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

分子进化的研究是基于近代物种特定蛋白质的氨基酸序列和编码它们的基因的核苷酸序列的数据。这项研究是世界上第一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试验。我选取了近代的鹦鹉螺（Nautilus pompilius）和刺尾螺（Atrina vexillum）的珍珠层，与北海道的白垩系鹦鹉螺（amonite）和刺尾螺（Inoceramus）的珍珠层进行比较。尽管化石贝壳散落在沉积物之间，但它们保存得很好，可以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馏。结果，在最近的样品中，在很宽的分子量范围内检测到几个条带。然而，在菊石中，SDS-PAGE没有检测到具有特定分子量的成分，只能看到较长的涂片，除了在凤尾鱼中有一条分子量为1 kDa的条带。为此，我们对该新种的全壳组分和一kDa组分进行了氨基酸序列分析。幸运的是，在鸡眼菌成分的n端区域确定了20个氨基酸残基，但这一序列与从黄芪中提取的成分没有同源性。这些结果表明，菊石的有机基质蛋白在7000万年的时间里几乎全部被分解消失，保存下来的成分可能被降解成非常小的肽，无法通过SDS-PAGE分离，不适合作为氨基酸序列分析的目标。为此，我尝试用TOF-MS分析Ammnonite的全脱钙组分，但由于缺少离子化组分，没有成功。其原因尚不清楚，未来需要进行氨基酸组成分析或LC-MS分析，以明确菊石壳中保存的有机物的组成。另一方面，雏菊壳中含有一种低分子量成分，但其是否来源于真正的有机基质尚不清楚。对阿特里纳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一问题的途径。少

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