Nuclear import of the stem-loop binding protein and localization during the cell cycle

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.     

Functional genomics of eukaryotic photosynthesis using insertional mutagenesis of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a widely used model organism for studies of oxygenic photosynthesis in eukaryotes. Here we describe the development of a resource for functional genomics of photosynthesis using insertional mutagenesis of the Chlamydomonas nuclear genome. Chlamydomonas cells were transformed with either of two plasmids conferring zeocin resistance, and insertional mutants were selected in the dark on acetate-containing medium to recover light-sensitive and nonphotosynthetic mutants. The population of insertional mutants was subjected to a battery of primary and secondary phenotypic screens to identify photosynthesis-related mutants that were pigment deficient, light sensitive, nonphotosynthetic, or hypersensitive to reactive oxygen species. Approximately 9% of the insertional mutants exhibited 1 or more of these phenotypes. Molecular analysis showed that each mutant line contains an average of 1.4 insertions, and genetic analysis indicated that approximately 50% of the mutations are tagged by the transforming DNA. Flanking DNA was isolated from the mutants, and sequence data for the insertion sites in 50 mutants are presented and discussed.     

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus.

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran''s nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.     

人颈淋巴结淋巴细胞酶超微结构定位与活性研究

应用电镜酶细胞化学方法研究了人颈淋巴结淋巴细胞ＡＴＰ酶、Ｇ－６－Ｐ酶、５’－Ｎａｓｅ的定位与活性。结果：ＡＴＰ酶主要定位在淋巴细胞膜及内质网、线粒体等膜相结构。Ｇ－６－Ｐ酶主要定位在内质网、线粒体等膜相结构。５’－Ｎａｓｅ主要定位在细胞膜外表面,在内质网与线粒体股外表面也可见此酶反应颗粒。３种酶定位准确、颗粒清晰,酶反应特异性强。结果提示应用此法可以检测以上酶活性,对判定机体免疫状态、对临床诊断与治疗具有一定意义。     

持续发展—人地系统优化的新思路

持续发展的本质是优化人与自然的关系和人与人的关系,环境对人类需求的支持能力是有一个自然极限的,在此极限范围内,人类文化调节可实现人类生态系统的持续性,可用生产力稳定性、恢复力、公平性、自立性和协调性等系统特性和原理来衡量人类生态系统的待续性,据此,提出人地系统优化的途径是把人类需求控制在系统承载力之内,使自然资源的再生产社会化,以市场机制协调资源的供需矛盾,政府干预与公众参与。     

真空负压ABC法在各种组织相关抗原检测的应用研究

在免疫组化染色过程中,如何把各组织中的相关抗原充分地显示出来,许多免疫组化工作者进行了各种方法的探讨。但是,其结果却不十分理想,主要问题是染色的背景深及组织切片容易脱离载片,造成染色的失败。真空负压在免疫组化染色过程中的应用,能减少步骤,缩短时间,消除非特异性染色,使背景清晰,易于判断结果。并且避免了组织切片脱落现象,还可提高某些抗体的稀释度。此法均可使用金属性或非金属性器皿,而且整个过程都可在室温中进行,比微波技术更为简便易行,安全可靠,实用性强,成本低,有较大的推广价值。     

白鹇距长与年龄关系的人工神经网络模型

注意到白鹇雄鸟距长在一定时期内随年龄增长的事实,运用人工神经网络的一典型模型──“反向传播”模型的改进形式,依据确知年龄和估计年龄的部分样本,建立了白鹇龄估计的神经网络模型,用此模型可以以实测距长初步估计标本和野外采集到的白鹇个体的年龄。