Sequence organization of barley centromeres

Sabina Hudakova 0 1 Wolfgang Michalek 0 1 Gernot G. Presting 0 1 Rogier ten Hoopen 0 1 Karla dos Santos 0 1 Zuzana Jasencakova 0 1 Ingo Schubert 0 1 0 Present addresses: Gernot G. Presting, Novartis Agriculture Discovery Institute , 3115 Marryfield Row, San Diego , CA 92121-U25, USA Karla dos Santos, Institut fr Pflanzenbau und Pflanzenzchtung , Universtt Gttingen, von Siebold Strasse 8, 37075 Gttingen, Germany 1 Institut fr Pflanzengenetik und Kulturpflanzenforschung (IPK) , Corrensstrasse 3, D-06466 Gatersleben, Germany By sequencing, fingerprinting and in situ hybridization of a centromere-specific large insert clone (BAC 7), the sequence organization of centromeric DNA of barley could be elucidated. Within 23 kb, three copies of the Ty3/gypsy-like retroelement cereba were present. Two elements of 7 kb, arranged in tandem, include long terminal repeats (LTRs) (1 kb) similar to the rice centromeric retrotransposon RIRE 7 and to the cereal centromeric sequence family, the primer binding site, the complete polygene flanked by untranslated regions, as well as a polypurine tract 5 of the downstream LTR. The high density (200 elements/centromere) and completeness of cereba elements and the absence of internally deleted elements and solo LTRs from the BAC 7 insert represent unique features of the barley centromeres as compared to those of other cereals. Obviously, the conserved cereba elements together with barley-specific G+C-rich satellite sequences constitute the major components of centromeric DNA in this species. - The centromere of monocentric chromosomes is morphologically recognizable as the primary constriction. Centromeres are essential for correct segregation into daughter cells of sister chromatids during mitosis and meiosis II and of homologous chromosomes during meiosis I (reviewed in 1,2). Although centromere function is highly conserved among eukaryotes, as are the kinetochore proteins including those of higher plants (3,4), centromeric DNA is rather variable. Centromeric DNA sequences have been described for several eukaryotes. However, except for some yeasts (5,6), their functional importance is at least controversial, the more so since for several species neocentromeric activities at noncentromeric positions have been reported, supporting the idea that the centromere location might be regulated epigenetically (7). While for some plants no centromere-specific repeats could be isolated (8) such sequences have been found in others. For instance, the 180 bp repeat of Arabidopsis (9), which forms large tandem arrays with the repeat 106B (10) interspersed therein, occupy the central domain of all five Arabidopsis centromeres (1113). A few years ago, two centromeric sequences were described for cereals. One is the cereal centromeric sequence (CCS1) family of Brachypodium that also occurs in wheat, rye, barley, maize and rice centromeres (14); the other is the Sau3A9 sequence of sorghum which also hybridized to the primary constriction of the above species (15). Thereafter, using a barley homolog of Sau3A9 as a probe, a clone (#9) from a genomic library was detected containing a cereba (centromeric retroelement of barley) element with high similarity to the Ty3/gypsy group of retrotransposons (16). This element hybridized to all barley centromeres. It contained a complete polygene, of which Sau3A9 represents the integrase encoding region, and flanking sequences similar to CCS1, supposed to represent long terminal repeats (LTRs) of cereba. Due to the additional presence of BARE retroelement sequences (dispersed along the chromosome arms of barley; 17) within the 9 clone and a DraI restriction pattern, which differed from that of genomic DNA when probed with the barley homolog of Sau3A9, we assumed that this clone might contain either sequences of a centromere-border or a chimeric insert not really representative of barley centromeres. Meanwhile, further conserved sequences representing parts of gypsy-like retroelements were found within the centromeres of several cereals [CentA in maize (18); pHind22 in sorghum, wheat, maize and rye (19); RCS1 in rice, rye, barley, sorghum and maize (20); RCB11 in rice and crwydryn in oats and rye (21,22); RIRE7 in rice (2325); R11H in wheat (26)] and even of Beta species [pBv26 and pBp10 (27)]. However, completeness and arrangement of these sequences have not yet been studied directly by complete sequencing of large insert clones. The aim of this work was to search for large insert clones harboring sequences representative of barley centromeres and to study the sequence organization of cereba elements and possibly associated centromere-specific sequences within the corresponding clone in comparison with sequences of other cereal centromeres. This should provide suitable candidate sequences for gel-shift assays with kinetochoric proteins to characterize interactions between DNA and proteinaceous components of the barley centromere/kinetochore complex. We describe the insert sequence of a selected barley BAC clone which yielded similar hybridization patterns as genomic DNA using the barley centromeric sequence (BCS2) (14) and the integrase encoding region as probes and which hybridized to all barley centromeres in situ. MATERIALS AND METHODS BAC library screening and fluorescent in situ hybridization (FISH) A BAC library of genomic DNA from Hordeum vulgare L. cultivar Morex (established at Clemson University) containing 313 344 clones (28) was transferred onto Hybond N+ filters (Amersham). Treatment of the filters, hybridization and washing conditions were as described (29,30). Of 10 BAC clones that hybridized with the integrase region (pGP7) of the polyprotein gene of Ty3/gypsy-like retrotransposon cereba labeled with [32P]dCTP [using a random primer extension kit (Amersham) as described previously (31)] only one (03J24, now called BAC 7) showed after FISH positive signals exclusively at the centromeric regions of all barley chromosomes. For FISH, BAC 7 DNA was isolated using a Qiagen Plasmid Mini Kit (100) and labeled with rhodamin-5-dUTP using a nick translation kit (Roche Biochemicals) according to the manufacturers instructions. The primers (AGGGAG)4 and (CTCCCT)4, representing the most frequent motif within the G+C-rich domain outside the cereba elements of the BAC 7 insert, were amplified without additional template sequence and biotin labeled by PCR as described previously (32) for FISH. Metaphase spreads from root tip meristems of the barley line MK 14/2034 (characterized by two homozygous reciprocal translocations between chromosomes 3H/4H and 7H/5H) were prepared as described (16), FISH and signal detection were performed as described (33). The size of the BAC 7 clone was measured by pulsed field gel electrophoresis (PFGE) using the CHEF-DR II electrophoresis system (Bio-Rad) with a 5 s pulsed time (5 V/cm) for 15 h on a 1% agarose gel (Gibco BRL) at 14C in 0.5 TBE buffer (45 mM Trisborate, 1 mM EDTA, pH (...truncated)