Shirkey Breanne, McMaster Nicole J, Smith Sue C, Wright Deborah J, Rodriguez Henry, Jaruga Pawel, Birincioglu Mustafa, Helm Richard F, Potts Malcolm
Virginia Tech Center for Genomics (VIGEN), Virginia Tech, Blacksburg, VA 24061, USA.
Nucleic Acids Res. 2003 Jun 15;31(12):2995-3005. doi: 10.1093/nar/gkg404.
Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modified during decades of desiccation. Amplification of gene loci from desiccated cells required pretreatment of DNA with N-phenacylthiazolium bromide, a reagent that cleaves DNA- and protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modified DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of amplification products were obtained with DNA from desiccated/rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible fingerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5'-GWCWATCGCC-3') based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection efficiency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation.
念珠藻(蓝细菌)的基因组DNA在数十年的干燥过程中发生了共价修饰。从干燥细胞中扩增基因位点需要用N-苯甲酰硫代溴化噻唑鎓对DNA进行预处理,该试剂可切割与DNA和蛋白质相连的晚期糖基化终产物。与市售小牛胸腺DNA相比,来自干燥13年的细胞的DNA在常见的氧化修饰DNA损伤生物标志物8-羟基鸟嘌呤、8-羟基腺嘌呤和5-羟基尿嘧啶方面没有显示出任何更高的水平。使用同一组引物,从干燥/复水细胞和干燥材料衍生的液体培养物的DNA中获得了不同的扩增产物模式。相比之下,使用基于基因组中高度重复的回文重复序列的引物(5'-GWCWATCGCC-3'),无论DNA复水时间如何,都能获得可重复的指纹图谱。在体外,cccDNA的干燥导致超螺旋丧失、聚集、琼脂糖凝胶电泳分辨率丧失以及转化和转染效率丧失。当DNA在海藻糖(念珠藻菌落中存在的一种非还原性二糖)存在下干燥和储存时,这些变化被最小化。念珠藻基因组对干燥的反应不同于蓝细菌和耐辐射球菌基因组对电离辐射的反应。