Fungal transposable elements and genome evolution

The transposable elements (TEs) identified in fungal genomes reflect the whole spectrum of eukaryotic transposable elements. Most of our knowledge comes from species representing different ecological situations: plant pathogens, industrial, and field strains, most of them lacking the sexual stage. A number of changes in gene structure and function has been shown to be TE-mediated: inactivation of gene expression upon insertion within or adjacent to a gene, DNA sequence variation through excision and probably extensive chromosomal rearrangements due to recombination between members of a particular family. Moreover, TEs may have other roles in evolution related to their ability to be horizontally transferred and to capture and transpose chromosomal host sequences, thus providing a mechanism for dispersing sequences to new sites. However, the activity of transposable elements and consequently their proliferation within a host genome can be affected, in some fungal species which undergo meiosis, by silencing processes. Our understanding of the biological effects of TEs on the fungal genome has increased dramatically in the past few years but elucidation of the extent to which transposons contribute to genetic variation in nature, providing the flexibility for populations to adapt successfully to environmental changes is an important area for future research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Site-specific retrotransposons of the trypanosomatid protozoa

Retrotransposons are mobile genetic elements that have invaded a wide variety of organisms. While these mobile elements share gene homologies and structural features with retroviruses, they have lost the ability to produce infectious particles. Typically these elements are 5-10 kilobases (kb) in length, are conserved in their structural organization and are present in many copies in the genomes into which they have integrated(1). Retrotransposons generally interrupt their host genome promiscuously and thus cause a variety of random effects. In general, their insertion results in mutations, inversions, deletions or rearrangements among host sequences. All of these changes are thought to add to the plasticity of the host genome and thus contribute to a faster pace of evolutionary development. However, because of the random nature of insertions, it has been difficult to attribute any one specific function to these diverse elements. Here, Serap Aksoy describes a newly recognized family of mobile elements that are different from most retrotransposons in that they have the ability to integrate into specific host sequences.     

插入序列共同区元件：细菌中新出现的一种基因捕获系统

摘要：插入序列共同区（Insertion sequence common region,ISCR）元件是一类在结构和功能上与IS91家族相似的特殊插入序列,特点是缺少了末端反向重复序列（Inverted repeats, IRs）,在插入位点不产生直接重复序列,并通过滚环式（Rolling circle, RC）进行转座。ISCR元件作为一种新的基因捕获系统,它可以移动邻近的任何DNA序列,为耐药基因在不同种属细菌间水平传播提供了高效的媒介。世界各地多种革兰氏阴性病原菌中已发现有19种ISCR元件,大部分ISCR元件同时携带了多种耐药基因,提示ISCR有可能会造成细菌多重耐药性的快速传播。本文就ISCR结构特征、类型、移动方式、起源及进化的研究进展进行了综述。     

Invasion of a multitude of genetic niches by mobile endonuclease genes

Persistence of a mobile DNA element in a population reflects a balance between the ability of the host to eliminate the element and the ability of the element to survive and to disseminate to other individuals. In each of the three biological kingdoms, several families of a mobile DNA element have been identified which encode a single protein that acts on nucleic acids. Collectively termed homing endonuclease genes (HEGs), these elements employ varied strategies to ensure their survival. Some members of the HEG families have a minimal impact on host fitness because they associate with genes having self-splicing introns or inteins that remove the HEGs at the RNA or protein level. The HEG and the intron/intein gene spread throughout the population by a gene conversion process initiated by the HEG-encoded endonuclease called 'homing' in which the HEG and intron/intein genes are copied to cognate alleles that lack them. The endonuclease activity also contributes to a high frequency of lateral transmission of HEGs between species as has been documented in plants and other systems. Other HEGs have positive selection value because the proteins have evolved activities that benefit their host organisms. The success of HEGs in colonizing diverse genetic niches results from the flexibility of the encoded endonucleases in adopting new specificities.     

