Landscape and evolutionary dynamics of Helitron transposons in plant genomes as well as construction of online database HelDB

Helitron transposons play an important role in host genome evolution due to their ability to capture genes and regulatory elements. In this study, we developed a pipeline to identify and annotate Helitrons systematically from 358 plant and 178 animal high-quality genomes. All these data were organized into HelDB, a database where Helitrons can be explored with a user-friendly Web interface and related software. Based on these data, further analysis showed that the number or the cumulative length of Helitrons is positively correlated with genome size. Helitrons had experienced two expansion periods in plants, with the first occurring 20–30 Ma and peaking at approximately 24 Ma. The second expansion occurred in the last 4 million years. The expansions might be due to stimulation of paleogeographic environment. Detailed investigation of gene capture by Helitrons in Brassicaceae and Solanaceae plants showed that the captured genes showed diverse functions. Interestingly, metal ion binding function was enriched in these captured genes in most species. This phenomenon might be due to the need for binding of divalent metal ions to the Rep domain required for Helitron transposition. This study improves our knowledge of the landscape and evolution of Helitron transposons in plants and paves a way for further functional studies of this kind of transposable element.     

The maize genome contains a helitron insertion

The maize mutation sh2-7527 was isolated in a conventional maize breeding program in the 1970s. Although the mutant contains foreign sequences within the gene, the mutation is not attributable to an interchromosomal exchange or to a chromosomal inversion. Hence, the mutation was caused by an insertion. Sequences at the two Sh2 borders have not been scrambled or mutated, suggesting that the insertion is not caused by a catastrophic reshuffling of the maize genome. The insertion is large, at least 12 kb, and is highly repetitive in maize. As judged by hybridization, sorghum contains only one or a few copies of the element, whereas no hybridization was seen to the Arabidopsis genome. The insertion acts from a distance to alter the splicing of the sh2 pre-mRNA. Three distinct intron-bearing maize genes were found in the insertion. Of most significance, the insertion bears striking similarity to the recently described DNA helicase-bearing transposable elements termed HELITRONS: Like Helitrons, the inserted sequence of sh2-7527 is large, lacks terminal repeats, does not duplicate host sequences, and was inserted between a host dinucleotide AT. Like Helitrons, the maize element contains 5' TC and 3' CTRR termini as well as two short palindromic sequences near the 3' terminus that potentially can form a 20-bp hairpin. Although the maize element lacks sequence information for a DNA helicase, it does contain four exons with similarity to a plant DEAD box RNA helicase. A second Helitron insertion was found in the maize genomic database. These data strongly suggest an active Helitron in the present-day maize genome.     

Helitrons on a roll: eukaryotic rolling-circle transposons

Rolling-circle eukaryotic transposons, known as Helitron transposons, were first discovered in plants (Arabidopsis thaliana and Oryza sativa) and in the nematode Caenorhabditis elegans. To date, Helitrons have been identified in a diverse range of species, from protists to mammals. They represent a major class of eukaryotic transposons and are fundamentally different from classical transposons in terms of their structure and mechanism of transposition. Helitrons seem to have a major role in the evolution of host genomes. They frequently capture diverse host genes, some of which can evolve into novel host genes or become essential for helitron transposition.     

Population and evolutionary dynamics of Helitron transposable elements in Arabidopsis thaliana

Helitrons, a recently discovered superfamily of DNA transposons that capture host gene fragments, constitute up to 2% of the Arabidopsis thaliana genome. In this study, we identified 565 insertions of a family of nonautonomous Helitrons, known as Basho elements. We aligned subsets of these elements, estimated their phylogenetic relationships, and used branch lengths to yield insight into the age of each Basho insertion. The age distribution suggests that 87% of Bashos inserted within 5 Myr, subsequent to the divergence between A. thaliana and its sister species Arabidopsis lyrata. We screened 278 of these insertions for their presence or absence in a sample of 47 A. thaliana accessions. With both phylogenetic and population frequency data, we investigated the effects of gene density, recombination rate, and element length on Basho persistence. Our analyses suggested that longer Basho copies are less likely to persist in the genome, consistent with selection against the deleterious effects of ectopic recombination between Basho elements. Furthermore, we determined that 39% of Basho elements contain fragments of expressed protein-coding genes, but all of these fragments were explained by only 5 gene-capture events. Overall, the picture of A. thaliana Helitron evolution is one of rapid expansion, relatively few gene-capture events, and weak selection correlated with element length.     

