Mouse ornithine decarboxylase gene: cloning, structure, and expression.
Mouse ornithine decarboxylase gene: cloning, structure, and expression.
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小鼠鸟氨酸脱羧酶基因：克隆、结构和表达。
DOI：
10.1073/pnas.85.7.2200
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发表时间：
1988
影响因子：
11.1
通讯作者：
Coffino,P
中科院分区：
文献类型：
--
作者：
Brabant,M;McConlogue,L;vanDaalenWetters,T;Coffino,P
我们使用分子克隆从细胞系中分离小鼠鸟氨酸脱羧酶（OrnDCase; L-鸟氨酸羧基裂解酶，EC 4.1.1.17）的功能基因，在该细胞系中该基因已被选择性扩增。mRNA的5'末端的位置被确定，并且编码序列被显示为在312或313个核苷酸（nt）的非翻译前导序列之前。后者是高度G + C丰富，特别是在其5 '最部分。前导序列具有广泛而稳定的二级结构。该基因的转录单位相对较小，从多聚腺苷酸化的起始位点到近端位点约等于6.2个酶（kb）。转录起始位置附近的DNA序列分析表明存在“TATA”盒，但不存在“CAAT”盒。通过将其转染到培养的细胞中来测试克隆基因的功能特性。推定的全长基因的表达有效地赋予鸟氨酸脱羧酶活性的受体突变体细胞缺乏这种活性。为了评估OrnDCase启动子区域的功能和强度并划定其边界，我们使用了瞬时表达测定。在细菌氯霉素乙酰转移酶基因的上游放置OrnDCase基因的一部分，包括假定的启动子区，跨越从转录起始位点的约等于3.0kb的5 ′端到转录物的前250 nt的区域。当在小鼠NIH 3 T3细胞中表达时，该OrnDCase基因组元件在强度上与劳斯肉瘤病毒长末端重复启动子相当。一个类似的构建体，截短以仅保留OrnDCase基因5'至转录起始位点的264个碱基对，产生未降低的表达水平。
We used molecular cloning to isolate a functional gene for mouse ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) from a cell line in which that gene had been selectively amplified. The position of the 5' terminus of the mRNA was identified, and the coding sequence was shown to be preceded by a 312- or 313-nucleotide (nt) untranslated leader. The latter is highly G + C rich, particularly in its 5'-most portion. The leader can be anticipated to have extensive and stable secondary structure. The transcription unit of the gene is of relatively small size, approximately equal to 6.2 kilobases (kb) from the start site to the proximal site of polyadenylylation. Sequence analysis of DNA near the transcription start position demonstrated the presence of a "TATA" box, but no "CAAT" box. Functional properties of the cloned gene were tested by transfecting it into cultured cells. Expression of the putative full-length gene efficiently conferred ornithine decarboxylase activity on recipient mutant cells deficient in that activity. To assess the function and strength of the OrnDCase promoter region and to delimit its boundaries, we used a transient expression assay. Upstream of a bacterial chloramphenicol acetyltransferase gene was placed a portion of the OrnDCase gene, including the presumed promoter region, spanning a region from approximately equal to 3.0 kb 5' of the site of transcription initiation to the first 250 nt of the transcript. When expressed in mouse NIH 3T3 cells, this OrnDCase genomic element was comparable in strength to the Rous sarcoma virus long terminal repeat promoter. A similar construct, truncated so as to retain only 264 base pairs of the OrnDCase gene 5' to the site of transcription start, yielded undiminished levels of expression.