Abstract
A mitotically stable linear extra chromosome obtained in a Leishmania donovani strain rendered mycophenolic acid-resistant has been physically mapped. This 290-kb chromosome has an inverted duplicated structure around a central inversion region, and is derived from a conservative amplification event of a approximately 140-kb subtelomeric end of chromosome 19. Large-sized targeted deletions of the central region were performed through homologous recombination using three specific transfection vectors. The size of the extra chromosome was thus successfully reduced from 290 to 260, 200 and 120 kb respectively. The mitotic stability of these chromosomes was then analysed in drug-free cultures over >140 days. Results differed according to the deletion created. By contrast with the smallest deletion the two largest deletions altered mitotic stability, leading to progressive loss of the size-reduced chromosomes with similar kinetics in both mutants. The 30-kb region common to both deletions may therefore be considered as involved in mitotic stability. A 44-kb contig covering this region could be assembled and sequenced. The analysis of this sequence did not reveal any sequence elements typical of centromeric DNA. By contrast, its enrichment in homopolymer tracts suggests that this region might contain an origin of replication.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Centromere / drug effects
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Centromere / genetics
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Chromosome Inversion
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Chromosome Segregation / drug effects
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Chromosome Segregation / genetics
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Chromosomes / drug effects*
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Chromosomes / genetics*
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Contig Mapping
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DNA Replication / drug effects
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DNA Replication / genetics
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Drug Resistance / genetics*
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Electrophoresis, Gel, Pulsed-Field
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Gene Amplification / drug effects
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Gene Amplification / genetics
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Genes, Duplicate / genetics
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Genetic Vectors / genetics
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IMP Dehydrogenase / antagonists & inhibitors
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IMP Dehydrogenase / genetics
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Kinetics
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Leishmania donovani / cytology
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Leishmania donovani / drug effects
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Leishmania donovani / genetics*
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Mitosis / drug effects*
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Mitosis / genetics
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Molecular Sequence Data
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Mycophenolic Acid / pharmacology*
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Recombination, Genetic / genetics
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Repetitive Sequences, Nucleic Acid / genetics
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Replication Origin / genetics
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Restriction Mapping
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Sequence Deletion / genetics*
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Sequence Homology, Nucleic Acid
Substances
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IMP Dehydrogenase
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Mycophenolic Acid
Associated data
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GENBANK/AF176312
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GENBANK/AF315645
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GENBANK/AF319040
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GENBANK/AF393161
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GENBANK/AF393162
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GENBANK/AF393163
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GENBANK/AF393164
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GENBANK/AF393165
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GENBANK/AF393166
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GENBANK/AF393167
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GENBANK/AF393168
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GENBANK/AF393169
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GENBANK/AF393170
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GENBANK/AF393171
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GENBANK/AF393172
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GENBANK/AF393173
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GENBANK/AF393174
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GENBANK/AF393175
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GENBANK/AF393176
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GENBANK/AF393177
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GENBANK/AF393178
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GENBANK/AF393179
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GENBANK/AF393180
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GENBANK/AF393181
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GENBANK/AF393182
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GENBANK/AY028171