感染性猪圆环病毒2型基因组DNA的分子克隆

本研究通过PCR扩增出猪圆环病毒2型(PCV—2)的全基因组(1768bp),克隆入pcDNA3载体的EcoR I酶切应点,获得含有PCV-2全基因组的重组质粒,命名为pcDNApcv2。将重组质粒大量扩增后,用EcoR I切出1768bp的PCV—2全基因组,在体外用T4DNA连接酶使其连接环化。用脂质体法将体外连接产物转染无PCV污染的PK—15细胞,经4次连续传代,用间接免疫荧光实验(IFA)及电镜观察证实已获得复制能力的PCV—2病毒。由此可见,本试验构建的环化的PCV—2全基因组DNA具有感染性。     

Annotating the genome of Medicago truncatula

Medicago truncatula will be among the first plant species to benefit from the completion of a whole-genome sequencing project. For each of these species, Arabidopsis, rice and now poplar and Medicago, annotation, the process of identifying gene structures and defining their functions, is essential for the research community to benefit from the sequence data generated. Annotation of the Arabidopsis genome involved gene-by-gene curation of the entire genome, but the larger genomes of rice, Medicago and other species necessitate the automation of the annotation process. Profiting from the experience gained from previous whole-genome efforts, a uniform set of Medicago gene annotations has been generated by coordinated international effort and, along with other views of the genome data, has been provided to the research community at several websites.     

Rats in the genomic era.

The rat genome project and the resources that it has generated are transforming the translation of rat biology to human medicine. The rat genome was sequenced to a high quality "draft," the structure and location of the genes were predicted, and a global assessment was published (Gibbs RA et al., Nature 428: 493-521, 2004). Since that time, researchers have made use of the genome sequence and annotations and related resources. We take this opportunity to review the currently available rat genome resources and to discuss the progress and future plans for the rat genome.     

Characterization of the genome of the murine papovavirus K.

The DNA genome of the murine papovavirus K virus (KV) was characterized and compared with the genome of polyoma virus. A physical map of the KV genome was constructed by analysis of the size of DNA fragments generated by sequential cleavage with combinations of restriction endonucleases. By using one of the three EcoRI sites in the KV genome as the 0 map position, the KV physical map was then oriented to the polyoma virus genome. Of 42 restriction sites mapped within the KV genome, 7 were localized within 0.01 map unit of their respective sites in the polyoma virus genome; an eighth site mapped within 0.02 map unit. KV replication was examined and found to be bidirectional, initiating at approximately 0.70 map unit. This corresponds well to the origin of replication within the polyoma virus genome and further supports the orientation of the KV physical map.     

A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

A resource of genome‐wide single‐nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel

Reduced representation genome sequencing such as restriction‐site‐associated DNA (RAD) sequencing is finding increased use to identify and genotype large numbers of single‐nucleotide polymorphisms (SNPs) in model and nonmodel species. We generated a unique resource of novel SNP markers for the European eel using the RAD sequencing approach that was simultaneously identified and scored in a genome‐wide scan of 30 individuals. Whereas genomic resources are increasingly becoming available for this species, including the recent release of a draft genome, no genome‐wide set of SNP markers was available until now. The generated SNPs were widely distributed across the eel genome, aligning to 4779 different contigs and 19 703 different scaffolds. Significant variation was identified, with an average nucleotide diversity of 0.00529 across individuals. Results varied widely across the genome, ranging from 0.00048 to 0.00737 per locus. Based on the average nucleotide diversity across all loci, long‐term effective population size was estimated to range between 132 000 and 1 320 000, which is much higher than previous estimates based on microsatellite loci. The generated SNP resource consisting of 82 425 loci and 376 918 associated SNPs provides a valuable tool for future population genetics and genomics studies and allows for targeting specific genes and particularly interesting regions of the eel genome.     