Distribution of split DnaE inteins in cyanobacteria

Inteins are genetic elements found inside the coding regions of different host proteins and are translated in frame with them. The intein-encoded protein region is removed by an autocatalytic protein-splicing reaction that ligates the host protein flanks with a peptide bond. This reaction can also occur in trans with the intein and host protein split in two. After translation of the two genes, the two intein parts ligate their flanking protein parts to each other, producing the mature protein. Naturally split inteins are only known in the DNA polymerase III alpha subunit (polC or dnaE gene) of a few cyanobacteria. Analysing the phylogenetic distribution and probable genetic propagation mode of these split inteins, we conclude that they are genetically fixed in several large cyanobacterial lineages. To test our hypothesis, we sequenced parts of the dnaE genes from five diverse cyanobacteria and found all species to have the same type of split intein. Our results suggest the occurrence of a genetic rearrangement in the ancestor of a large division of cyanobacteria. This event fixed the dnaE gene in a unique two-genes one-protein configuration in the progenitor of many cyanobacteria. Our hypothesis, findings and the cloning procedure that we established allow the identification and acquisition of many naturally split inteins. Having a large and diverse repertoire of these unique inteins will enable studies of their distinct activity and enhance their use in biotechnology.     

PRP8 inteins in species of the genus Botrytis and other ascomycetes

The mobile elements termed inteins have a sporadic distribution in microorganisms. It is unclear how these elements are maintained. Inteins are intervening protein sequences that autocatalytically excise themselves from a precursor. Excision is a post-translational process referred to as 'protein splicing' in which the sequences flanking the intein are ligated, reforming the mature host protein. Some inteins contain a homing endonuclease domain (HEG) that is proposed to facilitate propagation of the intein element within a gene pool. We have previously demonstrated that the HEG of the PRP8 intein is highly active during meiosis in Botrytis cinerea. Here we analysed the Prp8 gene status in 21 additional Botrytis species to obtain insight into the mode of intein inheritance within the Botrytis lineage. Of the 21 species, 15 contained a PRP8 intein whereas six did not. The analysis was extended to closely related (Sclerotiniaceae) and distantly related (Ascomycota) taxa, focussing on evolutionary diversification of the PRP8 intein, including their possible acquisition by horizontal transfer and loss by deletion. Evidence was obtained for the occurrence of genetic footprints of previous intein occupation. There is no compelling evidence of horizontal transfer among species. Three distinct states of the Prp8 allele were identified, distributed over different orders within the Ascomycota: an occupied allele; an empty allele that was never occupied; an empty allele that was presumably previously occupied, from which the intein was precisely deleted. The presence of the genetic footprint identifies 20 species (including Neurospora crassa, Magnaporthe oryzae and Fusarium oxysporum) that previously contained the intein but have lost it entirely, while only 18 species (including Podospora anserina and Fusarium graminearum) appear never to have contained a PRP8 intein. The analysis indicates that inteins may be maintained in an equilibrium state.     

Fungal transposable elements: Generators of diversity and genetic tools

Representatives of several classes of transposable elements (TEs) have been characterized in a broad range of fungal species. The studies indicate that these elements are ancient and ubiquitous components of fungal genomes. Some of these elements have been shown to actively affect gene structure and function in several ways: inactivation of gene expression upon insertion, modification of the nucleotide sequence through excision, and probably by inducing extensive chromosomal rearrangements. The ability of TEs to generate a high degree of genetic diversity may therefore be important in the evolution of the fungal genome. TEs also have many potential applications in genetic research, including insertional mutagenesis and population fingerprinting, as well as gene transfer within and between species. All these genetic approaches are important as tools in studies of molecular biology and evolution of fungal species, many of which lack a functional sexual cycle.     

