鱼类基因组结构研究——Ⅰ.染色体的限制性内切酶显带

本文报道了1种新的鱼类染色体研究方法——限制性内切酶显带技术。我们应用限制性内切酶Bsp631等分别对黄鳝和长春鳊的染色体进行显带处理,结果表明,Bsp631能使这两种鱼的染色体发生稳定的带纹分化:适度的处理产生多重的G-带状带型,而过度的处理则产生特殊的限制性内切酶抗性带型。根据显带结果分析,我们对鱼类染色体限制性内切酶显带的可行性和实用价值进行了讨论。     

东北梅花鹿染色体限制性内切酶显带

采用限制性内切酶Hae Ⅲ、Hind Ⅲ分别处理梅花鹿中期染色体标本,发现HaeⅢ处理标本21h左右,显出类似G带、C带带型,第1、4、11、18、27、28、31对染色体的结构异染色质区对HaeⅢ敏感而浅染,而HindⅢ处理标本18h左右能诱导梅花鹿中期染色体显出类似G带、C带,第2、6、15、18、29、32对染色体的结构异染色质区对HindⅢ较敏感而浅染,性染色体Y对HaeⅢ敏感呈浅染,性染色体X对HindⅢ敏感呈浅染。表明东北梅花鹿12对常染色体和两条性染色体结构异染色质在这两种酶处理时表现出异质性。     

三倍体草鱼染色体限制性内切酶带分析

本文报道运用Ｇｉｅｍｓａ染色、Ｃ－带显带、ＮＯＲ－显带和多种限制性内切酶消化对草鱼三倍体的染色体进行分析。其中,内切酶ＨａｅⅢ和ＨｉｎｄⅢ等使染色体产生Ｇ－带,其余内切酶产生与常规Ｃ－带不同的分化,即所谓修饰Ｃ－带。实验结果表明,限制性内切酶可使鱼类染色体产生多种带型,各种显带技术的结合使用,可探讨鱼类染色体及染色质结构与进化。     

用限制酶显带和CMA_3/DA/DAPI荧光染色研究中国小型白兔染色体结构异染色质

限制性内切酶(restriction endonuclease)是一类能识别并切开特定DNA序列的核酸内切酶,利用限制酶处理甲醇、冰醋酸固定后的哺乳动物中期染色体标本,再经Giemsa染色,能产生不同的特征性带纹,这是近几年发展的一种新的显带技术。目前,除用于人的中期染色体研究外,     

斑马鱼二价体制备与多重带显带的方法学探讨

采用碱性低渗结合高氯仿一步显带技术获得了斑马鱼二价体高分辨多重G带,所出现的带纹丰富,反差明显。采用地高辛为标记的以限制性内切酶AfuⅠ介导的原位切口平移技术使斑马鱼二价体上呈现出类G带的多重带,获得了明暗反差强烈的带纹结果。通过比较多个不同分裂相的多条染色体,发现带纹的分布具明显特征性．并且特征一致,带纹数目基本吻合。首次从方法学上对斑马鱼二价体的制备过程及多重带显带技术进行了分析、探讨和总结,力求将该技术程序化、系统化,使其具有可重复性和可操作性,并对显带的可能机理进行了讨论。     

毛冠鹿种内异染色质变化与染色体多态

采用原代和传代培养方法对8头毛冠鹿(Elaphodus cephalophus)的皮肤细胞进行了染色体研究,发现了一种核型与以前所报道的几种核型不一致,确定为一新核型。在该核型中,染色体众数2n=47,2条X染色体异型,一条为端着丝粒,另一条为近端着丝粒。C-带显示该核型中异染色质除了分布在2条X染色体长臂中之外,在第一对大的端着丝粒染色体中的一条近着丝粒区出现一异染色质“柄”。结合C-带及薄层扫描结果对毛冠鹿种内常染色体、性染色体中异染色质的含量和分布与染色体多态的关系进行了探讨。     

Comparative Analysis (Hippotragini versus Caprini, Bovidae) of X-Chromosome's Constitutive Heterochromatin by in situ Restriction Endonuclease Digestion: X-Chromosome Constitutive Heterochromatin Evolution

The Bovidae X-chromosome shows a considerable variation, in contrast to the preservative autosomal conservatism. The X-chromosome variation is mostly a consequence of the constitutive heterochromatin (CH) variation; in what respect to its amount and position. This is especially common among the non-Bovinae subfamilies and tribes. In order to characterize the X-chromosome CH in non-Bovinae species--Hippotragini and Caprini tribes--we have used restriction endonuclease digestion on fixed chromosomes and sequential C-banding. With these techniques we were able to distinguish between the two X-chromosome types (Hippotragini and Caprini) CH, in what respect to its position and molecular nature. Moreover, we define at least, six subclasses of CH in both X-chromosome types analyzed. Evolutionary considerations were draw based on the results obtained. The technology used here for the analysis of the Bovidae X-chromosome CH showed to be more evolutionary informative than the classical approaches.     

