Opened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica.

Opened brood chamber with embryos of different stages from the demosponge Amphimedon queenslandica. This poriferan species has its genome sequenced, assembled, and annotated and serves as a model organism for sponge developmental biology. Image supplied by Bernie Degnan (University of Queensland).     

PGV - Prokaryotic Genome Viewer

Recent sequencing of genomes of several microorganisms provides an opportunity to have access to huge volumes of data stored in various databases. This has resulted in the development of various computational and visualization tools to aid in retrieval and analysis of data. Development of user friendly genome data mapping and visualization tools facilitates researchers to closely examine various features of genes and make inferences from the displayed data efficiently. PGV - Prokaryotic Genome Viewer is a Java based web application tool capable of generating high quality interactive circular chromosome maps. With simple mouse roll over tasks on the interested region on the displayed map, the user is provided with features such as feature labeling, multi-fold zooming, image rotation and hyperlinking to different information resources. The tool is capable of instantaneously generating maps using user-supplied sequence data.     

Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism''s niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).     

Connexin Genes in the Mouse and Human Genome

Gap junctions serve for direct intercellular communication by docking of two hemichannels in adjacent cells thereby forming conduits between the cytoplasmic compartments of adjacent cells. Connexin genes code for subunit proteins of gap junction channels and are members of large gene families in mammals. So far, 17 connexin (Cx) genes have been described and characterized in the murine genome. For most of them, orthologues in the human genome have been found (see White and Paul 1999; Manthey et al. 1999; Teubner et al. 2001; Söhl et al. 2001). We have recently performed searches for connexin genes in murine and human gene libraries available at EMBL/Heidelberg, NCBI and the Celera company that have increased the number of identified connexins to 19 in mouse and 20 in humans. For one mouse connexin gene and two human connexin genes we did not find orthologues in the other genome. Here we present a short overview on distinct connexin genes which we found in the mouse and human genome and which may include all members of this gene family, if no further connexin gene will be discovered in the remaining non-sequenced parts (about 1-5%) of the genomes.     

草菇基因组中的tRNA分析

草菇(V olvariella volvacea),又名中国蘑菇,是一种生长于中国和东南亚的重要经济食用菌。本文基于本实验室的草菇基因组测序结果,分析了草菇基因组中的tRNA情况。草菇基因组的302个框架中,共发现了177个tRNA基因,其中有7个可能的假基因,有2个携带硒代半胱氨酸,一个抑制性tRNA基因和一个未知的异型结构tRNA。除了上述11个特殊的tRNA基因外,166个tRNA可按反密码子类型分成47类。与其它5种担子菌的基因组tRNA比较,草菇的tRNA数量处于第4位,少于鬼伞、裂褶菌、灵芝,多于双孢蘑菇和平菇。通过比较分析草菇及这5种大型真菌的基因组tRNA编码情况,首次报道了几种食用菌tRNA遵守修正的摆动假说的情况。另外,草菇的同功受体tRNA非编码子序列也具有差异性,对tRNA的分析将有助于进一步研究草菇的进化。     

A Novel Prokaryotic Promoter Identified in the Genome of Some Monopartite Begomoviruses

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.     

On the Informational Content of Overlapping Genes in Prokaryotic and Eukaryotic Viruses

In genetic language a peculiar arrangement of biological information is provided by overlapping genes in which the same region of DNA can code for functionally unrelated messages. In this work, the informational content of overlapping genes belonging to prokaryotic and eukaryotic viruses was analyzed. Using information theory indices, we identified in the regions of overlap a first pattern, exhibiting a more uniform base composition and more severe constraints in base ordering with respect to the nonoverlapping regions. This pattern was found to be peculiar to coliphage, avian hepatitis B virus, human lentivirus, and plant luteovirus families. A second pattern, characterized by the occurrence of similar compositional constraints in both types of coding regions, was found to be limited to plant tymoviruses. At the level of codon usage, a low degree of correlation between overlapping and nonoverlapping coding regions characterized the first pattern, whereas a close link was found in tymoviruses, indicating a fine adaptation of the overlapping frame to the original codon choice of the virus. As a result of codon usage correlation analysis, deductions concerning the origin and evolution of several overlapping frames were also proposed. Comparison of amino acid composition revealed an increased frequency of amino acid residues with a high level of degeneracy (arginine, leucine, and serine) in the proteins encoded by overlapping genes; this peculiar feature of overlapping genes can be viewed as a way with which they may expand their coding ability and gain new, specialized functions. Received: 28 October 1996 / Accepted: 29 January 1997     

Defining the Core of Nontransferable Prokaryotic Genes: The Euryarchaeal Core

If lateral gene transfer (LGT) has affected all genes over the course of prokaryotic evolution, reconstruction of organismal phylogeny is compromised. However, if a core of genes is immune to transfer, then the evolutionary history of that core might be our most reliable guide to the evolution of organisms. Such a core should be preferentially included in the subset of genes shared by all organisms, but where universally conserved genes have been analyzed, there is too little phylogenetic signal to allow determination of whether or not they indeed have the same history (Hansmann and Martin 2000; Teichmann and Mitchison 1999). Here we look at a more restricted set, 521 homologous genes (COGs) simultaneously present in four sequenced euryarchaeal genomes. Although there is overall little robust phylogenetic signal in this data set, there is, among well-supported trees, strong representation of all three possible four-taxon topologies. ``Informational' genes seem no less subject to LGT than are ``operational genes,' within the euryarchaeotes. We conclude that (i) even in this collection of conserved genes there has been extensive LGT (orthologous gene replacement) and (ii) the notion that there is a core of nontransferable genes (the ``core hypothesis') has not been proven and may be unprovable. Received: 7 November 2000 / Accepted: 20 February 2001     

