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Originally published as Genetics Published Articles Ahead of Print on December 30, 2005.
Genetics, Vol. 172, 1129-1137, February 2006, Copyright © 2006
doi:10.1534/genetics.105.047654
Cytogenetic and Molecular Evaluation of Centromere-Associated DNA Sequences From a Marsupial (Macropodidae: Macropus rufogriseus) X Chromosome
Kira Bulazel*,
Cushla Metcalfe*,
Gianni C. Ferreri*,
Jingwei Yu
,
Mark D. B. Eldridge
,
and
Rachel J. O'Neill*,1
* Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, Department of Laboratory Medicine, University of California, San Francisco, California 94107, Department of Biological Sciences, Macquarie University, New South Wales 2109, Australia and Evolutionary Biology Unit, Australian Museum, Sydney, New South Wales 2010, Australia
1 Corresponding author: Department of Molecular and Cell Biology, U-2131, 354 Mansfield Rd., Rm. 323, University of Connecticut, Storrs, CT 06269.
E-mail: rachel.oneill{at}uconn.edu
The constitution of the centromeric portions of the sex chromosomes of the red-necked wallaby, Macropus rufogriseus (family Macropodidae, subfamily Macropodinae), was investigated to develop an overview of the sequence composition of centromeres in a marsupial genome that harbors large amounts of centric and pericentric heterochromatin. The large, C-band-positive centromeric region of the X chromosome was microdissected and the isolated DNA was microcloned. Further sequence and cytogenetic analyses of three representative clones show that all chromosomes in this species carry a 178-bp satellite sequence containing a CENP-B DNA binding domain (CENP-B box) shown herein to selectively bind marsupial CENP-B protein. Two other repeats isolated in this study localize specifically to the sex chromosomes yet differ in copy number and intrachromosomal distribution. Immunocytohistochemistry assays with anti-CENP-E, anti-CREST, anti-CENP-B, and anti-trimethyl-H3K9 antibodies defined a restricted point localization of the outer kinetochore at the functional centromere within an enlarged pericentric and heterochromatic region. The distribution of these repeated sequences within the karyotype of this species, coupled with the apparent high copy number of these sequences, indicates a capacity for retention of large amounts of centromere-associated DNA in the genome of M. rufogriseus.
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