Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene.
Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene.
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通过普遍适用于任何很少表达的基因的方法对插入序列 IS10 转座酶基因表达进行定量。
DOI：
10.1073/pnas.83.6.1787
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发表时间：
1986
影响因子：
11.1
通讯作者：
Kleckner,N
中科院分区：
文献类型：
--
作者：
Raleigh,EA;Kleckner,N
我们发现，从合适的转座酶-LacZ基因融合的单个染色体拷贝的β-半乳糖苷酶活性判断，IS10转座酶的合成数量很少，平均每代细胞约0.15条多肽链。在渗透性全细胞实验中，融合基因的酶活性比细胞提取液中的酶活性低10倍。也许，大多数细胞含有不到四条多肽链，这些链只有在细胞破坏后才能组装成活性四聚体。这一解释允许建立酶活性与转录和翻译速率之间的两个方程，其解表明融合基因以每细胞每代仅0.25个转录本的平均速率表达，平均每个转录本仅有0.58个翻译产物。这种方法通常适用于每代细胞合成少于四条多肽链的任何基因的分析。
We have found that IS10 transposase is synthesized in tiny amounts, about 0.15 polypeptide chain per cell per generation on average, as judged from the beta-galactosidase activity of a single chromosomal copy of a suitable transposase-lacZ gene fusion. Enzymatic activity from the fusion gene is a factor of 10 lower in a permeabilized whole cell assay than in cell extracts. Probably, most cells contain fewer than four polypeptide chains, and these chains can assemble into active tetramers only after cell disruption. This interpretation permits formulation of two equations relating enzyme activities to transcription and translation rates, solution of which reveals that the fusion gene is expressed at the average rate of only 0.25 transcript per cell per generation, with an average of only 0.58 translation product per transcript. This methodology is generally applicable to analysis of any gene from which fewer than four polypeptide chains are synthesized per cell per generation.