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来自特定人类染色体的基因组文库的构建与表征

Construction and characterization of genomic libraries from specific human chromosomes.

作者信息

Krumlauf R, Jeanpierre M, Young B D

出版信息

Proc Natl Acad Sci U S A. 1982 May;79(9):2971-5. doi: 10.1073/pnas.79.9.2971.

DOI:10.1073/pnas.79.9.2971
PMID:6953442
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC346330/
Abstract

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.

摘要

通过使用荧光激活细胞分选仪（FACS II），从用溴化乙锭染色的中期染色体悬液中分离出高度纯化的人类21号和22号染色体片段。通过用EcoRI完全消化这些片段中的DNA并插入载体λgtWESλB，构建了代表21号和22号染色体的两个重组DNA文库。从22号染色体文库中随机选择的20个克隆与经EcoRI消化的人类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个单拷贝序列以及通过筛选22号染色体文库以寻找表达序列而分离出的一个编码8S RNA的克隆进行了详细表征。所有6个克隆与一组分选的染色体和杂交细胞系的杂交证实了这些序列定位于22号染色体。通过与从具有平衡易位t(17;22)(p13;q11)的细胞系中分选的易位染色体以及含有另一个易位t(X;22)(q13;q112)不同部分的杂交细胞系杂交，将这些序列定位到22号染色体的区域。5个克隆位于22号染色体长臂上q112和pter之间，而从22号染色体文库中分离出的一个克隆和一个18S rRNA基因位于pter和g112之间。通过这种方法构建染色体特异性文库具有直接且适用于几乎所有人类染色体的优点，并且在人类遗传疾病的分子分析中将具有重要意义。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/98a35b065564/pnas00448-0239-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/989f4e9eedf6/pnas00448-0238-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/13bf37ece11e/pnas00448-0238-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/80af852e3269/pnas00448-0238-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/7f5a9f6f61cc/pnas00448-0238-d.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/6049117f1cbd/pnas00448-0239-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/98a35b065564/pnas00448-0239-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/989f4e9eedf6/pnas00448-0238-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/13bf37ece11e/pnas00448-0238-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/80af852e3269/pnas00448-0238-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/7f5a9f6f61cc/pnas00448-0238-d.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/6049117f1cbd/pnas00448-0239-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d4c7/346330/98a35b065564/pnas00448-0239-b.jpg

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