Chromosome map of the phototrophic anoxygenic bacterium Chromatium vinosum

Abstract The genome of Chromatium vinosum has been characterized using pulsed field gel electrophoresis. Two restriction endonucleases, Ase I and Spe I, generated DNA fragments of size distributions suitable for mapping the genome of the anoxyphotobacterium C. vinosum DSM 180. Ase I produced 24 fragments ranging from 367 to 10.8 kb and Spe I yielded 13 fragments from 720 to 12 kb. A total genome size of 3.674 Mb was determined by summing the fragment lengths in each of the digests generated using the different restriction endonucleases. Intact total DNA from C. vinosum shows the presence of three extrachromosomal elements. Three rRNA regions were located in the strain DSM 180. Restriction patterns of the strain DSM 180 have been compared with ATCC 17899 and DSM 185 of the same species.     

Elucidation of the genome organization of tobacco mosaic virus

Proteins unique to tobacco mosaic virus (TMV)-infected plants were detected in the 1970s by electrophoretic analyses of extracts of virus-infected tissues, comparing their proteins to those generated in extracts of uninfected tissues. The genome organization of TMV was deduced principally from studies involving in vitro translation of proteins from the genomic and subgenomic messenger RNAs. The ultimate analysis of the TMV genome came in 1982 when P. Goelet and colleagues sequenced the entire genome. Studies leading to the elucidation of the TMV genome organization are described below.     

Comparative BAC‐based physical mapping of Oryza sativa ssp. indica var. 93–11 and evaluation of the two rice reference sequence assemblies

Reference sequences are sequences that are used for public consultation, and therefore must be of high quality. Using the whole‐genome shotgun/next‐generation sequencing approach, many genome sequences of complex higher plants have been generated in recent years, and are generally considered reference sequences. However, none of these sequences has been experimentally evaluated at the whole‐genome sequence assembly level. Rice has a relatively simple plant genome, and the genome sequences for its two sub‐species obtained using different sequencing approaches were published approximately 10 years ago. This provides a unique system for a case study to evaluate the qualities and utilities of published plant genome sequences. We constructed a robust BAC physical map embedding a large number of BAC end sequences forrice variety 93–11. Through BAC end sequence alignments and tri‐assembly comparisons of the 93–11 physical map and the two reference sequences, we found that the Nipponbare reference sequence generated using the clone‐by‐clone approach has a high quality but still contains small artifact inversions and missing sequences. In contrast, the 93–11 reference sequence generated using the whole‐genome shotgun approach contains many large and varied assembly errors, such as inversions, duplications and translocations, as well as missing sequences. The 93–11 physical map provides an invaluable resource for evaluation and improvements toward completion of both Nipponbare and 93–11 reference sequences.     

昆虫基因组及数据库研究进展

基因组序列为昆虫分子生物学研究提供丰富的数据资源,推动系统生物学在古老的昆虫学中蓬勃发展。昆虫基因组学研究已经成为当前的研究热点,目前在NCBI登录注册的昆虫基因组测序计划有494项,其中已提交原始测序数据的昆虫有225种,完成基因组拼接的有215种,具有基因注释的有65种,公开发表的昆虫基因组有43篇。本文综述了测序技术发展的历史及其对昆虫基因组研究的推动作用、昆虫基因组的组装和注释及其存在的问题、昆虫基因组测序进展、昆虫基因组数据库的发展及基因数据挖掘利用的基本思路和对策,以及昆虫基因大数据在害虫防治和资源昆虫利用中的应用前景。     

ACT: the Artemis Comparison Tool

The Artemis Comparison Tool (ACT) allows an interactive visualisation of comparisons between complete genome sequences and associated annotations. The comparison data can be generated with several different programs; BLASTN, TBLASTX or Mummer comparisons between genomic DNA sequences, or orthologue tables generated by reciprocal FASTA comparison between protein sets. It is possible to identify regions of similarity, insertions and rearrangements at any level from the whole genome to base-pair differences. ACT uses Artemis components to display the sequences and so inherits powerful searching and analysis tools. ACT is part of the Artemis distribution and is similarly open source, written in Java and can run on any Java enabled platform, including UNIX, Macintosh and Windows.     

Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose

We have established an improved large deletion method in Escherichia coli genome using a combination of two different recombination systems, lambda Red and Cre/loxP. The loxP site could be rapidly and efficiently integrated in the genome by lambda Red and large deletions of both 117- and 165-kbp regions could be generated in 100% efficiency by Cre/loxP. Comparative genomic hybridization microarray experiments of deletion strains indicated that deletions were generated only in expected regions of the genome. These results have demonstrated that the method is useful for genome engineering in E. coli.     

Genome Announcement: The Draft Genome of the Carrot Cyst Nematode Heterodera Carotae

Heterodera carotae, the carrot cyst nematode, is a significant pest affecting carrot globally. Here we present the draft genome of H. carotae, which was generated from short read libraries from Illumina HiSeq technology, and the corresponding genome annotation.     

A sequence of 'factishes': the media-metaphorical knowledge dynamics structuring the German press coverage of the human genome

This article deals with the cultural framing of the near sequencing of the human genome and its impact on the media coverage in Germany. It investigates in particular the way in which the weekly journal Die Zeit and the daily newspaper Frankfurter Rundschau reported this media event and its aftermath between June 2000 and June 2001. Both newspapers are quality papers that played an essential role in framing the human genome debate--alongside the Frankfurter Allgemeine Zeitung--which became the most prominent genomic forum. The decoding of the human genome prompted a huge controversy concerning the ethics of human engineering, research on stem cells and Preimplantation Genetic Diagnosis. The main aim of this article is to show how this controversy was structured by metaphor. The media coverage of the genome generated DNA-factishes--a neologism designating the ambivalence of something as fact (fait) and as a fetish (fetiche)--that mostly propagated images of a new DNA-scienticism or biological determinism. Mediated by cultural experiences, the human genome became a highly artificial and social construct of a 'NatureCulture'.     

Physical mapping of the Mycoplasma gallisepticum S6 genome with localization of selected genes.

We report the construction of a physical map of the Mycoplasma gallisepticum S6 genome by field-inversion gel electrophoresis of DNA fragments generated by digestion of genomic DNA with rare-cutting restriction endonucleases. The size of the M. gallisepticum S6 genome was calculated to be approximately 1,054 kb. The loci of several genes have been assigned to the map by Southern hybridization utilizing specific gene probes.     

Remi-RFLP Mapping in the Dictyostelium Genome

A set of 147 Dictyostelium discoideum strains was constructed by random integration of a vector containing rare restriction sites. The strains were generated by transformation using restriction enzymemediated integration (REMI) which results in the integration of linear DNA fragments into randomly distributed genomic restriction sites. Restriction fragment length polymorphism (RFLP) was generated in a single genomic site in each strain. These REMI-RFLP strains were used to confirm gene linkages previously supported by two other physical mapping techniques: yeast artificial chromosome (YAC) contig construction, and megabase-scale restriction mapping. New linkages were uncovered when two or more hybridization probes identified the same RFLP fragments. Probes for 100 genes have marked 53% of the RFLPs, representing greater than 22 Mb of the 40 Mb Dictyostelium genome. Alignment of these and other large fragments along each chromosome should lead to a complete physical map of the Dictyostelium genome.     

Mitochondrial DNA insertions in the nuclear horse genome

The insertion of mitochondrial DNA in the nuclear genome generates numts, nuclear sequences of mitochondrial origin. In the horse reference genome, we identified 82 numts and showed that the entire horse mitochondrial DNA is represented as numts without gross bias. Numts were inserted in the horse nuclear genome at random sites and were probably generated during the repair of DNA double-strand breaks. We then analysed 12 numt loci in 20 unrelated horses and found that null alleles, lacking the mitochondrial DNA insertion, were present at six of these loci. At some loci, the null allele is prevalent in the sample analysed, suggesting that, in the horse population, the number of numt loci may be higher than 82 present in the reference genome. Contrary to humans, the insertion polymorphism of numts is extremely frequent in the horse population, supporting the hypothesis that the genome of this species is in a rapidly evolving state.