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嗅觉标记蛋白cDNA的分子克隆与测序

Molecular cloning and sequencing of a cDNA for olfactory marker protein.

作者信息

Rogers K E, Dasgupta P, Gubler U, Grillo M, Khew-Goodall Y S, Margolis F L

出版信息

Proc Natl Acad Sci U S A. 1987 Mar;84(6):1704-8. doi: 10.1073/pnas.84.6.1704.

DOI:10.1073/pnas.84.6.1704
PMID:3470751
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC304505/
Abstract

cDNA clones corresponding to mRNA for rat olfactory marker protein (OMP) were isolated from a cDNA library. The library was constructed from olfactory mucosa poly(A)+ RNA enriched for OMP mRNA and cloned into a pBR322-derived plasmid, pMG5. OMP cDNA clones were detected by using a 17-base oligonucleotide probe that contained all 16 possible sequences coding for a known partial amino acid sequence of rat OMP. The identity of these clones was confirmed by hybrid-selected translation and nucleotide sequencing. The sequence of one clone was determined and contained the complete OMP coding region of 486 nucleotides followed by 1630 nucleotides of the 3' untranslated region. The 3' untranslated region included the polyadenylylation signal 16 nucleotides upstream of the poly(A) tail. No other ATG-initiated open reading frame larger than 20 codons was present in register. RNA blot analysis of olfactory mucosa poly(A)+ RNA using this clone as a probe indicated that the level of OMP mRNA, but not its size, declined significantly within a few days following olfactory bulbectomy. OMP mRNA was not detected in 14 nonolfactory rat tissues. Surprisingly, a small amount of OMP mRNA was observed in olfactory bulb. The presence of OMP mRNA in olfactory bulb was confirmed by in vitro translation and immunoprecipitation. These results suggest either that a previously undescribed population of neurons in the olfactory bulb synthesize OMP or that OMP mRNA is transported to the bulb by axonal transport.

摘要

从一个cDNA文库中分离出了与大鼠嗅觉标记蛋白(OMP)mRNA相对应的cDNA克隆。该文库是用富含OMP mRNA的嗅黏膜聚腺苷酸加尾RNA构建的,并克隆到一个源自pBR322的质粒pMG5中。通过使用一个17个碱基的寡核苷酸探针检测OMP cDNA克隆,该探针包含了编码大鼠OMP已知部分氨基酸序列的所有16种可能序列。这些克隆的身份通过杂交选择翻译和核苷酸测序得到了确认。确定了一个克隆的序列,其包含486个核苷酸的完整OMP编码区,随后是3'非翻译区的1630个核苷酸。3'非翻译区在聚腺苷酸尾上游16个核苷酸处包含聚腺苷酸化信号。没有其他大于20个密码子的由ATG起始的开放阅读框与之对齐。用该克隆作为探针对嗅黏膜聚腺苷酸加尾RNA进行RNA印迹分析表明,在嗅球切除术后几天内,OMP mRNA的水平显著下降,但其大小未变。在14种非嗅觉大鼠组织中未检测到OMP mRNA。令人惊讶的是,在嗅球中观察到少量的OMP mRNA。通过体外翻译和免疫沉淀证实了嗅球中存在OMP mRNA。这些结果表明,要么嗅球中存在以前未描述的神经元群体合成OMP,要么OMP mRNA是通过轴突运输被转运到嗅球的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/08a21b35eb23/pnas00271-0246-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/b66202e998f5/pnas00271-0244-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/32859b9aec76/pnas00271-0246-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/aee517590335/pnas00271-0246-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/08a21b35eb23/pnas00271-0246-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/b66202e998f5/pnas00271-0244-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/32859b9aec76/pnas00271-0246-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/aee517590335/pnas00271-0246-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/0731/304505/08a21b35eb23/pnas00271-0246-c.jpg

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