Microarray-based global differential expression profiling of P450 monooxygenases and regulatory proteins for signal transduction pathways in the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Whole genome sequencing of the model white rot basidiomycete Phanerochaete chrysosporium has revealed the largest P450 contingent known to date in fungi, along with related phase I and phase II metabolic genes and signaling cascade genes. As a part of their functional characterization, genome-wide expression profiling under physiologically distinct conditions, nutrient-limited (ligninolytic) and nutrient-rich (non-ligninolytic), was investigated using a custom-designed 70-mer oligonucleotide microarray developed based on 190 target genes and 23 control genes. All 150 P450 genes were found to be expressible under the test conditions, with 27 genes showing differential expression based on a >twofold arbitrary cut-off limit. Of these, 23 P450 genes were upregulated (twofold to ninefold) in defined high-nitrogen cultures whereas four genes were upregulated (twofold to twentyfold) in defined low-nitrogen cultures. Furthermore, tandem P450 member genes in ten of the 16 P450 genomic clusters showed nonassortative regulation of expression reflecting their functional diversity. Full-length cDNAs for two of the high-nitrogen upregulated genes pc-hn1 (CYP5035A1) and pc-hn2 (CYP5036A1) and partial cDNA for a low-nitrogen upregulated gene pc-ln1 (CYP5037A1) were cloned and characterized. The study provided first molecular evidence for the presence of active components of the cAMP- and MAP kinase-signaling pathways in a white rot fungus; four of these components (cpka and ste-12 of cAMP pathway and two MAP kinases, mps1 and sps1) were significantly upregulated (fourfold to eightfold) under nutrient-limited conditions, implying their likely role in the regulation of gene expression involved in secondary metabolism and biodegradation processes under these conditions.     

Triterpene functional genomics in licorice for identification of CYP72A154 involved in the biosynthesis of glycyrrhizin

Glycyrrhizin, a triterpenoid saponin derived from the underground parts of Glycyrrhiza plants (licorice), has several pharmacological activities and is also used worldwide as a natural sweetener. The biosynthesis of glycyrrhizin involves the initial cyclization of 2,3-oxidosqualene to the triterpene skeleton β-amyrin, followed by a series of oxidative reactions at positions C-11 and C-30, and glycosyl transfers to the C-3 hydroxyl group. We previously reported the identification of a cytochrome P450 monooxygenase (P450) gene encoding β-amyrin 11-oxidase (CYP88D6) as the initial P450 gene in glycyrrhizin biosynthesis. In this study, a second relevant P450 (CYP72A154) was identified and shown to be responsible for C-30 oxidation in the glycyrrhizin pathway. CYP72A154 expressed in an engineered yeast strain that endogenously produces 11-oxo-β-amyrin (a possible biosynthetic intermediate between β-amyrin and glycyrrhizin) catalyzed three sequential oxidation steps at C-30 of 11-oxo-β-amyrin supplied in situ to produce glycyrrhetinic acid, a glycyrrhizin aglycone. Furthermore, CYP72A63 of Medicago truncatula, which has high sequence similarity to CYP72A154, was able to catalyze C-30 oxidation of β-amyrin. These results reveal a function of CYP72A subfamily proteins as triterpene-oxidizing enzymes and provide a genetic tool for engineering the production of glycyrrhizin.     

CYP52 (cytochrome P450alk) multigene family in Candida maltosa: molecular cloning and nucleotide sequence of the two tandemly arranged genes

Southern blot analysis under low-stringency conditions using a previously isolated n-alkane-inducible cytochrome P450 (P450alk) gene as a probe revealed the presence of multiple P450alk-related genes in the genome of Candida maltosa. Nine P450alk-related genes (one reported previously and eight in the present report) were isolated from a genomic library constructed from this strain, and these were classified on the basis of sequence similarities into three pairs of putative allelic genes and three nonallelic genes. Two pairs of these alleles were tandemly arranged in the genome. The complete nucleotide sequences of one of these pairs were determined and compared to other members of this P450 family (CYP52) in C. maltosa and C. tropicalis. Northern blot analysis further showed that these genes were regulated by carbon sources. These results provide evidence for a P450alk (CYP52) multigene family in C. maltosa.     

Muscle force arises by actin filament rotation and torque in the Z-filaments

Cytochrome P450 partial sequences were isolated by PCR using genomic DNA from two hymenopteran insects of agronomical importance, Trichogramma cacoeciae, a parasitoid wasp, and Apis mellifera, the honeybee. Four new P450 genes were identified: one honeybee gene belongs to the CYP4 family and was named CYP4G11; the three other genes were from Trichogramma and belong to the CYP4 family (CYP4G12) and to a novel family, the CYP48 one (CYP48A1 and CYP48A2). The four genes contain a short intron (72-95 bp) at the same position as already described for other insect species. The two genes CYP48A1 and CYP48A2 have a supernumary intron (57-71 bp) upstream the first one. Only the two CYP4 genes were constitutively transcribed, at a high level for CYP4G12 and at a low level for CYP4G11. No expression was observed for CYP48A1 and CYP48A2.     