Mycoplasma genitalium: an efficient strategy to generate genetic variation from a minimal genome

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has smallest genome of any known free-living organism. The goal of this study was to investigate if and how M. genitalium uses a minimal genome to generate genetic variations. We analysed the sequence variability of the third gene (MG192 or mgpC) of the M. genitalium MgPa adhesion operon, demonstrated that the MG192 gene is highly variable among and within M. genitalium strains in vitro and in vivo, and identified MG192 sequence shifts in the course of in vitro passage of the G37 type strain and in sequential specimens from an M. genitalium-infected patient. In order to establish the origin of the MG192 variants, we examined nine genomic loci containing partial copies of the MgPa operon, known as MgPar sequences. Our analysis suggests that the MG192 sequence variation is achieved by recombination between the MG192 expression site and MgPar sequences via gene cross-over and, possibly, also by gene conversion. It appears plausible that M. genitalium has the ability to generate unlimited variants from its minimized genome, which presumably allows the organism to adapt to diverse environments and/or to evade host defences by antigenic variation.     

Target site recognition by a diversity-generating retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification.     

Conservation of the C-type lectin fold for accommodating massive sequence variation in archaeal diversity-generating retroelements

Background

Diversity-generating retroelements (DGRs) provide organisms with a unique means for adaptation to a dynamic environment through massive protein sequence variation. The potential scope of this variation exceeds that of the vertebrate adaptive immune system. DGRs were known to exist only in viruses and bacteria until their recent discovery in archaea belonging to the ‘microbial dark matter’, specifically in organisms closely related to Nanoarchaeota. However, Nanoarchaeota DGR variable proteins were unassignable to known protein folds and apparently unrelated to characterized DGR variable proteins.

Results

To address the issue of how Nanoarchaeota DGR variable proteins accommodate massive sequence variation, we determined the 2.52 Å resolution limit crystal structure of one such protein, AvpA, which revealed a C-type lectin (CLec)-fold that organizes a putative ligand-binding site that is capable of accommodating 10¹³ sequences. This fold is surprisingly reminiscent of the CLec-folds of viral and bacterial DGR variable protein, but differs sufficiently to define a new CLec-fold subclass, which is consistent with early divergence between bacterial and archaeal DGRs. The structure also enabled identification of a group of AvpA-like proteins in multiple putative DGRs from uncultivated archaea. These variable proteins may aid Nanoarchaeota and these uncultivated archaea in symbiotic relationships.

Conclusions

Our results have uncovered the widespread conservation of the CLec-fold in viruses, bacteria, and archaea for accommodating massive sequence variation. In addition, to our knowledge, this is the first report of an archaeal CLec-fold protein.

     

Mutator转座子及MULE在植物基因与基因组进化中的作用

Mutator（Mu）转座子是植物中已发现的转座最活跃的转座子,其高的转座频率及趋向于单拷贝功能基因转座的特性,使该转座子成为玉米功能基因克隆的主要方法.Mu转座子的同源类似因子广泛存在于被子植物基因组中,而且同一基因组中往往具有多种变异类型.它不仅具有其他DNA转座子在基因和基因组进化中的普遍作用,而且具有能够承载基因组内功能基因和基因片段的载体功能,这种载体Mu转座子（Pack-MuLEs）能够在基因组内移动众多的基因片段,从而对基因和基因组进化产生作用.Mu转座子的同源序列发生在水稻与狗尾草之间的水平转移提供了高等植物核基因水平转移的首个例证.对Mu转座子的了解促进了我们对动态基因组概念的认识.文章对Mutator转座子的发现、转座特征、基因标签应用等的研究进展进行了综述,对Mu转座子家族的同源序列进行了分类,讨论了该转座子在基因组进化中的作用,分析了应加强研究的问题.     

