CHARACTERIZATION OF YEAST ARTIFICIAL CHROMOSOMES FROM PLASMODIUM-FALCIPARUM - CONSTRUCTION OF A STABLE, REPRESENTATIVE LIBRARY AND CLONING OF TELOMERIC DNA FRAGMENTS
CHARACTERIZATION OF YEAST ARTIFICIAL CHROMOSOMES FROM PLASMODIUM-FALCIPARUM - CONSTRUCTION OF A STABLE, REPRESENTATIVE LIBRARY AND CLONING OF TELOMERIC DNA FRAGMENTS
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DOI：
10.1016/s0888-7543(05)80223-x
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发表时间：
1992-10-01
期刊：
影响因子：
4.4
通讯作者：
RAVETCH, JV
中科院分区：
文献类型：
--
作者：
DEBRUIN, D;LANZER, M;RAVETCH, JV
人类疟疾寄生虫恶性疟原虫的分子遗传学研究一直受到阻碍，部分原因是难以在细菌中稳定地克隆和繁殖寄生虫基因组DNA。这被认为是寄生虫DNA中不寻常的a + T偏置（bbb80 %）的结果。p的脉冲场凝胶电泳分离。恶性染色体研究表明，由于染色体末端DNA的缺失，经常发生大的染色体多态性。理解这种染色体多态性的生物学意义将需要对基因组的大区域，特别是端粒DNA进行分析。为了克服在细菌中克隆寄生虫DNA的局限性，我们从寄生虫中克隆了基因组DNA。恶性疟原虫FCR3在酵母中作为人工染色体。建立了一个平均插入大小约为100 kb的pYAC4文库，并发现单拷贝基因具有三到四倍的冗余。与细菌宿主不同，酵母菌稳定地维持和繁殖大量的寄生虫DNA。YAC克隆的远程限制性内切酶图谱表明，克隆的DNA与本地寄生虫的基因组DNA是连续和相同的。由于染色体端粒末端在YAC文库中代表性不足，我们通过克隆p丰富了这些序列。恶性肿瘤端粒DNA片段（从40到130 kb）在酵母中通过互补作为YACs。
Molecular genetic studies of the human malaria parasitePlasmodium falciparumhave been hampered in part due to difficulties in stably cloning and propagating parasite genomic DNA in bacteria. This is thought to be a result of the unusual A + T bias (>80%) in the parasite's DNA. Pulsed-field gel electrophoretic separation ofP. falciparumchromosomes has shown that large chromosomal polymorphisms, resulting from the deletion of DNA from chromosome ends, frequently occur. Understanding the biological implications of this chromosomal polymorphism will require the analysis of large regions of genomic, and in particular telomeric, DNA. To overcome the limitations of cloning parasite DNA in bacteria, we have cloned genomic DNA from theP. falciparumstrain FCR3 in yeast as artificial chromosomes. A pYAC4 library with an average insert size of approximately 100 kb was established and found to have a three to fourfold redundancy for single-copy genes. Unlike bacterial hosts, yeast stably maintain and propagate large tracts of parasite DNA. Long-range restriction enzyme mapping of YAC clones demonstrates that the cloned DNA is contiguous and identical to the native parasite genomic DNA. Since the telomeric ends of chromosomes are underrepresented in YAC libraries, we have enriched for these sequences by cloningP. falciparumtelomeric DNA fragments (from 40 to 130 kb) as YACs by complementation in yeast.