人类大脑中 KLK8 特异剪接体(Type Ⅱ)的进化遗传机制研究及其功能分析

可变性剪接在调节基因表达和增加蛋白多样化方面具有重要的作用。它也是高等生物基因组进化中产生新蛋白的主要机制之一。人类大脑――适应性进化的产物，则更加倾向运用可变性剪接的策略来行使其高度复杂的功能。因此，鉴定出人类中枢神经系统中的特异性剪接体对于我们理解人类认知的功能进化有着相当重要的意义。 KLK8 (Kallikrein 8， 又名 neuropsin)是中枢神经系统中参与大脑学习记忆的一种丝氨酸蛋白酶。以前的研究表明，这个基因的剪接形式在人和小鼠中是不同的，在人脑中表达一种特有的更长的剪接形式（type Ⅱ）。而序列分析又提示这个更长的剪接形式在灵长类中是最近起源的。本论文的研究目的：（1）揭示在人类大脑进化过程中，type Ⅱ特异剪接体产生的分子遗传机制；（2）了解type Ⅱ的蛋白异型体比原先KLK8 蛋白的typeⅠ异型体有何新的功能特点。我们发现type Ⅱ是一种人特有的剪接体，在其它非人灵长类的大脑中没有表达。运用体外剪接实验，我们证实：一个人类特有的T 到A 单碱基突变改变了KLK8 的剪接模式，使得type Ⅱ这一崭新的剪接体在人类大脑中产生。体外突变体的剪接实验则进一步证实了这一单碱基突变是type Ⅱ表达的充要条件。此外，运用突变实验我们也证实了多个位点参与削弱KLK8 原有组成性剪接位点的剪接效率。我们估计在灵长类进化过程中，新剪接位点的产生和原组成性剪接位点的削弱共同造成了KLK8 剪接的变化，这就表明KLK8 经过多个演化步骤最终才导致了type Ⅱ在人大脑中的表达。5’RACE，启动子区域序列分析以及启动子的活力检测则表明KLK8 的转录调节在进化过程中一直在动态变化。此项研究揭示了在人类进化过程中，中枢神经系统中通过可变剪接产生新蛋白的遗传学分子机制。除此之外，RT-PCR 和western blot 实验表明特异剪接体的表达是时空依赖性的，它的分泌效率也与细胞类型相关。生物化学和酶学实验则表明type Ⅱ 不仅能够产生原有KLK8 的活性形式蛋白，它还有type Ⅱ异型体特有的一个中间蛋白体，提示我们人类大脑中type Ⅱ蛋白的形成可能会对原有蛋白起一定的功能修饰作用

PASSIVATION OF COPPER BY LITHIUM IN P-TYPE GAAS

We report the passivation of two deep copper-related acceptor levels in Cu-diffused p-type GaAs by the group-I element lithium. The deep-level-transient-spectroscopy (DLTS) signals of the well-known Cu-related levels with apparent activation energies 0.15 eV and 0.40 eV disappear in Cu-diffused samples when they are diffused with Li, but can be reactivated by annealing. Photoluminescence measurements show a corresponding disappearance and reappearance of the copper-related luminescence at 1.36 eV. Also we observe with DLT'S an energy level at E(V) + 0.32 eV in the Cu-Li-diff-used samples. The level is neither present in the Cu-diffused samples before Li diffusion nor in Cu-Li-diffused samples after annealing. As the level is not observed in starting materials or solely Li-diffused samples we suggest that it is related to a Cu-Li complex.

RADIATIVE RECOMBINATION IN N-TYPE AND P-TYPE GAAS COMPENSATED WITH LI

We report fundamental changes of the radiative recombination in a wide range of n-type and p-type GaAs after diffusion with the group-I element Li. These optical properties are found to be a bulk property and closely related to the electrical conductivity of the samples. In the Li-doped samples the radiative recombination is characterized by emissions with excitation-dependent peak positions which shift to lower energies with increasing degree of compensation and concentration of Li. These properties are shown to be in qualitative agreement with fluctuations of the electrostatic potential in strongly compensated systems. For Li-diffusion temperatures above 700-800-degrees-C semi-insulating conditions with electrical resistivity exceeding 10(7) OMEGA cm are obtained for all conducting starting materials. In this heavy Li-doping regime, the simple model of fluctuating potentials is shown to be inadequate for explaining the. experimental observations unless the number of charged impurities is reduced through complexing with Li. For samples doped with low concentrations of Li, on the other hand, the photoluminescence properties are found to be characteristic of impurity-related emissions.

LITHIUM PASSIVATION OF ZN AND CD ACCEPTORS IN P-TYPE GAAS

We report lithium passivation of the shallow acceptors Zn and Cd in p-type GaAs which we attribute to the formation of neutral Li-Zn and Li-Cd complexes. Similar to hydrogen, another group-I element, lithium strongly reduces the concentration of free holes when introduced into p-type GaAs. The passivation is inferred from an increase of both the hole mobility and the resisitivity throughout the bulk of the sample. It is observed most clearly for Li concentrations comparable to the shallow-acceptor concentration. In addition, compensation of shallow acceptors by randomly distributed donors is present in varying degree in the Li-diffused samples. Unlike hydrogenation of n-type GaAs, Li doping shows no evidence of neutralizing shallow donors in GaAs.

A NEW TYPE OF SILICON-ON-INSULATOR WITH A PERFECT SURFACE SILICON LAYER

A novel silicon structure consisting of a silicon-on-defect layer (SODL), with enhanced surface Hall mobility in the surface layer on a buried defect layer (DL), has been discovered [J. Li, Nucl. Instr. and Meth. B59/60 (1991) 1053]. SODL material was formed by using proton implantation and subsequent two-step annealing. The implantation was carried out with a Varian 350D ion implanter. Based on the discovery, a standard measurement method (current-voltage curve method) was adopted to measure the true resistivity value of the DL in order to replace the spreading resistivity measurement by which the true resistivity in seriously defective silicon cannot be obtained. By adopting the current-voltage current method, the true resistivity value of the DL is measured to be 4.2 x 10(9) OMEGA cm. The SODL material was proved to be a silicon-on-insulator substrate.

NEW TYPE OF SILICON MATERIAL WITH VERY HIGH-QUALITY SURFACE-LAYER ON INSULATING DEFECT LAYER

A new-type silicon material, silicon on defect layer (SODL) was proved to have a very high quality surface microstructure which is necessary for commercially feasible high-density very large scale integrated circuits (VLSI). The structure of the SODL material was viewed by transmission electron microscopy. The SODL material was also proved to have a buried defect layer with an insulating resistivity of 5.7 x 10(10) OMEGA-cm.