Genome rearrangements, especially amplifications and deletions, have regularly been observed as responses to sustained application of the same strong selective pressure in microbial populations growing in continuous culture. We studied eight strains of budding yeast (Saccharomyces cerevisiae) isolated after 100 -500 generations of growth in glucose-limited chemostats. Changes in DNA copy number were assessed at single-gene resolution by using DNA microarray-based comparative genomic hybridization. Six of these evolved strains were aneuploid as the result of gross chromosomal rearrangements. Most of the aneuploid regions were the result of translocations, including three instances of a shared breakpoint on chromosome 14 immediately adjacent to CIT1, which encodes the citrate synthase that performs a key regulated step in the tricarboxylic acid cycle. Three strains had amplifications in a region of chromosome 4 that includes the high-affinity hexose transporters; one of these also had the aforementioned chromosome 14 break. Three strains had extensive overlapping deletions of the right arm of chromosome 15. Further analysis showed that each of these genome rearrangements was bounded by transposon-related sequences at the breakpoints. The observation of repeated, independent, but nevertheless very similar, chromosomal rearrangements in response to persistent selection of growing cells parallels the genome rearrangements that characteristically accompany tumor progression.

Lyssaviruses are unsegmented RNA viruses causing rabies. Their vectors belong to the Carnivora and Chiroptera orders. We studied 36 carnivoran and 17 chiropteran lyssaviruses representing the main genotypes and variants. We compared their genes encoding the surface glycoprotein, which is responsible for receptor recognition and membrane fusion. The glycoprotein is the main protecting antigen and bears virulence determinants. Point mutation is the main force in lyssavirus evolution, as Sawyer's test and phylogenetic analysis showed no evidence of recombination. Tests of neutrality indicated a neutral model of evolution, also supported by globally high ratios of synonymous substitutions (d S ) to nonsynonymous substitutions (d N ) (>7). Relative-rate tests suggested similar rates of evolution for all lyssavirus lineages. Therefore, the absence of recombination and similar evolutionary rates make phylogeny-based conclusions reliable. Phylogenetic reconstruction strongly supported the hypothesis that host switching occurred in the history of lyssaviruses. Indeed, lyssaviruses evolved in chiropters long before the emergence of carnivoran rabies, very likely following spillovers from bats. Using dated isolates, the average rate of evolution was estimated to be roughly 4.3 ؋ 10 ؊4 d S /site/year. Consequently, the emergence of carnivoran rabies from chiropteran lyssaviruses was determined to have occurred 888 to 1,459 years ago. Glycoprotein segments accumulating more d N than d S were distinctly detected in carnivoran and chiropteran lyssaviruses. They may have contributed to the adaptation of the virus to the two distinct mammal orders. In carnivoran lyssaviruses they overlapped the main antigenic sites, II and III, whereas in chiropteran lyssaviruses they were located in regions of unknown functions.

The genetic diversity of representative members of the Lyssavirus genus (rabies and rabies-related viruses) was evaluated using the gene encoding the transmembrane glycoprotein involved in the virus-host interaction, immunogenicity, and pathogenicity. Phylogenetic analysis distinguished seven genotypes, which could be divided into two major phylogroups having the highest bootstrap values. Phylogroup I comprises the worldwide genotype 1 (classic Rabies virus), the European bat lyssavirus (EBL) genotypes 5 (EBL1) and 6 (EBL2), the African genotype 4 (Duvenhage virus), and the Australian bat lyssavirus genotype 7. Phylogroup II comprises the divergent African genotypes 2 (Lagos bat virus) and 3 (Mokola virus). We studied immunogenic and pathogenic properties to investigate the biological significance of this phylogenetic grouping. Viruses from phylogroup I (Rabies virus and EBL1) were found to be pathogenic for mice when injected by the intracerebral or the intramuscular route, whereas viruses from phylogroup II (Mokola and Lagos bat viruses) were only pathogenic by the intracerebral route. We showed that the glycoprotein R333 residue essential for virulence was naturally replaced by a D333 in the phylogroup II viruses, likely resulting in their attenuated pathogenicity. Moreover, cross-neutralization distinguished the same phylogroups. Within each phylogroup, the amino acid sequence of the glycoprotein ectodomain was at least 74% identical, and antiglycoprotein virus-neutralizing antibodies displayed cross-neutralization. Between phylogroups, the identity was less than 64.5% and the cross-neutralization was absent, explaining why the classical rabies vaccines (phylogroup I) cannot protect against lyssaviruses from phylogroup II. Our tree-axial analysis divided lyssaviruses into two phylogroups that more closely reflect their biological characteristics than previous serotypes and genotypes.

