躯干连续性不同断面标本的制作方法

断面解剖学是通过制作不同方向各种断面的方法，研究正常人体形态、结构、位置及与其他器官组织相互关系的一门科学。它是医学影象诊断学的基础学科，由于它能在保护机体结构于原位情况下，准确的展示和表述诸结构的断面形态、位置及其毗邻、还可以利用串联的断面进行连续观察，为医学生理解和掌握各器官的形态结构及与其     

科学家用频谱法寻找基因变异获得成功

德国亚琛的一个科研小组目前成功利用频谱法寻找出人类单个基因片段中发生的变异 .据称 ,与现有使用的其它寻找基因变异的方法比较 ,新方法更加迅速准确 ,并有望在生物技术及医疗研究领域内获得广泛应用.在借助基因芯片对ＤＮＡ片段进行测定的过程中 ,使用频率为若干太拉 (ＴＥＲＡ ,10的 12次方 )赫兹的电磁波以特定方法对ＤＮＡ片段进行照射 .电磁波会引起ＤＮＡ片段中变异部分某种特殊形式的振荡 ,科学家再借助频谱分析仪进行分析 ,就可以准确定位变异发生的位置 .频谱分析法目前在天文学研究中应用较为广泛 ,但在生物技术领域中则尚属于…     

EST在克隆新基因中的应用

随着基因定位 (连锁图谱、物理图谱、转录图谱 )和DNA测序及生物信息技术的迅猛发展 ,EST已成为人类寻找新的未知基因以及克隆不同时空差异表达基因和疾病相关基因的重要标志物。随着EST数据库的进一步完善 ,网上克隆和定位候选克隆策略将成为克隆新基因的主要方法 ,并在虚拟的网上空间将模型生物基因组的研究成果成功地应用于人类基因组的研究中     

NOGO与中枢神经再生

NOGO基因编码一个抑制CNS轴突再生的蛋白 ,该蛋白有三种异构体 ,分别为NOGOA ,B ,C。由于掌握了这种完整蛋白及其基因 ,预计将准确地鉴定NOGO抑制轴突生长的部位、分子机制等。这为寻找更好的方法来阻止这种蛋白的作用创造了有利条件。     

多焦视网膜电流图中诱发成分的生理含义的解释和分离方法研究

诱发成分是指出现在mfERG一阶响应中较后的位置上的一个波形结构。本研究的第一个目的是要对诱发成分的生理含义作出准确描述，另一个目的是提出一个从一阶响应中完整，准确的分离诱发成分的方法。首先，通过分析诱发成分的各个组成信号的视网膜生理含义，指出诱发成分是一阶响应的一个固有成分，表现了视网膜的光适应机制，在波形和生理含义上与mfERG高阶响应相似但存在明显差别。其次，利用在不同刺激频率条件下得到的一阶响应之间的差异特性，设计实验从一阶响应中分离出诱发成分。实验结果还证实了本研究中关于诱发成分和高阶响应在波形上存在差异的推论。     

mRNA差别显示技术的研究进展

mRNA差别显示是继mRNA差减杂交技术之后又一种显示mRNA差异表达的新方法。它通过PCR随机引物与锚定引物的合理搭配,在特定PCR条件下,展示不同条件下真核细胞mRNA的差异扩增片断,为寻找未知基因提供了新途径。     

373A型DNA测序仪的应用体会

DNA序列测定是分子生物学领域最重要的技术之一，是了解基因结构和功能的基础。DNA测序技术的不断完善和仪器自动化程度的不断提高，为大型基因组测序奠定了基础，当今大多数蛋白质序列都是通过基因或。DNA的核着酸序列推导出来的。DNAN序方法主要有双脱氧链终止法（Seqer法）和化学降解法（Maxam－Gillsert法）。目前不管是传统的手工创序法，还是仪器自动测序法均使用前者，而仪器自动测序法以其快速、准确等优点得以广泛应用。我室于1995年，引进美国PE公司的373A型DNA序列测定仪，仪器专管共用，两年来为校内、外科研人员提供…     

