Cloning, characterization and chromosomal localization of a repeated sequence in Crypthecodinium cohnii, a marine...

Genomic DNA of Crypthecodinium cohnii has been extracted in the presence of cetylmethylammonium bromide and hydrolysed by 13 restriction enzymes. No typical ladder-like pattern or isolated band of satellite sequences were found with any of these enzymes. A "mini" genomic DNA library had been made and screened by reverse hybridization to isolate highly repeated sequences. Seven such DNA fragments were sequenced. The copy number of one of them (Cc18), 226 bp long, was estimated at around 25,000, representing 0.06% of the total genome. Cc18 was found to be included in a higher fragment of 3.0 kb by Southern blot analysis after cleavage by PstI. This higher molecular weight fragment could be composed either of tandemly repeated Cc18 sequences, or by only one or a very low copy number of Cc18. In this latter case, these fragments, also repeated 25,000 times would represent 1 to 2% of the total genome. Genomic localization of Cc18 by in situ hybridization on squashed C. cohnii cells showed that it was widely distributed on the different chromosomes. All the chromosomes observed displayed Cc18 labeling, which appeared homogeneously distributed. The ability of Cc18 to be a specific molecular marker to distinguish sibling C. cohnii species is discussed.