Hum Genet (1993) 90:577-583 human .. gene cs 9 Springer-Verlag 1993 Original investigations An alphoid DNA sequence conserved in all human and great ape chromosomes: evidence for ancient centromeric sequences at human chromosomal regions 2q21 and 9q13 Antonio Baldini 1,2, 3, Thomas Ried 1, Viji Shridhar 4, Keiko Ogura I, Leonardo D'Aiuto ~, Mariano Rocchi 6, David C. Ward 1 Department of Human Genetics, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA 2CNR Institute of Molecular Genetics, Porto Conte Research and Training Laboratories, Alghero, Italy 3Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London WC2A 3PX, UK Department of Molecular Biology and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA 5Istituto di Anatomia Umana Normale, Universita'di Modena, Modena, Italy 6Istituto di Genetica, Universita'di Bari, Bari, Italy Received: 15 April 1992 / Revised: 3 July 1992 Abstract. Using vector-CENP-B box polymerase chain reac- tion (PCR) we isolated and cloned from a human chromo- some 21-specific plasmid library, a 1 kb DNA sequence, named p~H21. In in situ hybridization experiments, p~H21 hybridized, under high stringency conditions, to the cen- tromeric region of all the human, chimpanzee, gorilla and orangutan chromosomes. On human chromosomes p~H21 also identified non-centromeric sequences at 2q21 (locus D2F33S1) and 9q13 (locus D9F33S2). The possible deriva- tion of these sequences from ancestral centromeres is dis- cussed. Sequence analysis confirmed the alphoid nature of the whole p~H21 insert. Introduction The centromeric region of primate chromosomes contains long arrays of tandemly repeated DNA sequences, referred to as alpha satellite DNA or alphoid DNA (Maio 1971; Kurnit and Maio 1973; Manuelidis 1978; Rosenberg et al. 1978; Willard and Waye 1987). The common feature of this DNA is the length of the repeat unit, which is about 170 bp. Alphoid repeats have been studied widely, especially in hu- man, where subsets highly specific for a single chromosome or shared by several chromosomes have been documented. Other subsets are distinguishable from each other using very high stringency conditions of hybridization or by restriction analysis (see Choo et al. 1991 for review). A human alphoid GenBank accession number, M64321 Correspondence to: A. Baldini, Institute for Molecular Genetics, Baylor College of Medicine, One Baylor Plaza, T936, Houston, TX 77030, USA DNA sequence that hybridizes to every human and great ape centromere under fairly high stringency conditions, has also been reported (clone p82H, Mitchell et al. 1985; Aleixandre et al. 1987; Miller et al. 1988). Chromosome-specific organi- zation and sequence conservation has been shown for a sin- gle gorilla- and a single chimpanzee-derived alphoid DNA sequence (Durfy and Willard 1990; Baldini et al. 1991). However, the evolutionary studies on alpha satellite DNA among human and great apes have not yet collected enough data to draw general conclusions. The highest sequence sim- ilarity so far reported between human and great ape alphoid sequences is 91% (Baldini et al. 1991), much lower than the expected similarity for selectively neutral sequences (see Goodman et al. 1989 for review), suggesting that this DNA is rapidly changing. However, sequence comparisons between tandem repeats from different species are of limited value owing to obvious difficulties in identifying orthologous DNA segments. No function has so far been assigned to the alpha satellite DNA. It contains a 17-bp protein binding domain, referred to as the "CENP-B box" owing to its ability to bind the CENP-B protein (reviews in Pluta et al. 1990; Rattner 1991). The CENP-B box is also present in the mouse minor satellite (Wong and Rattner 1988), which is located at the centromere of mouse chromosomes but is otherwise unre- lated to the alpha satellite DNA. We used the CENP-B box sequence as a primer, in combination with a vector-specific primer, to amplify alpha satellite sequences from the DNA of a chromosome-specific library, using the polymerase chain reaction (PCR). With this method, we isolated and cloned a 1-kb alphoid DNA sequence, named pc~H21, that hybridized, in situ, to the centromeric region of all the human and great ape chromosomes. Further, pooH21 identified non-cen- tromeric sequences on human chromosomes that could be the remnants of ancestral centromeres.