LncRNAs参与免疫调节及自身免疫性疾病的研究进展

在基因组非编码基因特异性的RNA分子表达产物中,长链非编码RNA(lncRNAs)在许多生物学过程中发挥了重要的调节作用,越来越多的研究发现,lncRNAs还参与了一些疾病的发生发展过程.目前较多的研究热点集中于lncRNAs和肿瘤的关系上,但是近年来人们发现lncRNAs还可能在免疫调节的不同时期发挥调控作用:在固有免疫应答中调节了免疫细胞的发生发展;在适应性免疫应答中调节效应免疫细胞的应答敏感性.这说明lncRNAs可能参与一些自身免疫性疾病的发病过程,了解近年来lncRNAs与免疫调节及自身免疫性疾病相关的研究成果很有意义.     

用外切酶Ⅲ降解和修复合成测定DNA序列的快速方法

由于快速简便DNA测序方法的发展,使最近对基因结构和功能的了解有了许多重大进展。Sanger等的双脱氧末端终止法便是一种既简便快速又准确的方法。但此法需要单链DNA模板及特异性引物。已经有一些报道简化了模板和引物的提取步骤。例如,把待测序的DNA片段克隆进双链复制型嗜菌体M13衍生物中,得到单链DNA模板,再与     

Toll样受体在适应性免疫中的作用与相关机制研究进展

TLRs是一类模式识别受体，能够被微生物的特异性成分和某些宿主分子所激活，在固有免疫系统中起着决定性的作用。同时大部分病原体通过TLPs激活抗原提呈细胞(主要是树突状细胞)上调其MHC分子表达，启动CD4^ T细胞向Th1型细胞分化。而当机体遭遇多细胞寄生虫感染或接触变应原时，CD4^ T细胞则向1112型细胞分化，这一类免疫反应可能是通过一系列不同于TLRs的固有识别系统进行调控，但究竟是通过目前未知的PRRs还是通过缺失自身标记的机制对病原体进行识别目前还不明了。进一步了解TLRs调节适应性免疫的过程不但能够发展治疗感染性疾病的新途径，而且可能为变态反应和某些自身免疫疾病发病机理深入研究以及治疗和防治提供新方法。     

Cloning and genetic analysis of subtilases in sapstaining fungi

In order to assess the genetic variance of a group of homologous subtilases in sapstaining fungi, molecular techniques were employed. First, PCR screening with degenerate primers and dot-blot analyses were used to screen 31 different isolates, representing nine species, of sapstaining fungi for the presence of subtilase-like sequences. Restriction fragment length polymorphism PCR and sequence analysis techniques were then used to determine the inter- and intraspecies variation of these genes. A labelled chemiluminescent probe was then used to screen an Ophiostoma floccosum 387N genomic library for subtilase genes. Over ten positive clones were found and one was subcloned and sequenced. Randomly amplified cDNA ends PCR techniques were then employed to obtain full-length subitilase gene sequence information from isolates of four different species. The obtained sequences were found to be homologous with other fungal subtilases and common structural features of the inferred proteins with proteinase K were apparent. Southern blot analysis was used to verify and determine the copy number of the subtilase genes in these fungi. In the work presented here, it was found that all of the isolates tested seemed to contain some sort of subtilase gene sequence and that there was inter- and intraspecies variation in the number and type of subtilase genes present. The data indicated that three distinct groups of subtilase genes are present in sapstaining fungi. However, individual isolates were found to contain only one or two of these gene types.     

Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species

Acinetobacter species are defined on the basis of several phenotypic characters, results of DNA-DNA homology, and more recently, similarities or dissimilarities in 16S rRNA gene sequences. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species. We used an RNA polymerase beta-subunit gene (rpoB)-based identification scheme for the delineation of species within the genus Acinetobacter, and towards that end, we determined the complete rpoB gene and flanking spacer (rplL-rpoB and rpoB-rpoC) sequences of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. By using complete gene sequences (4,089 bp), we clearly separated all species and grouped them into different clusters. A phylogenetic tree constructed using these sequences was supported by bootstrap values higher than those obtained with 16S rRNA or the gyrB or recA gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the rpoB gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. Each of these four variable sequences enabled us to delineate most species. Sequences of at least two polymorphic sequences should be used to distinguish Acinetobacter grimontii, Acinetobacter junii, Acinetobacter baylyi, and genomic species 9 from one another. Finally, 21 clinical isolates of Acinetobacter baumannii were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the rpoB gene and its flanking spacers.     

Analysis of the beak and feather disease viral genome indicates the existence of several genotypes which have a complex psittacine host specificity

Summary. A study was made of the phylogenetic relationships between fifteen complete nucleotide sequences as well as 43 nucleotide sequences of the putative coat protein gene of different strains belonging to the virus species Beak and feather disease virus obtained from 39 individuals of 16 psittacine species. The species included among others, cockatoos (Cacatuini), African grey parrots (Psittacus erithacus) and peach-faced lovebirds (Agapornis roseicollis), which were infected at different geographical locations, within and outside Australia, the native origin of the virus. The derived amino acid sequences of the putative coat protein were highly diverse, with differences between some strains amounting to 50 of the 250 amino acids. Phylogenetic analysis demonstrated that the putative coat gene sequences form six clusters which show a varying degree of psittacine species specificity. Most, but not all strains infecting African grey parrots formed a single cluster as did the strains infecting the cockatoos. Strains infecting the lovebirds clustered with those infecting such Australasian species as Eclectus roratus, Psittacula kramerii and Psephotus haematogaster. Although individual birds included in this study were, where studied, often infected by closely related strains, infection by highly diverged trains was also detected. The possible relationship between BFD viral strains and clinical disease signs is discussed.     

Use of genes encoding cellobiohydrolase-C and topoisomerase II as targets for phylogenetic analysis and identification of Fusarium

Molecular identification and phylogenetic studies rely to a large extent on rDNA sequence polymorphism. In the field of fungal taxonomy, despite the use of huge amounts of rDNA data available, some species within a given genus remain indistinguishable. Therefore, new target sequences need to be selected and validated. This is the case for Fusarium, which includes numerous species most of which are involved in both animal and plant pathologies. In addition to the rDNA fragment encompassing the internal transcribed spacers ITS1 and ITS2 and the 5.8 S sequence, two newly characterized genes were used as molecular markers for Fusarium species genotyping. The cellobiohydrolase-C (cbh-C) and the topoisomerase II (topII) gene parts were cloned and sequenced for at least one isolate of each of the eleven different species of our collection. Both cbh-C and topII were found to be single copy genes. DNA fragments amplified by PCR in order to establish phylogenetic trees range from 1123 to 1157 bp for rDNA and from 327 to 344 bp for cbh-C (this part contains one intron). The topII gene part encoding the carboxy-terminus of the ATP binding domain of the enzyme is constant in length with a value of 724 bp. PAUP-generated phylogenetic analyses based either on cbh-C or topII data enabled all species to be distinguished, and were more informative than those resulting from rDNA sequences. Furthermore, a combination of the three datasets enhanced the accuracy of the analyses and open up new possibilities for rapid molecular identification and evolution studies within the Fusarium genus.     

Application of RNA polymerase beta-subunit gene (rpoB) sequences for the molecular differentiation of Legionella species

