The objective of this proposal is to examine the formation of human centromeres, which ensure proper chromosome segregation during cell division. Errors in chromosome segregation lead to aneuploidy and mosaicism, resulting in birth defects and neoplasias. Normal human centromeres contain large amounts of repetitive alpha satellite DNA, which presents certain limitations to the analysis of their formation. However, neocentromeres, found on mitotically stable rearranged chromosomal fragments separated from normal centromeres, do not contain repetitive sequences and can be found localized to low or single copy genomic DNA. Thus, neocentromeres provide a novel approach to investigate current models that centromere formation requires either a distinct primary DNA sequence, e.g. alpha satellite or neocentromere DNA, or largely sequence independent epigenetic modifications, e.g. a distinct chromatin structure or temporal differences in DNA replication in DNA replication. Thus, the following three Specific Aims examine a unique collection of seven independent cell lines that each contain a supernumerary inversion/duplication 13q chromosome with a neocentromere. 1) Molecular cytogenetic FISH mapping will determine the positions of the neocentromeres and the inversion breakpoints to individual or overlapping cosmids from chromosome 13q. At least four neocentromeres in this collection have been cytogenetically localized to chromosome band 13q32. This FISH mapping will potentially define regions in 13q with a high propensity for neocentromere formation and/or a relationship to inversion breakpoints. 2) The role of primary DNA sequence in centromere formation will be addressed by isolation and sequence analysis of neocentromere DNA, using two complementary approaches. Modified extended chromatin techniques that retain the kinetochore protein CENP-C will be used to localize genomic clones to neocentromeres to high resolution. Neocentromere sequences will be isolated by immunoprecipitation of centromeric chromatin using antibodies to the centromere-specific histone CENP-1. 3) The role of DNA replication timing in centromere formation will be addressed by examination of the replication timing of neocentromere DNA and comparison to corresponding DNA sequences in homologous chromosomes, using two approaches. BrdU incorporation will permit assessment of replication of specific chromosomal regions in neocentromeric and non-centromeric states. In situ replication timing will permit assessment of the relative replication timing of specific genomic sequences at neocentromeres and on normal chromosomes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM061150-01
Application #
6088959
Study Section
Mammalian Genetics Study Section (MGN)
Program Officer
Carter, Anthony D
Project Start
2000-04-01
Project End
2002-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
1
Fiscal Year
2000
Total Cost
$121,951
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
114400633
City
New York
State
NY
Country
United States
Zip Code
10029
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Alonso, Alicia; Fritz, Bjorn; Hasson, Dan et al. (2007) Co-localization of CENP-C and CENP-H to discontinuous domains of CENP-A chromatin at human neocentromeres. Genome Biol 8:R148
Qin, N; Bartley, J; Wang, J-C et al. (2007) A neocentromere derived from a supernumerary marker deleted from the long arm of chromosome 6. Cytogenet Genome Res 119:154-7
Cardone, Maria Francesca; Alonso, Alicia; Pazienza, Michele et al. (2006) Independent centromere formation in a capricious, gene-free domain of chromosome 13q21 in Old World monkeys and pigs. Genome Biol 7:R91
Knegt, A C; Li, S; Engelen, J J M et al. (2003) Prenatal diagnosis of a karyotypically normal pregnancy in a mother with a supernumerary neocentric 13q21 -->13q22 chromosome and balancing reciprocal deletion. Prenat Diagn 23:215-20
Alonso, Alicia; Mahmood, Radma; Li, Shulan et al. (2003) Genomic microarray analysis reveals distinct locations for the CENP-A binding domains in three human chromosome 13q32 neocentromeres. Hum Mol Genet 12:2711-21
Assumpcao, Juliana Godoy; Berkofsky-Fessler, Windy; Viguetti Campos, Nilma et al. (2002) Identification of a neocentromere in a rearranged Y chromosome with no detectable DYZ3 centromeric sequence. Am J Med Genet 113:263-7
Li, Shulan; Malafiej, Paul; Levy, Brynn et al. (2002) Chromosome 13q neocentromeres: molecular cytogenetic characterization of three additional cases and clinical spectrum. Am J Med Genet 110:258-67
Warburton, P E (2001) Epigenetic analysis of kinetochore assembly on variant human centromeres. Trends Genet 17:243-7
Levy, B; Papenhausen, P; Tepperberg, J et al. (2000) Prenatal molecular cytogenetic diagnosis of partial tetrasomy 10p due to neocentromere formation in an inversion duplication analphoid marker chromosome. Cytogenet Cell Genet 91:165-70

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