikojenin glikoz yapı taşlarına dönüşmesini kataliz eder ve elde edilen glikoz daha sonra hücresel farklılaşmanın son safhasında kullanılacak olan ürünlerin sentezlenmesinde kullanılır. Bu genin ifade edilmesi organizmanın gelişim evrelerinde düzenlendiği için, seçici gen ekspresyonunu anlamak açısından ideal bir adaydır. Bu yüzden bu çalışmada, gp2 geninin transkripsiyonunda görev alabilecek, promoter bölgesinde yer alan DNA dizileri (cis-davranan diziler) footprint analiz yöntemiyle belirlenmiştir. Dictyostelium’un promoter bölgelerinde çok sayıda bulunan Adenin ve Timin (AT) nükleotidlerinden dolayı, footprint deneyleri kalıp DNA, spesifik olmayan kompetitör, protein ekstrakt kaynakları ve DNaz I ile kesme koşullarına bağlı olarak yoğun bir optimizasyon gerektirdi. Bir ucundan radyoaktif olarak işaretlenen ve 7 adet tekrarlanan sekansları (3 TA box [TAATTATA], 2 TAG box [TAAAAATGGT] ve 2 C box [ACCCACT]) içeren DNA fragmanlarının kullanımıyla, çeşitli gelişimsel nükleer ekstraktların DNA’ya bağlanma aktiviteleri test edildi. Sonuçta; TAG ve C box bölgelerinde küçük ayakizleri tespit edildi. Bununla beraber, primer uzatımının çoklu döngülerini içeren daha duyarlı bir ayakizi stratejisinin kullanımıyla, daha büyük ayakizleri elde edildi. Her iki durumda da ayakizlerinin varlığı gp2 geninin bilinen transkripsiyonel aktivitesi ile uyum içindedir. Bütün bu bilgiler ışığında varılan sonuç, TAG ve C box DNA bölgelerinin gp2 geninin ifade edilmesinde rol alan cis- davranan elementler olduğudur

Dictyostelium discoideum‘daki glikojen fosforilaz 2 geninin kontrol bölgesinde yer alan cis-davranan dizilerin DNaz I footprint yöntemiyle belirlenmesi

Glycogen phosphorylase 2 (encoded by gp2) is a key enzyme expressed during the development of Dictyostelium discoideum. The Gp2 enzyme breaks down glycogen into glucose monomers that are subsequently used to synthesize the terminal end products of cellular differentiation. This gene is an ideal candidate for studying the process of selective gene expression because its product figures so prominently in the development of this organism, implying a dependable control mechanism responsible for its developmentally regulated expression. In this report we present the identification of several putative cis-acting elements of gp2 as revealed through footprint analysis. Due to the extreme AT-bias characteristic of Dictyostelium promoters, footprinting conditions required intensive optimization with respect to template, nonspecific competitor, source of protein extract and DNase I digestion. Using an end-labeled fragment containing seven repeated sequences (3 TA boxes [TAATTATA], 2 TAG boxes [TAAAAATGGT] and 2 C boxes [ACCCACT]), several developmental nuclear extracts were tested for DNA binding activity. Small footprints were observed on the TAG and C boxes of the promoter for the protein sources. However, using a more sensitive footprint strategy involving multiple rounds of primer extension, larger footprints spanning the same promoter regions were detected. In both cases, the appearance of the footprints coincided with the documented transcriptional activity of the gene. It is concluded that the TAG and C boxes are cis-acting elements involved in the regulation of gp2 expression.

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