人脆性组氨酸三联体（FHIT）基因的克隆及在HeLa细胞中表达的研究

目的：构建人脆性组氨酸三联体（fragile histidine triad gene,fhit）基因与报告基因egfp的双顺反子表达载体及融合表达载体,研究fhit基因过表达对HeLa细胞生长及凋亡的影响。方法：根据已知fhit基因的mRNA序列,RT-PCR得到493bp的cDNA序列,将其构建到pMD18-T中,筛选目的片段插入正确的重组质粒,经限制性内切酶切割,回收目的片段分别插入双顺反子表达载体pIRES2-EGFP和融合表达载体pEGFP-C1的多克隆位点中。再以脂质体介导转染HeLa细胞,G418筛选稳定转染的克隆,分别通过基因组PCR、RT-PCR、免疫细胞化学及TUNEL法在DNA、RNA和蛋白质水平检测其表达情况。结果：转基因细胞基因组PCR获得493bp片段,证明目的基因已整合到细胞基因组中;RT-PCR检测其在RNA水平得到表达;免疫细胞化学及TUNEL法分析表明fhit基因在HeLa细胞内实现了稳定遗传与表达。结论：实验证明fhit基因过表达可有效促进HeLa细胞的凋亡。     

A Spherical Model For Color Vision

An analysis of the general principles of the organization of analyzers as systems consisting of detectors shows that in such a system a stimulus may be presented as an n-dimension vector whose components are the respective excitations in selected detectors. Then the difference between two stimuli is defined by the angle between the two vectors representing them. Accordingly, the whole range of stimuli that neurons are able to discriminate may be plotted on an n-dimension sphere [4]. If we assume that for some stimuli, the difference discriminable by neurons will be directly correlated with the magnitude of the subjective difference between the stimuli, the assessment of the difference between two stimuli in a psychophysical experiment may be regarded as an arc on an n-dimension sphere of constant radius.     

New 3D graphical representation of DNA sequence based on dual nucleotides

We introduce a 3D graphical representation of DNA sequences based on the pairs of dual nucleotides (DNs). Based on this representation, we consider some mathematical invariants and construct two 16-component vectors associated with these invariants. The vectors are used to characterize and compare the complete coding sequence part of beta globin gene of nine different species. The examination of similarities/dissimilarities illustrates the utility of the approach.     

Characterization of neutralizing epitopes within the major capsid protein of human papillomavirus type 33

Background

Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains a predicted sequence/structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79–81 that are essential for the incorporation of in vitro generated ssRNAs into new reovirus progeny viral particles. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA.

Results

The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members.

Conclusion

This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.     

Mycoplasma gallisepticum 16S rRNA genes

Abstract The genome of Mycoplasma gallisepticum A5969 contains a truncated pseudogene for 16S rRNA in addition to a single unsplit rRNA-operon and a second discontinuous set of rRNA genes. Other M. gallisepticum strains tested do not posses the truncated gene. This gene is almost identical to full-size isolated 16S rRNA gene starting from at least 500 nucleotides upstream of the coding sequence and ending at the 977th nucleotide within the structural part of 16S rRNA.     

The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.     

Construction and characterization of a bovine BAC library with four genome-equivalent coverage

A bovine artificial chromosome (BAC) library of 105 984 clones has been constructed in the vector pBeloBAC11 and organized in 3-dimension pools and high density membranes for screening by PCR and hybridization. The average insert size, determined after analysis of 388 clones, was estimated at 120 kb corresponding to a four genome coverage. Given the fact that a male was used to construct the library, the probability of finding any given autosomal and X or Y locus is respectively 0.98 and 0.86. The library was screened for 164 microsatellite markers and an average of 3.9 superpools was positive for each PCR system. None of the 50 or so BAC clones analysed by FISH was chimeric. This BAC library increases the international genome coverage for cattle to around 28 genome equivalents and extends the coverage of the ruminant genomes available at the Inra resource center to 15 genome equivalents.     

濒危植物羊踯躅全基因组SSR标记开发与鉴定研究

该研究利用基于全基因组限制性酶切位点简化基因组测序技术（RAD seq技术）,开发濒危植物羊踯躅（Rhododendron molle G. Don）全基因组SSR标记,并对3个群体共63份羊踯躅材料进行验证鉴定,为进一步研究羊踯躅的遗传多样性和群体遗传结构以及保护利用提供技术支持。结果显示：（1）羊踯躅基因组测序获得原始数据7.653G bp,过滤后为7.513G bp;经组装发现,羊踯躅171.534 M bp的基因组分布在498 252 contigs中。（2）通过SSR检测,在11 961 SSR位点中获得了11 687对SSR分子标记,并且二核苷酸为基序的重复类型最丰富,达51.76%。（3）随机选取128对SSR标记在6个羊踯躅株系中进行PCR扩增,获得20对高多态性的SSR标记。（4）用所选的20对多态性SSR标记对3个群体共63份羊踯躅材料进行验证分析发现,这些多态性SSR标记位点的等位基因数为4~16个,期望杂合度（H_e）为0.489~0.908。 研究表明,羊踯躅的SSR丰度适中,且二核苷酸为羊踯躅中最丰富的重复序列,该实验进一步证明RAD seq技术是一种经济有效的基因测序方法,实验中开发的SSR引物将有助于进一步研究羊踯躅和其他近缘种的群体结构和多样性。     

