Munjaal R P, Chandra T, Woo S L, Dedman J R, Means A R
Proc Natl Acad Sci U S A. 1981 Apr;78(4):2330-4. doi: 10.1073/pnas.78.4.2330.
Calmodulin mRNA has been partially purified from a total nucleic acid extract of the electroplax of Electrophorus electricus by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. A 9- to 10S fraction was determined to contain 39% calmodulin mRNA by translation in a reticulocyte lysate followed by immunoprecipitation with antibodies to calmodulin. Double-stranded cDNA was synthesized from the RNA fraction by using reverse transcriptase from avian myeloblastosis virus. The double-stranded cDNA was joined to pBR322 linearized by restriction endonuclease Pst I and used to transform Escherichia coli RRI. DNAs from 60 tetracycline-resistant cloned hybridized to [32P]cDNA synthesized from the partially purified calmodulin mRNA fraction. By direct DNA sequence analysis, one of these clones, pCM109, was shown to contain calmodulin-specific sequences corresponding to amino acid residues 93--148 of calmodulin or approximately 38% of the peptide-coding region of the calmodulin structural gene sequence. pCM109 was hybridized to DNA isolated from three vertebrate and one plant species by the procedure of Southern. Positive hybridization bands were noted regardless of the DNA source. These data suggest thaat calmodulin gene sequences are evolutionarily conserved, as has been shown to be the case for the primary amino acid sequence.
通过寡聚(dT)-纤维素色谱法和蔗糖梯度离心法,从电鳗(Electrophorus electricus)的电板总核酸提取物中部分纯化了钙调蛋白mRNA。通过在网织红细胞裂解物中进行翻译,然后用抗钙调蛋白抗体进行免疫沉淀,确定9至10S的级分含有39%的钙调蛋白mRNA。使用禽成髓细胞瘤病毒的逆转录酶从RNA级分合成双链cDNA。将双链cDNA连接到经限制性内切酶Pst I线性化的pBR322上,并用于转化大肠杆菌RR1。来自60个四环素抗性克隆的DNA与从部分纯化的钙调蛋白mRNA级分合成的[32P]cDNA杂交。通过直接DNA序列分析,其中一个克隆pCM109被证明含有与钙调蛋白93-148位氨基酸残基相对应的钙调蛋白特异性序列,约占钙调蛋白结构基因序列肽编码区的38%。通过Southern印迹法,将pCM109与从三种脊椎动物和一种植物物种中分离的DNA杂交。无论DNA来源如何,均观察到阳性杂交带。这些数据表明,钙调蛋白基因序列在进化上是保守的,正如其一级氨基酸序列所示。