Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P-postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity - fluorescence labeling of nucleotides followed by CE-LIF detection and (32)P-postlabeling. DNA of Hydra was digested either to deoxyribonucleoside-5'-monophosphates or deoxyribonucleoside-3'-monophosphates selectively conjugated with the fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE-LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5-methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the (32)P-postlabeling method.