Evidence for genomic rearrangements mediated by human endogenous retroviruses during primate evolution.

Human endogenous retroviruses (HERVs), which are remnants of past retroviral infections of the germline cells of our ancestors, make up as much as 8% of the human genome and may even outnumber genes. Most HERVs seem to have entered the genome between 10 and 50 million years ago, and they comprise over 200 distinct groups and subgroups. Although repeated sequence elements such as HERVs have the potential to lead to chromosomal rearrangement through homologous recombination between distant loci, evidence for the generality of this process is lacking. To gain insight into the expansion of these elements in the genome during the course of primate evolution, we have identified 23 new members of the HERV-K (HML-2) group, which is thought to contain the most recently active members. Here we show, by phylogenetic and sequence analysis, that at least 16% of these elements have undergone apparent rearrangements that may have resulted in large-scale deletions, duplications and chromosome reshuffling during the evolution of the human genome.     

Susceptibility to testicular germ-cell tumours in a 129.MOLF-Chr 19 chromosome substitution strain.

The identification of genes that control susceptibility to testicular germ-cell tumours (TGCTs), the most common cancer affecting young men, has been difficult. In laboratory mice, TGCTs arise from primordial germ cells in only the 129 inbred strains, and susceptibility is under multigenic control. The spontaneously arising mutation Ter (ref. 5) on mouse chromosome 18 (Refs 6,7) increases TGCT frequency on a 129/Sv background. We originally used Ter in genetic crosses to identify loci that control tumorigenesis. A genome scan of tumour-bearing progeny from backcrosses between the 129/Sv-Ter/+ and MOLF/Ei strains provided modest evidence that MOLF-derived alleles on chromosome 19 enhance development of bilateral TGCTs (ref. 9). To obtain independent evidence for linkage to the MOLF chromosome, we made an autosomal chromosome substitution strain (CSS; or 'consomic strain') in which chromosome 19 of 129/Sv+/+ was replaced by its MOLF-derived homologue. The unusually high frequency of TGCTs in this CSS (even in the absence of the Ter mutation) provides evidence confirming the genome survey results, identifies linkage for a naturally occurring strain variant allele that confers susceptibility to TGCTs and illustrates the power of CSSs in complex trait analysis.     

Human Alu element retrotransposition induced by genotoxic stress

Alu elements are found exclusively in primate species and comprise over 10% of the human genome. To better define the mechanisms responsible for Alu replication, we introduced a human Alu element into mouse cells. We report that Alu retrotransposition can be induced in mouse cells by exposure to the topoisomerase II inhibitor etoposide and is mediated in trans by endogenous mouse long interspersed elements (LINEs).     

The Arabidopsis lyrata genome sequence and the basis of rapid genome size change

We report the 207-Mb genome sequence of the North American Arabidopsis lyrata strain MN47 based on 8.3× dideoxy sequence coverage. We predict 32,670 genes in this outcrossing species compared to the 27,025 genes in the selfing species Arabidopsis thaliana. The much smaller 125-Mb genome of A. thaliana, which diverged from A. lyrata 10 million years ago, likely constitutes the derived state for the family. We found evidence for DNA loss from large-scale rearrangements, but most of the difference in genome size can be attributed to hundreds of thousands of small deletions, mostly in noncoding DNA and transposons. Analysis of deletions and insertions still segregating in A. thaliana indicates that the process of DNA loss is ongoing, suggesting pervasive selection for a smaller genome. The high-quality reference genome sequence for A. lyrata will be an important resource for functional, evolutionary and ecological studies in the genus Arabidopsis.     

Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates as an intracellular parasite of amoebae and persists in the environment as a free-living microbe. Here we have analyzed the complete genome sequences of L. pneumophila Paris (3,503,610 bp, 3,077 genes), an endemic strain that is predominant in France, and Lens (3,345,687 bp, 2,932 genes), an epidemic strain responsible for a major outbreak of disease in France. The L. pneumophila genomes show marked plasticity, with three different plasmids and with about 13% of the sequence differing between the two strains. Only strain Paris contains a type V secretion system, and its Lvh type IV secretion system is encoded by a 36-kb region that is either carried on a multicopy plasmid or integrated into the chromosome. Genetic mobility may enhance the versatility of L. pneumophila. Numerous genes encode eukaryotic-like proteins or motifs that are predicted to modulate host cell functions to the pathogen's advantage. The genome thus reflects the history and lifestyle of L. pneumophila, a human pathogen of macrophages that coevolved with fresh-water amoebae.     

Reductive evolution suggested from the complete genome sequence of a plant-pathogenic phytoplasma

The minimal gene set essential for life has long been sought. We report the 860-kb genome of the obligate intracellular plant pathogen phytoplasma (Candidatus Phytoplasma asteris, OY strain). The phytoplasma genome encodes even fewer metabolic functions than do mycoplasma genomes. It lacks the pentose phosphate cycle and, more unexpectedly, ATP-synthase subunits, which are thought to be essential for life. This may be the result of reductive evolution as a consequence of life as an intracellular parasite in a nutrient-rich environment.     

Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells

Mammalian genomes contain two main classes of retrotransposons, the well-characterized long and short interspersed nuclear elements, which account for approximately 30% of the genome, and the long terminal repeat (LTR) retrotransposons, which resemble the proviral integrated form of retroviruses, except for the absence of an envelope gene in some cases. Genetic studies confirmed mobility of the latter class of elements in mice, with a high proportion of phenotypic mutations consequent to transposition of the intracisternal A particle (IAP) family of LTR retrotransposons. Using the mouse genome sequence and an efficient ex vivo retrotransposition assay, we identified functional, master IAP copies that encode all the enzymatic and structural proteins necessary for their autonomous transposition in heterologous cells. By introducing mutations, we found that the three genes gag, prt and pol are all required for retrotransposition and identified the IAP gene products by electron microscopy in the form of intracellular A-type particles in the transfected cells. These prototypic elements, devoid of an envelope gene, are the first LTR retrotransposons autonomous for transposition to be identified in mammals. Their high rates of retrotransposition indicate that they are potent insertional mutagens that could serve as safe (noninfectious) genetic tools in a large panel of cells.     

Rapid gene mapping in Caenorhabditis elegans using a high density polymorphism map

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene.     

Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast.

We have constructed a new generation yeast artificial chromosome (YAC) library from female C57BL/10 mice in a recombination-deficient strain of Saccharomyces cerevisiae carrying a mutation in the RAD52 gene. The YAC library contains 41,568 clones with an average insert size of 240 kilobases, representing a greater than threefold coverage of the mouse genome. Currently, the library can be screened by polymerase chain reaction and we have isolated positive clones at a number of loci in the mouse genome. This rad52 library should enable a long-term assessment of the effect of one of the yeast recombination pathway genes on both, genome-wide YAC clone stability and the frequency of chimaeric clones.     

Emergence of a new disease as a result of interspecific virulence gene transfer

New diseases of humans, animals and plants emerge regularly. Enhanced virulence on a new host can be facilitated by the acquisition of novel virulence factors. Interspecific gene transfer is known to be a source of such virulence factors in bacterial pathogens (often manifested as pathogenicity islands in the recipient organism) and it has been speculated that interspecific transfer of virulence factors may occur in fungal pathogens. Until now, no direct support has been available for this hypothesis. Here we present evidence that a gene encoding a critical virulence factor was transferred from one species of fungal pathogen to another. This gene transfer probably occurred just before 1941, creating a pathogen population with significantly enhanced virulence and leading to the emergence of a new damaging disease of wheat.     

The complete genome sequence of Francisella tularensis, the causative agent of tularemia

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.