家蚕浓核病毒DNV-3（中国株）的VD2基因组序列分析

浓核病毒BmDNV-3(中国株)基因组中含有两种不同的单链线形DNA分子(VD1,VD2)。该病毒的VD2被分离、纯化、克隆到pUC119载体上,并完成了VD2全基因组序列的测定。序列分析显示:VD2全基因组长为6022个核苷酸,末端拥有524个核苷酸反向重复序列(ITRs)。VD2基因组正链含有2个大的开放阅读框,负链含有1个小的开放阅读框。计算机分析推测该基因组正链上开放阅读框(ORF1)及负链上开放阅读框(ORF3)主要编码病毒的非结构蛋白,而正链上开放阅读框(ORF2)主要编码病毒的结构蛋白。比较BmDNV-3 VD2和BmDNV-2(Yamanashiisolate)VD2基因组全序列,两者同源性达97.7%,并且有132个碱基的替代1、1个碱基的删除和2个碱基插入。研究结果显示BmDNV-3 VD2和BmDNV-2 VD2有很近的亲缘关系,但也发生一定的变异,这为更好地理解浓核病毒种类的多样性,也为研究家蚕浓核病毒进化提供了有益的线索。     

Substitution of the gene for chloroplast RPS16 was assisted by generation of a dual targeting signal

Organelle (mitochondria and chloroplasts in plants) genomes lost a large number of genes after endosymbiosis occurred. Even after this major gene loss, organelle genomes still lose their own genes, even those that are essential, via gene transfer to the nucleus and gene substitution of either different organelle origin or de novo genes. Gene transfer and substitution events are important processes in the evolution of the eukaryotic cell. Gene loss is an ongoing process in the mitochondria and chloroplasts of higher plants. The gene for ribosomal protein S16 (rps16) is encoded in the chloroplast genome of most higher plants but not in Medicago truncatula and Populus alba. Here, we show that these 2 species have compensated for loss of the rps16 from the chloroplast genome by having a mitochondrial rps16 that can target the chloroplasts as well as mitochondria. Furthermore, in Arabidopsis thaliana, Lycopersicon esculentum, and Oryza sativa, whose chloroplast genomes encode the rps16, we show that the product of the mitochondrial rps16 has dual targeting ability. These results suggest that the dual targeting of RPS16 to the mitochondria and chloroplasts emerged before the divergence of monocots and dicots (140-150 MYA). The gene substitution of the chloroplast rps16 by the nuclear-encoded rps16 in higher plants is the first report about ongoing gene substitution by dual targeting and provides evidence for an intermediate stage in the formation of this heterogeneous organelle.     

The initiation of translation in E. coli: apparent base pairing between the 16srRNA and downstream sequences of the mRNA.

Bacteriophage T7's gene 0.3, coding for an antirestriction protein, possesses one of the strongest translation initiation regions (TIR) in E. coli. It was isolated on DNA fragments of differing length and cloned upstream of the mouse dihydrofolate reductase gene in an expression vector to control the translation of this gene's sequence. The TIR's efficiency was highly dependent on nucleotides +15 to +26 downstream of the gene's AUG. This sequence is complementary to nucleotides 1471-1482 of the 16srRNA. Similar sequences complementary to this rRNA region are present in other efficient TIRs of the E. coli genome and those of its bacteriophages. There seems to be a correlation between this sequence homology and the efficiency of the initiation signals. We propose that this region specifies a stimulatory interaction between the mRNA and 16srRNA besides the Shine-Dalgarno interaction during the translation initiation step.     

Analyses of copy number variation of GK rat reveal new putative type 2 diabetes susceptibility loci

Large efforts have been taken to search for genes responsible for type 2 diabetes (T2D), but have resulted in only about 20 in humans due to its complexity and heterogeneity. The GK rat, a spontanous T2D model, offers us a superior opportunity to search for more diabetic genes. Utilizing array comparative genome hybridization (aCGH) technology, we identifed 137 non-redundant copy number variation (CNV) regions from the GK rats when using normal Wistar rats as control. These CNV regions (CNVRs) covered approximately 36 Mb nucleotides, accounting for about 1% of the whole genome. By integrating information from gene annotations and disease knowledge, we investigated the CNVRs comprehensively for mining new T2D genes. As a result, we prioritized 16 putative protein-coding genes and two microRNA genes (rno-mir-30b and rno-mir-30d) as good candidates. The catalogue of CNVRs between GK and Wistar rats identified in this work served as a repository for mining genes that might play roles in the pathogenesis of T2D. Moreover, our efforts in utilizing bioinformatics methods to prioritize good candidate genes provided a more specific set of putative candidates. These findings would contribute to the research into the genetic basis of T2D, and thus shed light on its pathogenesis.     