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Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.Members of the Lyssavirus genus are nonsegmented negative-strand RNA viruses belonging to the Mononegavirales order, Rhabdoviridae family. On the basis of phylogenetic studies, seven genotypes have been distinguished among which genotype 1 (rabies virus, PV strain) and genotype 3 (Mokola virus) are the most divergent (5, 44). These enveloped viruses are responsible for rabies encephalomyelitis. Usually transmitted mechanically by bite, injury, or aerosol, lyssaviruses are highly neurotropic, migrating from inoculation point to the central nervous system (CNS) through peripheral nerves. Their viral cycle takes place in the cytoplasm, where the viral genetic information exclusively found in the form of a ribonucleoprotein (RNP) complex serves as a template for two distinct RNA synthesis functions: transcription of a leader RNA and 5Ј capped and polyadenylated mRNAs encoding the different viral proteins (nucleoprotein [N], phosphoprotein [P], matrix protein [M], glycoprotein [G], and RNA polymerase [L]) and viral replication occurring in anti-genomic and new genomic RNA molecule synthesis. Transcription and replication are insured by the RNP complex composed of the L protein associated with the P protein and the genomic RNA tightly enwrapped by the N protein. The P protein via N:P complexes prevents nonspecific N protein aggregation while the L protein, considered the catalytic core, attaches to the N:RNA template through interactions with P. Thus, the P protein is considered to play a dual and pivotal role in this regulation. The P protein (297 amino acids [aa], PV strain [genotype 1]; 303 aa, Mokola virus [genotype 3]) is thought to be composed of two conserved domains: one is NH 2 terminal (the first 60 aa residue...

A key problem in evolutionary biology has been distinguishing the contributions of current and historical processes to the maintenance of genetic variation. Because alleles at self-recognition genes are under balancing selection, they exhibit extended residence times in populations and thus may provide unique insight into population demographic history. However, evidence for balancing selection and extended residence times has almost exclusively depended on identification of transspecific polymorphisms; polymorphisms retained in populations through speciation events. We present a broadly applicable approach for detecting balancing selection and apply it to the b1 mating type gene in the mushroom fungus Coprinus cinereus. The comparison of neutral molecular variation within and between allelic classes was used to directly estimate the strength of balancing selection. Different allelic classes are defined as encoding different mating compatibility types and are thus potentially subject to balancing selection. Variation within an allelic class, where all alleles have the same mating compatibility type, provided an internal standard of neutral evolution. Mating compatibility in this organism is determined by the complex A mating type locus, and b1 is one of several redundantly functioning genes. Consequently, we conducted numerical simulations of a model with two subloci and varying levels of recombination to show that balancing selection should operate at each sublocus. Empirical data show that strong balancing selection has indeed occurred at the b1 locus. The widespread geographic distribution of identical b1 alleles suggests that their association with differing A mating types is the result of recent recombination events.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

A molecular epidemiological study of the rabies virus currently prevalent in France was carried out by directly sequencing polymerase chain reaction-amplified genes. The rabies virus pseudogene ~' was chosen as the most divergent genomic area, and as such the best 'clock' for measuring virus evolution. Sequence comparisons between 12 wild rabies virus isolates indicated strong conservation whatever the host and wherever the virus had been isolated. This holds true for a unique wild reservoir, the fox. On the other hand, a good correlation between genetic and geographical criteria indicates a slow evolution of the wild virus in parallel with the spatio-temporal progression of the epizootic. In contrast to their intrinsic homogeneity (about 2% divergence), the wild isolate sequences showed a marked divergence from those of vaccine seed strains (about 14-7 %). This finding invites world-wide molecular epidemiological studies, particularly in countries in which vaccination failures have been reported.