Organization of the 1.9-kb repeat unit RCE1 in the centromeric region of rice chromosomes

This paper presents the first report on the structure of a 14-kb centromere sequence in a cereal genome that includes 1.9-kb direct repeats. The cerea l centromeric sequence (CCS1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which serves as the binding site f or the centromere-specific protein CENP-B in human. To isolate centromeric units from rice (Oryza sativa L.), we performed PCR using the CENP-B box-li ke sequences (CBLS) as primers. A 264-bp clone was amplified by this method , and called RCS1516. It appeared to be a novel member of the CCS I family, sharing about 60% identity with the CCS1 sequences of other cereals. Then, a 14-kb genomic clone, lambda RCB11, carrying the RCS1516 sequence was iso lated and sequenced. It was found to contain three copies of a 1.9-kb direc t repeat, RCE1, separated by 5.1- and 1.7-kb. A 300-bp sequence at the 3' e nd of RCE1 is highly conserved in all three copies (>90%) and is almost ide ntical to the RCS1516 sequence including the CBLS motif. The copy number of RCE1 was estimated to range from 10(2) to 10(3) in the haploid genome of r ice. Cloned RCE1 units were used for fluorescent in situ hybridization (FIS H) analysis, and signals were observed on almost every primary constriction of rice chromosomes. Thus it was concluded that RCE1 is a significant comp onent of the rice centromere. The lambda RCB11 clone contained at least fou r A/T-rich regions, which are candidate for matrix attachment regions (MARs ), in the sequences between the RCEI repeats. Other elements that are homol ogous to the short centromeric repetitive sequences pSau3A9 and pRG5, detec ted in both sorghum and rice, were also found in the clone.