The overall objective of this proposal is to develop rapid, sensitive and reproducible in situ hybridization methods suitable for the analysis of both entire chromosome domains and individual genes directly in interphase cells and to apply these techniques to the diagnosis of inherited genetic disorders and neoplastic diseases which exhibit numerical or structural chromosome abnormalities. Our specific goals are: 1) to use cloned DNA subsets that hybridize uniquely to specific human chromosomes or discrete subchromosomal regions to identify chromosomal territories in interphase cells by non-isotopic in situ hybridization. 2) to develop a simple and automated screening test to detect the major trisomic disorders (chromosomes 13, 18, 21 and X) directly in interphase cells from amniotic fluids or chorionic villi, using probes sets labeled with different reporter molecules that can be distinguished by fluorescence or colonmetric methods. 3) to detect chromosomal translocations in interphase cells that are diagnostic for specific inherited genetic diseases or neoplastic disorders using chromosome specific probe sets in combination with 3-D optical imaging techniques. 4) to improve the routine detection sensitivity of non-isotopically labeled probes in situ to the single gene level (less than or equal to 1 kb), using fluorescence and chemiluminescence detection methods that generate quantitative results, and 5) to apply the techniques developed by aim 4 to determine the intranuclear location of specific genes before and after transcriptional activation and to monitor translocations or other genetic rearrangements in interphase nucleic using defined gene probes.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG000246-03
Application #
3333291
Study Section
Special Emphasis Panel (SSS (A))
Project Start
1989-04-01
Project End
1994-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Haaf, T; Ward, D C (1996) Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains. Exp Cell Res 224:163-73
Cupelli, L; Renault, B; Leblanc-Straceski, J et al. (1996) Assignment of the human myogenic factors 5 and 6 (MYF5, MYF6) gene cluster to 12q21 by in situ hybridization and physical mapping of the locus between D12S350 and D12S106. Cytogenet Cell Genet 72:250-1
Haaf, T; Mater, A G; Wienberg, J et al. (1995) Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific alpha-satellite DNA. J Mol Evol 41:487-91
Haaf, T; Ward, D C (1995) Rabl orientation of CENP-B box sequences in Tupaia belangeri fibroblasts. Cytogenet Cell Genet 70:258-62
Ashley, T; Lieman, J; Ward, D C (1995) Multicolor FISH with a telomere repeat and Sry sequences shows that Sxr (Sex reversal) in the mouse is a new type of chromosome rearrangement. Cytogenet Cell Genet 71:217-22
Davies, R L; Yoon, S J; Weissenbach, J et al. (1995) Physical mapping of the human ELA1 gene between D12S361 and D12S347 on chromosome 12q13. Genomics 29:766-8
Sipley, J D; Menninger, J C; Hartley, K O et al. (1995) Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8. Proc Natl Acad Sci U S A 92:7515-9
Haaf, T; Ward, D C (1995) Higher order nuclear structure in mammalian sperm revealed by in situ hybridization and extended chromatin fibers. Exp Cell Res 219:604-11
LeBlanc-Straceski, J M; Montgomery, K T; Kissel, H et al. (1994) Twenty-one polymorphic markers from human chromosome 12 for integration of genetic and physical maps. Genomics 19:341-9
Lu-Kuo, J M; Le Paslier, D; Weissenbach, J et al. (1994) Construction of a YAC contig and a STS map spanning at least seven megabasepairs in chromosome 5q34-35. Hum Mol Genet 3:99-106

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