Polymorphism of Mitochondrial DNAs of Yunnan Domestic Water Buffaloes, Bubalus bubalis, in China, Based on Restriction Endonuclease Cleavage Patterns

Restriction endonuclease cleavage patterns of mitochondrial DNA(mtDNA) of three local types of Yunnan native water buffalo were analyzed using 18 enzymes which recognize six nucleotides. Among the 12 animals analyzed, 3 of 18 enzymes, BamHI, EcoRI, and ScaI, revealed polymorphisms. Three mtDNA types were identified. The results indicate that a relatively low level of mtDNA variation exists in Yunnan domestic water buffaloes. The origin of Chinese buffalo derived from Yunnan province of China is discussed.     

Genome Analysis of Bacteroides by Pulsed-field Gel Electrophoresis: Chromosome Sizes and Restriction Patterns

The chromosomal DNAs of nine strains of seven Bacteroides speciesincluding B. fragilis, the type species of the genus Bacteroides,were digested with rare-cutting restriction enzymes I-Ceu I,Not I, and Asc I and analysed by pulsed-field gel electrophoresis.The genome sizes of B. fragilis, B. distasonis, B. eggerthii,B. ovatus, B. thetaiotaomicron, B. uniformis, and B. vulgatuswere determined to be 5.3, 4.8, 4.4, 6.9, 4.8, 4.6, and 5.1Mbp, respectively. B. distasonis and B. vulgatus, and also B.uniformis and B. eggerthii, showed simillar I-Ceu I restrictionprofiles. I-Ceu I cut B. uniformis and B. eggerthii genomesinto four, B. ovatus into five, B. fragilis and B. thetaiotaomicroninto six, and B. distasonis and B. vulgatus into seven fragments.On the basis of genome size, restriction profile, and I-CeuI fragment number, a phylogenetic tree of the Bacteroides specieswas proposed. This was in overall agreement with the previousphylogenetic tree obtained by 16S rRNAdata, with the exceptionsof B. distasonis and B. ovatus.     

Differential digestion of the centromeric heterochromatic regions of the 5-azacytidine-decondensed human chromosomes 1, 9, 15, and 16 by NdeII and Sau3AI restriction endonucleases

A study on the factors involved in chromosome digestion by restriction endonuclease was carried out on 5-azacytidine treated and untreated human chromosomes 1, 9, 15 and 16 by using NdeII and Sau3AI isoschizomers. After treatment with 5-azacytidine, chromosomes 1, 9, 15, and 16 showed two differentiated areas at the centromeric regions: the centromere, fully condensed, and the pericentromeric heterochromatin, decondensed. Chromosomes not treated with 5-azacytidine after digestion with Sau3AI and NdeII showed all the centromeric regions undigested, except pair number 1, digested at the pericentromeric area. Digestion of the 5-azacytidine decondensed chromosomes with Sau3AI and NdeII showed the centromeres undigested in the four chromosome pairs while the pericentromeric heterochromatin appeared largely digested. Other factors, different to target distribution, are necessary to explain the pattern of restriction endonuclease digestion observed in this communication.     

Evolutionary chromosomal differentiation among four species of Conoderus Eschscholtz, 1829 (Coleoptera, Elateridae, Agrypninae, Conoderini) detected by standard staining, C-banding, silver nitrate impregnation, and CMA3/DA/DAPI staining

The speciose Brazilian Elateridae fauna is characterized by high karyotypic diversity, including one species (Chalcolepidius zonatus Eschscholtz, 1829) with the lowest diploid number within any Coleoptera order. Cytogenetic analysis of Conoderus dimidiatus Germar, 1839, C. scalaris (Germar, 1824,) C. ternarius Germar, 1839, and C. stigmosus Germar, 1839 by standard and differential staining was performed with the aim of establishing mechanisms of karyotypic differentiation in these species. Conoderus dimidiatus, C. scalaris, and C. ternarius have diploid numbers of 2n(♂) = 17 and 2n(♀) = 18, and a X0/XX sex determination system, similar to that encountered in the majority of Conoderini species. The karyotype of C. stigmosus was characterized by a diploid number of 2n=16 and a neoXY/neoXX sex determination system that was highly differentiated from other species of the genus. Some features of the mitotic and meiotic chromosomes suggest an autosome/ancestral X chromosome fusion as the cause of the neoXY system origin in C. stigmosus. C-banding and silver impregnation techniques showed that the four Conoderus species possess similar chromosomal characteristics to those registered in most Polyphaga species, including pericentromeric C band and autosomal NORs. Triple staining techniques including CMA₃/DA/DAPI also provided useful information for differentiating these Conoderus species. These techniques revealed unique GC-rich heterochromatin associated with NORs in C. scalaris and C. stigmosus and CMA₃-heteromorphism in C. scalaris and C. ternarius.     

Augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/X1 and 7a/X4 proteins through NF-kappaB activation

Severe acute respiratory syndrome (SARS) is characterized by rapidly progressing respiratory failure resembling acute/adult respiratory distress syndrome (ARDS) associated with uncontrolled inflammatory responses. Here, we demonstrated that, among five accessory proteins of SARS coronavirus (SARS-CoV) tested, 3a/X1 and 7a/X4 were capable of activating nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK), and significantly enhanced interleukin 8 (IL-8) promoter activity. Furthermore, 3a/X1 and 7a/X4 expression in A549 cells enhanced production of inflammatory chemokines that were known to be up-regulated in SARS-CoV infection. Our results suggest potential involvement of 3a/X1 and 7a/X4 proteins in the pathological inflammatory responses in SARS.