Chromosome Research 1995, 3, 124-127 Ordered mapping of three alpha satellite DNA subsets on human chromosome 22 Rachele Antonacci, Mariano Rocchi, Nicoletta Archidiacono & Antonio Baldini Received 11 August 1994; received in revised form 15 September 1994 Accepted for publication by R. Baker 15 September 1994 We report the physical order of three aiphoid DNA subsets on human chromosome 22 determined by a combination of low- and high-resolution cytological mapping. Multicolor fluorescence in situ hybridization was performed on metaphase chromosomes, inter- phase nuclei and extended chromatin preparations. The results visually demonstrate the presence of three distinct alphoid DNA domains at the centromeric re- gion of chromosome 22. Two domains appear adjacent by extended chromatin hybridization, while the third one is separated by DNA that does not hybridize with any of our probes. Our data demonstrate the applic- ability of interphase mapping for ordering alpha satellite DNA repeat arrays. However, in our experi- ments, the relationship between the extremities of re- peat arrays could only be studied by extended chromatin experiments. Key words: e-satellite DNA, centromere, chromatin mapping, chromosome 22 Introduction Centromeric regions of primate chromosomes contain long arrays of tandemly repeated monomers (about 170 bp) of alpha satellite DNA (Willard & Waye 1987). Sequence divergence between subsets located in different human chromosomes is usually great enough to ensure chromosome-specific hybridization. Alphoid probes specific for almost every human chro- mosome have been reported (reviewed in Choo et al. 1991). A single chromosome can accommodate distinct subsets of alphoid DNA (Waye et al. 1987). The genomic organization of alphoid subsets located in the same chromosome has been investigated with different ex- perimental approaches: Southern blot analysis of parti- ally digested genomic DNA (Waye et al. 1987), pulsed- field gel electrophoresis (PFGE) (TroweU et al. 1993) or fluorescence in situ hybridization (FISH) (Haaf & Ward, 1994). In all studied cases (chromosomes 7, 13, 14 and 21) the results suggested that alphoid subsets were not interspersed but organized in distinct domains and their order could be derived (Trowell et al. 1993). In this paper we report the physical order of three alphoid subsets located at the centromeric region of human chromosome 22, using high-resolution cytological map- ping. Materials and methods For these studies we used three probes identifying three different alphoid subsets on chromosome 22: probe pi90.22 (locus D22Z4; Rocchi et al. 1991), probe p22/1 (locus D22Z2; McDermid et al. 1986) and probe p14.1 (M. Rocchi et al. unpublished). The three probes identify alphoid DNA sequences that have different genomic organization (M. Rocchi, unpublished data). Chromosome spreads were obtained from a male donor using standard methods. For interphase mapping ex- periments, we used G0/Gl-synchronized nuclei from a chromosome 22-only hybrid cell line (GM10888A, Can- dem Cell Repository, NJ, USA). We chose to use hybrid cells because probe p14.1 also hybridizes to chromo- some 14. Extended chromatin preparations were ob- tained from the same hybrid nuclei preparations as described previously (Fidlerova et al. 1994). Differential labeling of probes was obtained usingbiotin-14-dATP (BRL) [detected with avidin-fluorescein isothiocyanate (FITC) from Vector], digoxigenin-11-dUTP (Boehringer) (detected with anti-digoxigenin antibodies conjugated with rhodamine) and direct fluorescence labeling with AMCA-6-dUTP (Boehringer). Detailed procedures for FISH experiments have been reported previously (D'Aiuto et al. 1993). Images of FISH experiments were recorded using a fluorescence microscope equipped R. Antonacci and A. Baldini (corresponding author) are at the Department of Molecular and Human Genetics, BayIor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel: (+1) 713 798 6519; Fax: (+1) 713 798 5386. R. Antonacci, M. Rocchi and N. Archidiacono are at the Istituto di Genetica, Universit~ di Bari, Via Amendola 165/A, 70126 Bari, Italy. 124 Chromosome Research Vol 3 1995 © 1995 Rapid Communications of Oxford Ltd