Contrasting definitive hosts as determinants of the genetic structure in a parasite with complex life cycle along the south‐eastern Pacific

The spatial genetic structure (and gene flow) of parasites with complex life cycles, such as digeneans, has been attributed mainly to the dispersion ability of the most mobile host, which most often corresponds to the definitive host (DH). In this study, we compared the genetic structure and diversity of adult Neolebouria georgenascimentoi in two fish species (DHs) that are extensively distributed along the south‐eastern Pacific (SEP). The analysis was based on the cytochrome oxidase subunit I gene sequences of parasites collected between 23°S and 45°S. In total, 202 sequences of N. georgenascimentoi in Pinguipes chilensis isolated from nine sites and 136 sequences of Prolatilus jugularis from five sites were analysed. Our results showed that N. georgenascimentoi is a species complex that includes three different parasite species; however, in this study, only lineage 1 and 2 found in P. chilensis and P. jugularis, respectively, were studied because they are widely distributed along the coastline. Lineage 1 parasites had two common haplotypes with wide distribution and unique haplotypes in northern sites. Lineage 2 had only one common haplotype with wide distribution and a large number of unique haplotypes with greater genetic diversity. Both lineages have experienced recent population expansion. Only lineage 1 exhibited a genetic structure that was mainly associated with a biogeographical break at approximately 30°S along the SEP. Our finding suggests that host access to different prey (=intermediate hosts) could affect the genetic structure of the parasite complex discovered here. Consequently, difference between these patterns suggests that factors other than DH dispersal are involved in the genetic structure of autogenic parasites.     

Expression and structure of cartilage proteins.

Cartilage has unique physical characteristics attributable to the presence of an unusually high content of proteoglycan embedded in the network of collagen fibrils. Advances in understanding the structure of these components and how their synthesis is regulated have been greatly assisted by the application of molecular biology. For example, an immortalized rat chondrocyte cell line was obtained by infection with a recombinant retrovirus encoding the myc gene product. Several positive and negative DNA regulatory elements of the collagen II gene have been identified that appear to be important in the regulation of this gene in chondrocytes. The complete primary structure of the cartilage proteoglycan (aggrecan) core protein deduced from cDNA sequence displays a complex multidomain structure including numerous repeats of Ser-Gly sequences and sequence homologies with link protein and animal lectins. Such studies advance our understanding of normal morphogenetic events and lay the groundwork for determining the basis of molecular and genetic defects.     

Comparative genome analysis of filamentous fungi reveals gene family expansions associated with fungal pathogenesis

Fungi and oomycetes are the causal agents of many of the most serious diseases of plants. Here we report a detailed comparative analysis of the genome sequences of thirty-six species of fungi and oomycetes, including seven plant pathogenic species, that aims to explore the common genetic features associated with plant disease-causing species. The predicted translational products of each genome have been clustered into groups of potential orthologues using Markov Chain Clustering and the data integrated into the e-Fungi object-oriented data warehouse (http://www.e-fungi.org.uk/). Analysis of the species distribution of members of these clusters has identified proteins that are specific to filamentous fungal species and a group of proteins found only in plant pathogens. By comparing the gene inventories of filamentous, ascomycetous phytopathogenic and free-living species of fungi, we have identified a set of gene families that appear to have expanded during the evolution of phytopathogens and may therefore serve important roles in plant disease. We have also characterised the predicted set of secreted proteins encoded by each genome and identified a set of protein families which are significantly over-represented in the secretomes of plant pathogenic fungi, including putative effector proteins that might perturb host cell biology during plant infection. The results demonstrate the potential of comparative genome analysis for exploring the evolution of eukaryotic microbial pathogenesis.     

A retrotransposon-like element and its occurrence in British Columbia populations of Chondrostereum purpureum

Repetitive DNA elements discovered in the basidiomycete Chondrostereum purpureum were characterized and validated for use as genetic markers. Regions of these marker sequences were similar to retrotransposon and retrotransposon-like sequences, as indicated by BLAST searches of NCBI databases. These sequences occur in multiple DNA fragments of variable length in a given C. purpureum isolate, and thus can serve as strain-specific genetic markers. The segregation of the markers within a progeny set demonstrated their stability through meiosis. The population structure of C. purpureum was assessed using the markers. There was no evidence of a barrier to gene flow between C. purpureum populations separated by 1400 km and no indication of population sub-structuring based on host or geographical source of isolate. Repetitive fragments were amplified from four other species, suggesting the occurrence of these retrotransposon-like elements in other basidiomycetes and the potential utility of these markers for other fungi.