LINE-related component of mouse heterochromatin and complex chromocenters' composition.
Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3' untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.
Institute of Cytology RAS, St Petersburg, 194064, Russia.|St. Petersburg State University, St Petersburg, 199034, Russia.|School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.
Inna S
IS
Kuznetsova
Institute of Cytology RAS, St Petersburg, 194064, Russia. necroforus@gmail.com.
Dmitrii I
DI
Ostromyshenskii
Institute of Cytology RAS, St Petersburg, 194064, Russia.|St. Petersburg State University, St Petersburg, 199034, Russia.
Alexei S
AS
Komissarov
A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.
Andrei N
AN
Prusov
Institute of Cytology RAS, St Petersburg, 194064, Russia.
Irina S
IS
Waisertreiger
Institute of Cytology RAS, St Petersburg, 194064, Russia.
Anna V
AV
Gorbunova
Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, 630090, Russia.
Vladimir A
VA
Trifonov
Cambridge Resource Centre for Comparative Genomics, University of Cambridge, Cambridge, CB3 0ES, UK.
Malcolm A
MA
Ferguson-Smith
Institute of Cytology RAS, St Petersburg, 194064, Russia.|St. Petersburg State University, St Petersburg, 199034, Russia.|Far Eastern Federal University, Vladivostok, 690922, Russia.
Olga I
OI
Podgornaya
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D015342:DNA Probes / Q000235:genetics; D004276:DNA, Satellite / Q000235:genetics; D030541:Databases, Genetic; D005347:Fibroblasts / Q000166:cytology; D016678:Genome / Q000235:genetics*; D006570:Heterochromatin / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D020084:Long Interspersed Nucleotide Elements / Q000235:genetics*; D051379:Mice; D008809:Mice, Inbred C3H; D016366:Open Reading Frames / Q000235:genetics; D017422:Sequence Analysis, DNA; D020080:Tandem Repeat Sequences / Q000235:genetics*
D020413:3' Untranslated Regions; D015342:DNA Probes; D004276:DNA, Satellite; D006570:Heterochromatin
Bioinformatics analysis; Fluorescent in situ hybridization (FISH); Heterochromatin; LINE; Mouse genome; Tandem repeat