Genetic and Physical Analyses of the Centromeric and Pericentromeric Regions of Human Chromosome 5: Recombination across 5cen JacquesPuechberty, Anne-Marie Laurent, Sylvie Gimenez, Alain Billault,* Marie-Elisabeth Brun-Laurent, Alphonse Calenda, Bertrand Marc ¸ais, Catherine Prades, Panos Ioannou,† Yuri Yurov, and Ge ´rard Roize `s 1 Se ´quences re ´pe ´te ´es et Centrome `res Humains, Institut de Ge ´ne ´tique Humaine, CNRSUPR1142, Institut de Biologie, 4 Boulevard Henri IV, 34060 Montpellier Cedex, France; * Centre d’Etude du Polymorphisme Humain, 27 Rue Juliette Dodu, 75010 Paris, France; and †Gene Therapy Group, The Murdoch Institute for Research into Birth Defects, Royal Children’s Hospital, Flemington Road, Parkville Melbourne, Victoria 3052, Australia Received November 2, 1998; accepted January 4, 1999 Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel elec- trophoresis after cutting genomic DNA with restric- tion endonucleases NcoI (chromosome 5) and BamHI (chromosome 19). We could thus define a highly poly- morphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junc- tion of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymor- phic and could therefore be used as a second centro- meric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected be- tween the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen in- deed exerts a direct negative effect on meiotic recom- bination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the en- tire chromosome 5. Rates then become close to the average when one goes further within the arms. Fi- nally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombi- nation around 5cen is essentially occurring in the fe- male lineage. © 1999 Academic Press INTRODUCTION Although centromeres are unambiguously defined at the cytogenetic level as primary constrictions of chro- mosomes, their definition is not as clear at the molec- ular DNA level, as the DNA sequences necessary and sufficient to ensure proper centromere function in both mitosis and meiosis in higher eukaryotes remain un- known. A certain amount of alpha satellite DNA is, however, always detected at primary constrictions and can, therefore, be considered as spanning centromeres in all human chromosomes. Other satellite DNAs and sometimes other alphoid sequence subsets map close to the primary constrictions on the proximal p or q arms, a situation that is highly variable among the chromo- somes and the satellite DNAs considered (Tagarro et al., 1994a,b; Lee et al., 1997). Exceptional situations are also found with the so-called neo-centromeres, where no alpha satellite is present (Voullaire et al., 1993; du Sart et al., 1997). These observations suggest Sequence data from this article have been deposited with the EMBL/GenBank/DDBJ Data Libraries under Accession Nos. AJ007751–AJ007762. The accession number for D5S2930 is AF023528. Genotypic data for D5S2930 and D5S2934 are available from the CEPH database http://www.cephb.fr/cephdb). 1 To whom correspondence should be addressed. Telephone: 33 (0) 4.67.66.35.54. Fax: 33 (0) 4.67.66.03.06. E-mail: roizes@igh.cnrs.fr. Genomics 56, 274 –287 (1999) Article ID geno.1999.5742, available online at http://www.idealibrary.com on 274 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.