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利用隐蔽剪接和聚腺苷酸化位点，在耐甲氨蝶呤小鼠细胞中对人工二氢叶酸还原酶转录单位进行选择性扩增。

Selective amplification in methotrexate-resistant mouse cells of an artificial dihydrofolate reductase transcription unit making use of cryptic splicing and polyadenylation sites.

作者信息

Breathnach R

出版信息

EMBO J. 1984 Apr;3(4):901-8. doi: 10.1002/j.1460-2075.1984.tb01903.x.

DOI:10.1002/j.1460-2075.1984.tb01903.x
PMID:6202514
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC557445/
Abstract

We have constructed a recombinant ( pMOP ) which is based on the bacterial plasmid pML2 and contains bovine papilloma virus type 1 (BPV 1) sequences linked to an artificial mouse dihydrofolate reductase (DHFR) transcription unit. This unit consists of the SV40 early gene promoter, a complete DHFR coding sequence and splice and polyadenylation sites from a rabbit beta-globin gene. Intact pMOP or a fragment thereof devoid of pML2 sequences will transform mouse cells to methotrexate resistance. The lines obtained contain approximately 200 copies of the DHFR transcription unit. In no case, however, did we find evidence of extrachromosomal maintenance of BPV 1 or DHFR sequences. One line, when selected for resistance to elevated levels of methotrexate, contained amplified copies of a 'novel' DHFR transcription unit resulting from fusion of two normal units. This line contained the DHFR RNA species present in the parent line plus some additional species. The structure of these various RNA species was determined and evidence found for the use of cryptic splice and polyadenylation sites.

摘要

我们构建了一种重组体（pMOP），它基于细菌质粒pML2，并包含与人工小鼠二氢叶酸还原酶（DHFR）转录单位相连的1型牛乳头瘤病毒（BPV 1）序列。该单位由SV40早期基因启动子、完整的DHFR编码序列以及来自兔β-珠蛋白基因的剪接和聚腺苷酸化位点组成。完整的pMOP或其不含pML2序列的片段将使小鼠细胞对甲氨蝶呤产生抗性。获得的细胞系含有约200个DHFR转录单位拷贝。然而，在任何情况下，我们都没有发现BPV 1或DHFR序列染色体外维持的证据。一个细胞系在被选择对高水平甲氨蝶呤产生抗性时，含有由两个正常单位融合产生的“新”DHFR转录单位的扩增拷贝。该细胞系含有亲本细胞系中存在的DHFR RNA种类以及一些其他种类。确定了这些各种RNA种类的结构，并发现了使用隐蔽剪接和聚腺苷酸化位点的证据。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/3cb781408b3a/emboj00308-0205-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/4a9362c9f671/emboj00308-0201-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/694c9fd5dd5c/emboj00308-0202-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/0cf24114280f/emboj00308-0204-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/15407806ddc5/emboj00308-0204-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/3cb781408b3a/emboj00308-0205-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/4a9362c9f671/emboj00308-0201-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/694c9fd5dd5c/emboj00308-0202-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/0cf24114280f/emboj00308-0204-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/15407806ddc5/emboj00308-0204-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0b33/557445/3cb781408b3a/emboj00308-0205-a.jpg

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引用本文的文献

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本文引用的文献

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EMBO J. 1983;2(9):1487-92. doi: 10.1002/j.1460-2075.1983.tb01612.x.
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Multiple initiation and polyadenylation sites for the chicken ovomucoid transcription unit.鸡卵类粘蛋白转录单位的多个起始和聚腺苷酸化位点。
J Mol Biol. 1982 Dec 5;162(2):345-64. doi: 10.1016/0022-2836(82)90531-9.
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Plasmid screening at high colony density.高菌落密度下的质粒筛选
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Corrected splicing of a chicken ovalbumin gene transcript in mouse L cells.小鼠L细胞中鸡卵清蛋白基因转录本的校正剪接
Proc Natl Acad Sci U S A. 1980 Feb;77(2):740-4. doi: 10.1073/pnas.77.2.740.
5
Recombinant DNA molecules comprising bovine papilloma virus type 1 DNA linked to plasmid DNA are maintained in a plasmidial state both in rodent fibroblasts and in bacterial cells.包含与质粒DNA相连的1型牛乳头瘤病毒DNA的重组DNA分子在啮齿动物成纤维细胞和细菌细胞中均以质粒状态维持。
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Plasmids for the cloning and expression of full-length double-stranded cDNAs under control of the SV40 early or late gene promoter.
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Transformation and replication in mouse cells of a bovine papillomavirus--pML2 plasmid vector that can be rescued in bacteria.一种可在细菌中拯救的牛乳头瘤病毒-pML2质粒载体在小鼠细胞中的转化与复制。
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