超抗原融合基因VEGF-SEA的克隆、原核表达及蛋白纯化

根据Genebank报道的VEGF(NM-003376)、SEA(A28664)基因序列,对密码子进行优化,合成血管内皮细胞生长因子和超抗原基因序列,将VEGF-SEA基因片段插入质粒pET22b构建重组质粒pET22b-VEGF-SEA.重组质粒经序列分析正确,转化大肠杆菌BL21(DE3)进行IPTG诱导表达,表达产物经SDS-PAGE分析蛋白条带与预期一致,证明融合蛋白原核表达成功.产物经His·Bind Buffer kit试剂盒纯化,纯度达到90%,这一成果为进一步研究VEGF-SEA融合蛋白的活性及其功能,探讨超抗原抑制肿瘤生长作用奠定了基础.     

Cytochrome P450 Genes from the Sacred Lotus Genome

Cytochrome P450 monooxygenases (P450s) in the sacred lotus (Nelumbo nucifera) genome have been identified and named according to systematic P450 nomenclatures. Comparisons of these sequences with those in the papaya and grape CYPomes have indicated that gene blooms exist in the CYP89, CYP94, CYP96 and CYP714 families and that less dramatic expansions exist in the CYP71 and CYP72 families. Expansions in the CYP94 and CYP96 families may be associated with generation of the extremely hydrophobic leaf surfaces associated with the “lotus effect” in this water-adapted species, since these families are known to hydroxylate fatty acids and alkanes in the wax biosynthetic pathways of other plant species. Evolution of the CYP719 and CYP80 families may be associated with production of a number of benzylisoquinoline and aporphine alkaloids. Structures for anonaine and roemerine, two of the most abundant aporphine alkaloids in lotus leaves and seeds, contain methylenedioxy bridges that are known to be generated by members of the CYP719 family. With only one CYP719A22 gene existing in the lotus genome, it is likely that it is involved in making aporphine alkaloids. The fact that CYP719 has not previously been seen in angiosperm phylogeny below the order of Ranunculales suggests that its presence in lotus (in the Proteales) presents an evolutionary terminus prior to its loss in more recent eudicot species. With several CYP80 family genes existing in the lotus genome, there are multiple candidates for those involved in conducting benzylisoquinoline alkaloid synthesis.     

Genome Scanning in Haemophilus influenzae for Identification of Essential Genes

We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented.     

Extensive Error in the Number of Genes Inferred from Draft Genome Assemblies

Current sequencing methods produce large amounts of data, but genome assemblies based on these data are often woefully incomplete. These incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. In this paper we investigate the magnitude of the problem, both in terms of total gene number and the number of copies of genes in specific families. To do this, we compare multiple draft assemblies against higher-quality versions of the same genomes, using several new assemblies of the chicken genome based on both traditional and next-generation sequencing technologies, as well as published draft assemblies of chimpanzee. We find that upwards of 40% of all gene families are inferred to have the wrong number of genes in draft assemblies, and that these incorrect assemblies both add and subtract genes. Using simulated genome assemblies of Drosophila melanogaster, we find that the major cause of increased gene numbers in draft genomes is the fragmentation of genes onto multiple individual contigs. Finally, we demonstrate the usefulness of RNA-Seq in improving the gene annotation of draft assemblies, largely by connecting genes that have been fragmented in the assembly process.     

氧化亚铁硫杆菌中铜代谢相关两个基因的差异

目的:氧化亚铁硫杆菌(Acidithiobocllius ferrooxidans)在微生物冶金中发挥着重要的作用,研究其铜代谢机理有着十分典型的意义.在A.ferrooxidans全基因组序列数据库中,4个基因被注释与铜代谢相关.其中两个基因,Afe0454和Afe1073目前为止未发现有实验报道.本文旨在研究Afe0454和Afe1073与铜代谢的相关性.方法:通过一系列的方法如实时定量PER、反转录PER、序列分析,将基因导入抗铜基因缺陷的大肠杆菌(Escherichia coli)菌株中等,研究了Afe0454和Afe1073.结果:与Afe0454相比,Afe1073的表达对铜压力更敏感;Afe1073作为一个转录子单独转录,而Afe0454与Afe0453一起转录;序列分析显示Afe1073表达一种典型的重金属离子泵P1b1型ATP酶,而Afe0454表达一种未知功能的跨膜蛋白;不像Afe0454,Afe1073能使P1b型ATP酶敲除的大肠杆菌菌株铜抗性提高.结论:单独转录的Afe1073比Afe0454在铜代谢中发挥的作用更加明显.