Münger K, Germann U A, Lerch K
Biochemisches Institut Universität Zürich, Switzerland.
Experientia Suppl. 1987;52:393-400. doi: 10.1007/978-3-0348-6784-9_37.
The N. crassa CuMT gene has been cloned and its nucleotide sequence determined. To this end an MT specific undecanucleotide was synthesized and used for cDNA synthesis with enriched MT mRNA as a template. Sequence analysis of the cDNA obtained allowed the synthesis of a unique 21mer which was used as a hybridization probe to screen a genomic DNA library of N. crassa. Several positive clones were isolated and subjected to restriction and sequence analysis. In agreement with the published amino acid sequence, the gene codes for a polypeptide of 26 amino acid residues in length. The coding region is interrupted by a small intron. Compared to the structure of mammalian MT genes the intron-exon boundaries are located in different sequence positions. The induction of MT mRNA was studied by Northern analysis. Maximum levels of MT mRNA were detected about 1 hour after addition of copper ions to mycelium of N. crassa. The half-life time of the messenger was estimated as 2.5 hours. The CuMT amounts reach a maximum level at 3 hours after induction and thereafter remain constant.
粗糙脉孢菌的铜金属硫蛋白（CuMT）基因已被克隆并测定了其核苷酸序列。为此，合成了一种金属硫蛋白特异性的十一核苷酸，并以富集的金属硫蛋白信使核糖核酸（mRNA）为模板用于互补脱氧核糖核酸（cDNA）合成。对所获得的cDNA进行序列分析后，合成了一种独特的21聚体，该21聚体用作杂交探针来筛选粗糙脉孢菌的基因组DNA文库。分离出了几个阳性克隆，并对其进行了限制性酶切和序列分析。与已发表的氨基酸序列一致，该基因编码一个长度为26个氨基酸残基的多肽。编码区被一个小内含子中断。与哺乳动物金属硫蛋白基因的结构相比，内含子-外显子边界位于不同的序列位置。通过Northern分析研究了金属硫蛋白mRNA的诱导情况。在向粗糙脉孢菌的菌丝体中添加铜离子后约1小时检测到金属硫蛋白mRNA的最高水平。信使核糖核酸的半衰期估计为2.5小时。铜金属硫蛋白的量在诱导后3小时达到最高水平，此后保持恒定。
