Structure analysis of the organic matrix components in Ammonite shells through gene cloning

通过基因克隆分析菊石壳中有机基质成分的结构

• 批准号: 11640468
• 负责人: SAMATA Tetsuro
• 金额: $ 2.18万
• 依托单位: Azabu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11640468/
• 关键词: fossil molluscan shell; organic matrix; cDNA cloning; amino acid sequence analysis; SDS-PAGE; Mass analysis; alterative change; molecular evolution; 殻体真珠層; cDNA クローニング; アミノ酸配列; 化石; オームガイ; アンモナイト

The research of molecular evolution has been based on data of the amino acid sequences of the specific proteins and nucleotide sequences of the genes that encode them of recent species. This study is the first trial in the world to examine molecular evolution on the basis of the amino acid sequences of the proteins preserved in the fossil hard tissues.I selected the nacreous layer of recent Nautilus pompilius and Atrina vexillum for comparison with the nacreous layer of Cretaceous Ammonite and Inoceramus from Hokkaido. Although the fossil shells lied scattered between sediments as pieces, preservation of them were so well to separate the prismatic layer and nacreous layer. I fractionated the organic matrix in the decalcified components of these samples by SDS-PAGE.As a result, several bands were detected in the wide range of molecular weight in the recent samples. However, no component with specific molecular weight could be detected in Ammonite by SDS-PAGE and only long smear was visi … More ble except for a band with molecular weight of I kDa in Inoceramus. On this account, amino acid sequence analysis was carried out on the whole components in shells of the recent species and 1 kDa component in Inoceramus. Fortunately, 20 amino acid residues were determined for the N-terminus region of the Inoceramus component but this sequence showed no homology with those of the components extracted from Atrina.These results imply that almost all portion of the organic matrix protein of Ammonite was decomposed and disappeard during periods for 70 million years, and preserved components may be degraded into very small peptides which can not be separated by SDS-PAGE and it is unsuitable for a target of amino acid sequence analysis. On this account, I tried TOF-MS analysis of whole decalcified component of Ammnonite, but it was unsuccessful because of the lack of the ionized components. The reason of this is unclear, and amino acid compositional analysis or LC-MS analysis should be carried out to make clear the composition of organic materials preserved in the shell of Ammonite in future. On the other hand, a low molecular weight component was contained in the shells of Inoceramus but the judgment whether it originates from the real organic matrix or not still remains unclear. The analysis of the complete primary structures of the organic matrix proteins in the nacreous layer of Atrina will show us a way to solve this problem. Less

分子进化的研究一直基于特定蛋白质的氨基酸序列和编码这些蛋白质的基因的核苷酸序列的数据。这项研究是世界上首次根据保存在化石硬组织中的蛋白质的氨基酸序列来研究分子进化的尝试。我选择了近代的蓬贝鹦鹉螺和涡虫珍珠层与北海道白垩纪的菊石和伊诺神经节珍珠层进行比较。尽管化石贝壳散落在沉积物之间，但它们的保存很好，将棱柱层和珍珠层分开。我用SDS-PAGE对这些样品脱钙组分中的有机基质进行了分级，结果在最近的样品中在较宽的相对分子质量范围内检测到了几条带。但未检测到特定相对分子质量的成分，只有较长的涂片可见Visi…。在Inocerus中，除一条相对分子质量为I kDa的条带外，其余条带均为BLE。在此基础上，对新种贝壳的全组分和Inocerus的1 kDa组分进行了氨基酸序列分析。幸运的是，肌球蛋白组分的N-末端有20个氨基酸残基，但这一序列与从Atrina中提取的组分的氨基酸序列没有同源性，这表明Ammonite的有机基质蛋白的几乎所有部分在7000万年的周期中被分解和消失，保存下来的组分可能被降解成很小的多肽，不能用SDS-PAGE进行分离，不适合作为氨基酸序列分析的目标。有鉴于此，我尝试了用TOF-MS分析法分析氨锰矿的全部脱钙组分，但由于缺少电离组分，所以没有成功。原因尚不清楚，今后应进行氨基酸组成分析或LC-MS分析，以明确保存在铵盐壳中的有机物质的组成。另一方面，Inocerus的壳层中含有一个低分子量组分，但它是否来自真正的有机基质仍不清楚。对Atrina珍珠层中有机基质蛋白的完整一级结构的分析将为我们提供解决这个问题的方法。较少