Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.
Structure of the Spec1 gene encoding a major calcium-binding protein in the embryonic ectoderm of the sea urchin, Strongylocentrotus purpuratus.
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海胆（Strongylocentrotus purpuratus）胚胎外胚层中编码主要钙结合蛋白的 Spec1 基因的结构。
DOI：
10.1016/0022-2836(85)90101-9
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发表时间：
1985
影响因子：
5.6
通讯作者：
Klein,WH
中科院分区：
文献类型：
--
作者：
Hardin,SH;Carpenter,CD;Hardin,PE;Bruskin,AM;Klein,WH
我们已经确定和特点的Spec 1基因的结构在海胆Strongylocentrotus purpuratus。在早期的研究中，我们证明了一个小的信使RNA家族，称为S。purpuratusectodermal mRNA在受精后20小时开始在海胆胚胎的外胚层细胞中积累。Spec mRNA编码属于肌钙蛋白C超家族的一组低分子量蛋白质。Spec 1转录本是该家族的主要mRNA，其3′端非翻译序列是异质的，但编码一种蛋白质，最近被证明是一种钙结合蛋白。使用Spec互补DNA克隆从两个λ文库中分离基因组克隆。这些基因组克隆包含一个41 kb（kb = 103个碱基或碱基对）的S. Purpuratus基因组中含有与另一个Spec基因Spec 2c紧密连锁的Spec 1基因。Spec 1基因全长10.3kb，包含6个外显子。基于限制性片段长度差异和使用来自Spec 1 3′非翻译区的探针的杂交强度差异，可将含有Spec 1基因的基因组克隆分为两组。通过用Spec 1互补DNA克隆的3′非翻译序列探测不同个体精子DNA的基因组DNA印迹，获得了这些组可能对应于Spec 1基因的两个等位基因的证据。这些印迹显示，我们已经鉴定的Spec 1 mRNA中有两个，可能还有第三个，是Spec 1基因的等位基因。因此，在海胆基因组中似乎有一个多态性Spec 1基因。采用S1保护和引物延伸技术对Spec 1基因5′端进行定位。这些实验的结果表明，Spec 1 mRNA的转录起始于距第一外显子3′端220个碱基的A残基。为支持这一说法，在该位点上游分别发现了指示许多真核启动子的典型T-A-T-A和C-A-A-T序列，分别为23个碱基和60个碱基。对Spec 1基因几kb内的序列分析表明，在该基因附近有5个重复序列家族成员，3个上游和2个下游。另一种SpecmRNA的5′前导序列Spec 2a也含有该重复序列家族的一个成员。Spec 1基因结构相比，其他四个基因的肌钙蛋白C超家族，虽然核苷酸序列有很大的分歧，我们发现惊人的相似之处，所有这些基因的结构。有趣的是，这些基因编码的蛋白质的钙结合结构域并不对应于外显子/内含子的边界，这表明必须有其他的原因保持基因结构的相似性。
We have identified and characterized the structure of the Spec1 gene in the sea urchinStrongylocentrotus purpuratus. In earlier studies we demonstrated that a small family of messenger RNAs, termed Spec mRNAs forS. purpuratusectodermal mRNAs, begins to accumulate 20 hours after fertilization in ectoderm cells of the sea urchin embryo. The Spec mRNAs code for a group of low molecular weight proteins belonging to the troponin C superfamily. Spec1 transcripts, the predominant mRNAs of the family, are heterogeneous in their 3′ untranslated sequences but code for a single protein, recently shown to be a calcium-binding protein. Spec complementary DNA clones were used to isolate genomic clones from two λ libraries. These genomic clones comprise a 41 kb (kb = 103bases or base-pairs) region of theS. purpuratusgenome and contain a Spec1 gene closely linked to another Spec gene, Spec2c. The Spec1 gene is 10.3 kb in length and contains six exons. The genomic clones containing the Spec1 gene can be placed into two groups based on restriction fragment length differences and differences in hybridization strengths using probes derived from Spec1 3′ untranslated regions. Evidence that these groups probably correspond to two alleles of the Spec1 gene was obtained by probing genomic DNA blots of sperm DNA from different individuals with 3′ untranslated sequences of Spec1 complementary DNA clones. These blots show that two of the Spec1 mRNAs we have characterized, and probably a third, are alleles of the Spec1 gene. Thus, there appears to be a single polymorphic Spec1 gene in the sea urchin genome. We used S1protection and primer extension procedures to map the 5′ end of the Spec1 gene. Results from these experiments indicate that the initiation of transcription of the Spec1 mRNA begins at an A residue 220 bases from the 3′ end of the first exon. Adding support to this claim, cannonical T-A-T-A and C-A-A-T sequences, indicative of many eukaryotic promoters, are found 23 bases and 60 bases upstream from this site, respectively. Analysis of sequences within a few kb of the Spec1 gene show that there are five members of a repetitive sequence family near the gene, three upstream and two downstream. The 5′ leader sequence of another Spec mRNA, Spec2a, also contains a member of this repeat family. The Spec1 gene structure is compared to four other genes of the troponin C superfamily and, although the nucleotide sequences have diverged greatly, we find striking similarities among the structures of all of these genes. Interestingly, the calcium-binding domains of the proteins encoded by these genes do not correspond to the exon/intron boundaries, suggesting that there must be other reasons for maintaining the similarities in gene structure.