Theor Appl Genet (1996) 93:477-484 9Springer-Verlag 1996 A. Houben 9 A. Brandes 9 U. Pich 9 R. Manteuffel I. Schubert Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex Received: 12 January 1996 / Accepted: 19 January 1996 Abstract The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by de- generate oligonucleotide-primed polymerase chain reac- tion (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome com- plement of Viciafaba was labelled by these probes except for the nucleolus organizing region (NOR) and the inter- stitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA frag- ments amplified from centromeric and acentric regions no sequences specifically located at the field bean centro- meres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved ~mong higher plants and are - at least in species with large genomes - intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary con- strictions by stereophysical parameters rather than by their base sequence. Key words Microdissection of plant centromeres 9 Subtractive hybridization 9 Communicated by F. Mechelke A. Houben. A. Brandes - U. Pich, R. Manteuffel. I. Schubert ([]) Institut ffir Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, D-06466 Gatersleben, Germany Fluorescence in situ hybridization 9 Indirect immunofluorescence - Immunoadsorption Introduction The centromere, usually the primary constriction, is the chromosomal domain of monocentric eukaryotic chromo- somes and is responsible for their correct segregation dur- ing mitotic and meiotic nuclear divisions. Centromeres form the final points of adhesion for sister chromatids at the metaphase:anaphase transition, the attachment points for mitotic spindle fibers and the location for motor pro- teins mediating active chromosome movement during mi- tosis (for review see Earnshaw and Mackay 1994; Pluta et al. 1995; Sunkel and Coelho 1995). The kinetochore, pre- viously a term synonymous to the centromere, now refers to a button-like proteinaceous structure resting on the sur- face of the centromeric chromatin, into which most of the microtubuli of the mitotic spindle attaching to the centro- meric region are inserted (for review see Earnshaw 1991). At present, detailed knowledge about DNA components essential for the functioning of centromeres is available only for yeasts. Centromeres of Saccharomyces cerevisiae and KIuyveromyces lactis consist of three to four conserved centromeric DNA elements (CDEs) of about 250 bp all to- gether, while in the fission yeast Schizosaccharomyces pombe the essential centromeric DNA consists of 44-100 kb with a central core of 4-7 kb (Clarke et al. 1993). These components are species-specific and do not function when transferred into another species (Heus et al. 1993). For several insects and vertebrates satellite sequences are known to be located at centromeric regions. Murphy and Karpen (1995) have found a 220-kb island of complex DNA to be essential for the functioning of the centromere of a Drosophila minichromosome. For complete mitotic and meiotic stability this core had to be flanked on either side by approximately 200 kb of satellite DNA (AATAT). Despite extensive studies, especially ones on human alphoid sequences (see Haaf et al. 1992; Tyler-Smith et al.