A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species.
A cloned calmodulin structural gene probe is complementary to DNA sequences from diverse species.
复制标题
克隆的钙调蛋白结构基因探针与不同物种的 DNA 序列互补。
DOI：
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发表时间：
1981
影响因子：
11.1
通讯作者：
A. Means
中科院分区：
文献类型：
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作者：
R. Munjaal;T. Chandra;S. Woo;J. Dedman;A. Means
用Oligo（dT）-纤维素层析和蔗糖梯度离心从Electrophorus electricus的电泳板总核酸提取物中部分纯化了钙调素mRNA。通过在网织红细胞裂解物中翻译，然后用钙调蛋白抗体进行免疫沉淀，确定9至10 S级分含有39%的钙调蛋白mRNA。通过使用来自禽成髓细胞瘤病毒的逆转录酶从RNA组分合成双链cDNA。将双链cDNA连接到用限制性内切酶Pst I线性化的pBR 322上，并用于转化大肠杆菌RRI。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[32 P]cDNA杂交。通过直接DNA序列分析，这些克隆之一，pCM 109，显示含有钙调蛋白特异性序列，其对应于钙调蛋白的氨基酸残基93- 148或钙调蛋白结构基因序列的肽编码区的约38%。用Southern杂交法将pCM 109与从三种脊椎动物和一种植物中分离的DNA进行杂交。无论DNA来源如何，均观察到阳性杂交条带。这些数据表明，钙调素基因序列是进化保守的，如已被证明是一级氨基酸序列的情况。
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.