1997 Fiscal Year Final Research Report Summary
The Construction of A sequence-ready Map of Human Chromosome 2l and Gene Hunting Involved in Down Syndrome
Project/Area Number |
07558295
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Research Category |
Grant-in-Aid for Scientific Research (A)
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Allocation Type | Single-year Grants |
Section | 展開研究 |
Research Field |
Biomedical engineering/Biological material science
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Research Institution | The Institute of Physical and Chemical Research (RIKEN) (1996-1997) Okayama University (1995) |
Principal Investigator |
SOEDA Eiichi RIKEN,Gene Bank, Senior Researcher, ジーンバンク室, 副主任研究員 (00039330)
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Co-Investigator(Kenkyū-buntansha) |
HOSOKAWA Keiichi Kawasaki Medical School, Professor, 医学部, 教授 (00104787)
KATAYAMA Yasuhito Medical School of Okayama, Lecturer, 医学部, 講師 (40033352)
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Project Period (FY) |
1995 – 1997
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Keywords | sequence-ready map / CpG-islands / chromosome walking / CpG-tagged sequence / PAC contigs / 筋萎縮側鎖硬化症 / PAC |
Research Abstract |
A sequence-ready map or the human genome will be generated from PAC contigs because they have provided unique materials for high resolution map and sequencing. We have developed rapid screening and chromosome walking with which almost all gaps were able to be sealed with use of a half million PAC clones, representing approx. 20-fold equivalents of the total genome. We extracted 1440 DNAs from 384-well plates which have been arrayd with PAC clones, and prepared "PCR screening kits" for the plate selection. For the isolation of clone from the plate, we devised "ditch plates" for mixing bacteria for PCR with which enabled us to isolate any PAC with STS or PCR-primers within a couple of days. For chromosome walking, we applied direct sequencing from the ends of PAC inserts and read consistently more than 500 bases from which we designed PCR-primers for the confirmation of the existing contigs and screening of next PACs. With this system, we have completed a 7-Mb high resolution map from SOD
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l to ETS2. The full integrity of PAC and high dense cosmid overlap map shows the fidelity in cloning with the bacterial system. Approximately 60 % of the expressed genes of the human genome is known to be associated with CpG islands, most of which are able to be detected with BssHII, EagI and SacII.To help isolate and identify genes, we have sought CpG-islands in the cosmid/PAC contigs with these diagnostic enzymes, assuming close clustering (within l kb) of two or more cutting sites as potential CpG islands. The sequences around the sites have revealed known and unknown genes in this region. Of those, the sequences in PAC 25P16 clone identified.CBR and its analogue, respectively. To verify this approach, we completed the sequence of this clone in shotgun strategies and defined seven CpG islands with which three known and three unknown genes were associated. This approach, designated as CpG-tagged sequence, is rapid and convenient for searching genes and will provide a scaffold for the construction of a transcription map. Less
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Research Products
(15 results)
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[Publications] Osoegawa, K., Susukida, R., Okano, S., Kudoh, J., Minoshima, S., Shimizu, N., de Jong, P., Groet, J., Ives, J., Lehrach, H., Nizetic, D.and Soeda ; E.: "An integrated map with cosmid/PAC contigs of 4-Mb Down syndrome critical region." Genomics. 32. 375-387 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] 0soegawa, K., Susukida, R., 0kano, S,Kato, Y., Lehrach, H., Nizetic, D.and Soeda, E.: "Potential CpG-rich islands clustering around single-minded gene in Down syndrome critical region." Mammalian Genome. 7. 46l-463 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] 0hta, T., Nakano, M., Tsujita, T., Abe, K., 0soegawa, K., Yamagata, T., Yoshira, K., Jinno, Y., Soeda, E., Nakamura, Y.and Nikawa, N.: "Isolation of a cosmid clone corresponding to an inv (21) breakpoint of a patient with transient abnormal myelopoiesis." Am.J.Hum.Genet. 58. 544-550 (1996)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Matsumoto, N., Soeda, E., Ohashi, H., Fujimoto, M., Kato, R., Tsujita T., Tomita, H., Kondo, S., Fukushima Y.and Niikawa N.: "A l.2-Megabase BAC/PAC contig spanning the 14ql3 breakpoint of t (2 ; 14) in a mirror-image polydactyly patient" Genomics. 45. 11-16 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Matsumoto, N., 0hashi, H., Tsukahara, M., Chang Kim, K., Soeda, E.and Niikawa, N.: "Possible narrowed assignment of the loci of monosomy 21-associated microcephaly and intrauterine growth retardation to a 1.2-Mb segment at 21q22.2" Am.J.Hum.Genet. 60. 1997-999 (1997)
Description
「研究成果報告書概要(欧文)」より
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[Publications] Kitamura, E., Hosoda, F., Fukushima, M., Asakawa, S., Shimizu, N., Imai, T., Soeda, E.and 0hki, M.: "A 3-Mb sequence-ready contig map encompassing the multiple disease gene cluster on chromosome 11ql3.1-ql3.3" DNA Reserch. 4. 281-289 (1997)
Description
「研究成果報告書概要(欧文)」より