Construction and characterization of genomic libraries from specific human chromosomes.
Construction and characterization of genomic libraries from specific human chromosomes.
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特定人类染色体基因组文库的构建和表征。
DOI：
10.1073/pnas.79.9.2971
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发表时间：
1982
影响因子：
11.1
通讯作者：
B. Young
中科院分区：
文献类型：
--
作者：
R. Krumlauf;M. Jeanpierre;B. Young
从用溴化乙锭染色的中期染色体悬浮液中分离高度纯化的人类染色体21和22的组分，通过使用荧光激活细胞分选仪（FACS II）。代表染色体21和22的两个重组DNA文库通过用EcoRI完全消化来自这些级分的DNA并插入载体λ gtWES λ B中而构建。从22号染色体文库中随机选择的20个克隆与EcoRI消化的人DNA杂交，其中5个克隆与大小与噬菌体插入片段相同的单一条带杂交。对这5个单拷贝序列和通过筛选22号染色体文库的表达序列分离的编码8 S RNA的克隆进行了详细表征。将所有六个克隆与一组分选的染色体和杂交细胞系杂交，证实了序列分配至染色体22。通过与从具有平衡易位t（17;22）（p13;q11）的细胞系和含有另一易位t（X;22）（q13;q112）的各个部分的杂交细胞系分选的易位染色体杂交，将序列定位于染色体22的区域。5个克隆位于22号染色体长臂上q112和pter之间，而1个克隆和从22号染色体文库中分离的18 S rRNA基因位于pter和g112之间。利用该方法构建的染色体特异性文库具有直接、适用于几乎所有人类染色体的优点，在人类遗传病的分子分析中具有重要意义。
Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.