Creusot F, Fouilloux E, Dron M, Lafleuriel J, Picard G, Billault A, Le Paslier D, Cohen D, Chabouté M E, Durr A
Laboratoire de Phytopathologie Moléculaire, URA CNRS 1128, Université Paris-sud, Orsay, France.
Plant J. 1995 Nov;8(5):763-70. doi: 10.1046/j.1365-313x.1995.08050763.x.
A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.
一个新的拟南芥（生态型哥伦比亚）基因组文库已构建在酵母人工染色体中：即CIC文库（用于CEPH、法国国家农业研究院和法国国家科学研究中心）。植物培养条件和原生质体制备的优化使得能够回收大量有活力的原生质体。通过从包埋在琼脂糖中的原生质体中分离DNA，将DNA的机械剪切降至最低。通过包括两个连续的大小分级步骤（部分EcoRI消化后和与载体臂连接后），有利于大插入片段的克隆，这两个步骤选择了大于350 kb的DNA片段。该文库由1152个克隆组成，平均插入片段大小为420 kb。已鉴定出携带叶绿体DNA和各种核重复序列的克隆。发现21%的克隆含有叶绿体DNA。因此，该文库代表了约四个核基因组当量。含有5S rDNA基因、18S - 25S rDNA序列和180 bp着丝粒旁重复元件的克隆分别占3.6%、8.9%和5.8%。仅发现一个克隆携带160 bp着丝粒旁重复元件。鉴于携带拟南芥重复DNA的克隆较小，其余克隆的平均大小约为480 kb。使用与分散在基因组中的序列对应的引物对通过PCR扩增对该文库进行筛选。从76对引物中，有70对鉴定出了1至7个酵母人工染色体克隆。因此，CIC文库中至少代表了92%的基因组。对该文库中含有不连锁DNA序列的克隆进行的调查表明，嵌合克隆的比例低于10%。