The nucleotide sequences of the partial rpoB gene were determined from 38 Legionella species, including 15 serogroups of Legionella pneumophila. These sequences were then used to infer the phylogenetic relationships among the Legionella species in order to establish a molecular differentiation method appropriate for them. The sequences (300 bp) and the phylogenetic tree of rpoB were compared to those from analyses using 16S rRNA gene and mip sequences. The trees inferred from these three gene sequences revealed significant differences. This sequence incongruence between the rpoB tree and the other trees might have originated from the high frequency of synonymous base substitutions and/or from horizontal gene transfer among the Legionella species. The nucleotide variation of rpoB enabled more evident differentiation among the Legionella species than was achievable by the 16S rRNA gene and even by mip in some cases. Two subspecies of L. pneumophila (L. pneumophila subsp. pneumophila and subsp. fraseri) were clearly distinguished by rpoB but not by 16S rRNA gene and mip analysis. One hundred and five strains isolated from patient tissues and environments in Korea and Japan could be identified by comparison of rpoB sequence similarity and phylogenetic trees. These results suggest that the partial sequences of rpoB determined in this study might be applicable to the molecular differentiation of Legionella species.     

The structure and evolution of immunoglobulin kappa chain constant region genes in the genus Rattus

We have cloned and sequenced the C-kappa (Ck) genes from seven species and subspecies of rats which have diverged over the past few million years in Australia. Comparisons of these sequences with each other and the Ck genes of the laboratory rat, Rattus norvegicus, indicate noncoding regions have accumulated fewer mutations than adjacent coding sequences, and amino acid replacing nucleotide substitutions in the coding regions have accumulated at a rate at least as great as silent changes. Exactly the opposite of both of these findings is observed when comparisons are made between Ck or other genes from more distantly related species, indicating that these features may be characteristic of Ck short-term evolutionary gene divergence. Changes in the coding regions of these genes result in a non-random distribution of amino acid substitutions on the three-dimensional alpha-carbon backbone of the Ck domain in the most serologically distinct forms of Ck. While phylogenetic relationships inferred from the Ck nucleotide sequences are in general agreement with those derived from other data, considerable differences are seen in rates of accumulation of Ck gene nucleotide substitutions vs rates of accumulation of enzyme polymorphisms.     

Human repetitive and unique sequences coexist in a large circulating DNA species found in cryoprecipitates from SLE patients.

Cryoprecipitates from systemic lupus erythematosus (SLE) patients with high levels of anti DNA antibodies show a sharply migrating large circulating DNA species of about 17-20 kb (M. Rieber et al., Clin. exp. Immunol. (1986) 66, 61). We have now used Southern blot analysis of circulating DNA from different individuals to analyse the relative cross-hybridization of circulating DNA from different individuals, as well as their homology with genomic DNA from different species. Molecular hybridization showed significant homology of the various circulating DNA examined, only with human genomic DNA, but limited cross-reactivity among circulating DNA from different individuals. This suggests that the circulating DNA is composed of sequences repeated in human genomic DNA and by specific sequences unique to circulating DNA from some individuals. Our data suggests the possibility of using probes derived from the specific sequences now reported in the circulating DNA, in gene typing and in the analysis of susceptibility to disease.     

Comparative genome analysis across a kingdom of eukaryotic organisms: specialization and diversification in the fungi

The recent proliferation of genome sequencing in diverse fungal species has provided the first opportunity for comparative genome analysis across a eukaryotic kingdom. Here, we report a comparative study of 34 complete fungal genome sequences, representing a broad diversity of Ascomycete, Basidiomycete, and Zygomycete species. We have clustered all predicted protein-encoding gene sequences from these species to provide a means of investigating gene innovations, gene family expansions, protein family diversification, and the conservation of essential gene functions-empirically determined in Saccharomyces cerevisiae-among the fungi. The results are presented with reference to a phylogeny of the 34 fungal species, based on 29 universally conserved protein-encoding gene sequences. We contrast this phylogeny with one based on gene presence and absence and show that, while the two phylogenies are largely in agreement, there are differences in the positioning of some species. We have investigated levels of gene duplication and demonstrate that this varies greatly between fungal species, although there are instances of coduplication in distantly related fungi. We have also investigated the extent of orthology for protein families and demonstrate unexpectedly high levels of diversity among genes involved in lipid metabolism. These analyses have been collated in the e-Fungi data warehouse, providing an online resource for comparative genomic analysis of the fungi.