CYP719A subfamily of cytochrome P450 oxygenases and isoquinoline alkaloid biosynthesis in <Emphasis Type="Italic">Eschscholzia californica</Emphasis>

Eschscholzia californica produces various types of isoquinoline alkaloids. The structural diversity of these chemicals is often due to cytochrome P450 (P450) activities. Members of the CYP719A subfamily, which are found only in isoquinoline alkaloid-producing plant species, catalyze methylenedioxy bridge-forming reactions. In this study, we isolated four kinds of CYP719A genes from E. californica to characterize their functions. These four cDNAs encoded amino acid sequences that were highly homologous to Coptis japonica CYP719A1 and E. californica CYP719A2 and CYP719A3, which suggested that these gene products may be involved in isoquinoline alkaloid biosynthesis in E. californica, especially in methylenedioxy bridge-forming reactions. Expression analysis of these genes showed that two genes (CYP719A9 and CYP719A11) were preferentially expressed in plant leaf, where pavine-type alkaloids accumulate, whereas the other two showed higher expression in root than in other tissues. They were suggested to have distinct physiological functions in isoquinoline alkaloid biosynthesis. Enzyme assay analysis using recombinant proteins expressed in yeast showed that CYP719A5 had cheilanthifoline synthase activity, which was expected based on the similarity of its primary structure to that of Argemone mexicana cheilanthifoline synthase (deposited at DDBJ/GenBanktrade mark/EMBL). In addition, enzyme assay analysis of recombinant CYP719A9 suggested that it has methylenedioxy bridge-forming activity toward (R,S)-reticuline. CYP719A9 might be involved in the biosynthesis of pavine- and/or simple benzylisoquinoline-type alkaloids, which have a methylenedioxy bridge in an isoquinoline ring, in E. californica leaf.     

The limited distribution of Helitrons to vesper bats supports horizontal transfer

Transposable elements (TEs) have the unique ability to move and replicate within the genome and therefore engender dramatic changes to genome architecture. Among different types of TEs, rolling-circle transposons (Helitrons) are well known for their ability to capture and amplify host gene fragments. Bioinformatic analysis revealed that Helitrons constitute ~3% of the Myotis lucifugus, (little brown bat) genome, while no Helitrons were found in any of the other 44+ sequenced mammalian genomes. Recently horizontal transfer has been implicated for some of the M. lucifugus Helitrons, in part explaining this disparate distribution among mammals. The purpose of this work is to determine both the distribution of Helitrons among bats and to estimate the number of independent invasions. We employed a combination of in silico, PCR and hybridization based techniques to identify Helitrons from diverse bat species belonging to ten different families. This work reveals that Helitrons invaded the vesper bat lineage, at least once. Indeed, Helitrons were not identified in the sister taxa 'Miniopterus', which suggests that the amplification of Helibat occurred (30-36 mya) only in the vesper bat lineage. The estimated age of amplification of the Helibats and the rapid radiation of vesper bats are roughly coincidental and suggest that the invasion and amplification of these elements might have influenced their evolutionary trajectory potentially contributing to phenotypic and genotypic diversity.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.     

Isolation and phylogeny of novel cytochrome P450 genes from tunicates (Ciona spp.): a CYP3 line in early deuterostomes?

Cytochromes P450 (CYPs) form a gene superfamily involved in the biotransformation of numerous endogenous and exogenous natural and synthetic compounds. In humans, CYP3A4 is regarded as one of the most important CYPs due to its abundance in liver and its capacity to metabolize more than 50% of all clinically used drugs. It has been suggested that all CYP3s arose from a common ancestral gene lineage that diverged between 800 and 1100 million years ago, before the deuterostome-protostome split. While CYP3s are well known in mammals and have been described in lower vertebrates, they have not been reported in non-vertebrate deuterostomes. Members of the genus Ciona belong to the tunicates, whose lineage is thought to be the most basal among the chordates, and from which the vertebrate line diverged. Here we describe the cloning, exon-intron structure, phylogeny, and estimated expression of four novel genes from Ciona intestinalis. We also describe the gene structure and phylogeny of homologous genes in Ciona savignyi. Comparing these genes with other members of the CYP clan 3, show that the Ciona sequences bear remarkable similarity to vertebrate CYP3A genes, and may be an early deuterostome CYP3 line.