Construction of bacteriophage luminal diameterX174 mutants with maximum genome sizes.

The bacteriophage phi X174 strain ins6 constructed previously was used to investigate the maximum genome size that could be packaged into the icosahedral phage without concomitant loss of phage viability. The J-F intercistronic region of ins6, which already contains an insert of 117 base pairs with a unique PvuII site, was enlarged further by insertion of HaeIII restriction fragments of the plasmid pBR322 into that PvuII site. By using a biochemical approach for the site-specific mutagenesis as well as selection of mutant genomes, a series of mutants was isolated with genomes of up to 5,730 nucleotides, 6.4% larger than that of the wild-type DNA. Phages with genomes larger than 5,550 nucleotides were highly unstable and were rapidly outgrown by spontaneously occurring deletion mutants. The data predict that genomes of at least 6,090 nucleotides could be constructed and, most likely, packaged, but the resulting phages would not grow well. We speculate that the volume of the phage capsid is not the limiting factor of genome size or is not the only limiting factor.     

A novel method of characterizing genetic sequences: genome space with biological distance and applications

Background

Most existing methods for phylogenetic analysis involve developing an evolutionary model and then using some type of computational algorithm to perform multiple sequence alignment. There are two problems with this approach: (1) different evolutionary models can lead to different results, and (2) the computation time required for multiple alignments makes it impossible to analyse the phylogeny of a whole genome. This motivates us to create a new approach to characterize genetic sequences.

Methodology

To each DNA sequence, we associate a natural vector based on the distributions of nucleotides. This produces a one-to-one correspondence between the DNA sequence and its natural vector. We define the distance between two DNA sequences to be the distance between their associated natural vectors. This creates a genome space with a biological distance which makes global comparison of genomes with same topology possible. We use our proposed method to analyze the genomes of the new influenza A (H1N1) virus, human rhinoviruses (HRV) and mammalian mitochondrial. The result shows that a triple-reassortant swine virus circulating in North America and the Eurasian swine virus belong to the lineage of the influenza A (H1N1) virus. For the HRV and mammalian mitochondrial genomes, the results coincide with biologists'' analyses.

Conclusions

Our approach provides a powerful new tool for analyzing and annotating genomes and their phylogenetic relationships. Whole or partial genomes can be handled more easily and more quickly than using multiple alignment methods. Once a genome space has been constructed, it can be stored in a database. There is no need to reconstruct the genome space for subsequent applications, whereas in multiple alignment methods, realignment is needed to add new sequences. Furthermore, one can make a global comparison of all genomes simultaneously, which no other existing method can achieve.     

Prediction of mitochondrial proteins using support vector machine and hidden Markov model

Mitochondria are considered as one of the core organelles of eukaryotic cells hence prediction of mitochondrial proteins is one of the major challenges in the field of genome annotation. This study describes a method, MitPred, developed for predicting mitochondrial proteins with high accuracy. The data set used in this study was obtained from Guda, C., Fahy, E. & Subramaniam, S. (2004) Bioinformatics 20, 1785-1794. First support vector machine-based modules/methods were developed using amino acid and dipeptide composition of proteins and achieved accuracy of 78.37 and 79.38%, respectively. The accuracy of prediction further improved to 83.74% when split amino acid composition (25 N-terminal, 25 C-terminal, and remaining residues) of proteins was used. Then BLAST search and support vector machine-based method were combined to get 88.22% accuracy. Finally we developed a hybrid approach that combined hidden Markov model profiles of domains (exclusively found in mitochondrial proteins) and the support vector machine-based method. We were able to predict mitochondrial protein with 100% specificity at a 56.36% sensitivity rate and with 80.50% specificity at 98.95% sensitivity. The method estimated 9.01, 6.35, 4.84, 3.95, and 4.25% of proteins as mitochondrial in Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, mouse, and human proteomes, respectively. MitPred was developed on the above hybrid approach.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

一种快速非比对的蛋白质序列相似性与进化分析方法

本文提出了一种新的快速非比对的蛋白质序列相似性与进化分析方法。在刻画蛋白质序列特征时,首先将氨基酸的10种理化性质通过主成分分析浓缩为6个主成分,并且将每条蛋白质序列里的氨基酸数目作为权重对主成分得分值进行加权平均,然后再融合氨基酸的位置信息构成一个26维的蛋白质序列特征向量,最后利用欧式距离度量蛋白质序列间的相似性及进化关系。通过对3个蛋白质序列数据集的测试表明,本文提出的方法能将每条蛋白质序列准确聚类,并且简便快捷,说明了该方法的有效性。