Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: CLONING OF LARGE DNA FRAGMENTS FROM NICOTIANA PLUMBAGINIFOLIA
INTO A YEAST ARTIFICIAL CHROMOSOME SUITABLE FOR CHROMOSOME
WALKING
CLONING OF LARGE DNA FRAGMENTS FROM NICOTIANA PLUMBAGINIFOLIA
INTO A YEAST ARTIFICIAL CHROMOSOME SUITABLE FOR CHROMOSOME
WALKING.
Chung-Mong Chen, Institute of Botany, Academia Sinica, Taipei,
Taiwan, ROC.
DNA fragments bigger than 2 megabase pairs have been
isolated from the leaf protoplasm of Nicotiana plumbaginifolia.
These DNA molecules were partially digested with EcoRl,
size-fractionated by pulse field gel electrophoresis, ligated
with a yeast artificial chromosome pYACCMC5 and then transformed
into yeast to get YAC clones. Analysis of 14 YAC clones by
Southern hybridization reveal that each of them contains a cloned
DNA fragment with a size ranging from 90 kb to 600 kb. In a
pilot experiment both end fragments of the insert in clone NPYAC1
(contains about 450 kb insert DNA) were subcloned into bacteria
by plasmid rescue. Both subcloned fragments hybridized with DNA
from the original clone NPYAC1 and from. N. plumbaginifolia but
not from the host yeast AB1380, indicating that the insert in
clone NPYAC1 comes from N. plumbaginifolia. It is also
suggesting that most (if not all) YAC clones contain DNA from
this plant and both end fragments of any NPYAC clone can be
reisolated by plasmid rescue. End fragments from each YAC clone
are useful for chromosome walking to identify overlapping YAC
clones and to set up a physical map of this plant genome.
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