Characterization of Cyclops kolensis inter-simple sequence repeats in germline and postdiminution somatic cells

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337 Chromatin diminution is programmed elimination of a part of the genome from presumptive somatic cells during early embryogenesis of some animal species. Chromatin diminution was described for a fairly small number of species of different groups of animals, such as ascarides, myxines, dipterans, Cyclops , and protozoans [2, 3, 6, 8, 10, 13, 14]. Although a great variety of chromatin diminution forms is known, it seems most promising to study this process in Cyclops kolensis Lill., a member of the family Cyclopidae. This freshwater crustacean contains a record-breaking (~94%) amount of eliminated DNA (eDNA) compared to other multicellular animals. In this species, DNA is eliminated from chromosomes of somatic cells during the fourth cleavage division, with the diploid chromosome number remaining unchanged [2, 3]. In this study, we demonstrated that chromatin diminution in C. kolensis is not accompanied by total elimination of nonfunctional genomic regions. This finding broadens our notion on chromatin diminution in C. kolensis . Currently, the molecular mechanisms ensuring diminution and determine the eDNA type remain largely obscure. However, it was shown that the C. kolensis eDNA contains noncoding sequences, some of which have an intricate mosaic structure of repeats [7]. It is known that Mesocyclops edax , another member of the family Cyclopidae, loses a considerable part of satellite DNA sequences during chromatin diminution [9]. The so-called microsatellite sequences are also classified with the satellite DNA. Microsatellite, or short tandem repeats (simple sequence repeats, SSRs), are a characteristic component of the eukaryotic genome. Today, an ample body of evidence regarding the structure of this component and the mechanisms that ensure their size polymorphism and increase in the number of their copies has been accumulated [15]. The consistent patterns of microsatellite distribution over the genome has been studied and their predominant concentration in the noncoding region of the genome has been shown [11]. An average distance between microsatellites in heterochromatin regions varies from several hundreds to several thousands of nucleotide pairs [12], which makes it possible to amplify the DNA fragments located between microsatellites (the so-called inter-simple sequence repeats, or ISSRs sequences) by polymerase chain reaction (PCR). It was shown earlier that, in some species, the majority of heterochromatin regions in chromosomes is eliminated from the genome of somatic cells in the course of chromatin diminution [10, 13]. It can be assumed that a considerable part of heterochromatin, including the microsatellite sequences, is eliminated from the C. kolensis genome during chromatin diminution. In this study, we amplified, cloned, and sequenced five C. kolensis DNA sequences located between the (GA) n microsatellite sequences. A comparative analysis of the nucleotides comprising the cloned sequences was performed using special software programs. Unique internal primer pairs selected for four out of five cloned sequences made it possible to amplify by PCR the corresponding DNA fragments initially located between the (GA) n microsatellites. We performed a comparative analysis of the amplification products of these sequences in C. kolensis before and after chromatin diminution. It was found that three out of four intermicrosatellite loci studied remain in the genome of somatic cells after chromatin diminution and only one of them is eliminated during diminution. Female C. kolensis caught in the pond situated on Vorob’evy Gory (Moscow) were determined according to Monchenko [4]. The prediminution stage of