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Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units.

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

Automation↗

Is self-fertilization an evolutionary dead end? Revisiting an old hypothesis with genetic theories and a macroevolutionary approach.

G. Ledyard Stebbins suggested that self-fertilization (selfing) may be an evolutionary dead end because it may result in the loss of genetic diversity and consequently preclude adaptation to changing environments. While the basic premise of selfing as a dead end is widely accepted, there have been few rigorous evaluations of the hypothesis. We examine the foundations of the dead-end hypothesis by considering theoretical advances in the study of mating-system evolution. We discuss theories predicting the irreversibility of self-fertilization and the extinction of selfing lineages through the loss of adaptive potential and genetic degradation. In the second portion of the review, focusing on the irreversibility of selfing, we summarize the contribution of phylogenetic studies of mating-system evolution to determine if evolutionary history supports this well-established hypothesis. Most studies are in accord with the hypothesis; no single study unequivocally demonstrates the transition from highly selfing to outcrossing lineages. Finally, we discuss the problems encountered when phylogenetic studies rely on reconstruction of ancestral mating systems. To avoid some of these problems, we applied likelihood ratio tests of irreversibility of mating-system evolution to several data sets and found that current data sets are probably too small for this test.

Journal Article↗

Genetic study of Coris julis (Osteichthyes, Perciformes, Labridae) evolutionary history and dispersal abilities.

Microsatellite markers have been used to study the genetic variability of rainbow wrasse (Coris julis) Mediterranean and Atlantic populations. Differentiation tests failed to reveal any significant genetic differentiation among samples from continental Portugal and the Azores, despite more than 1800 km of geographical separation. Preliminary results tended to indicate a significant genetic differentiation among Atlantic and Mediterranean samples. It also supported the specific status of Cape Verde populations (Coris atlantica). We compare these results with previous mtDNA analyses and propose a biogeographic scenario that could explain our results.

Animals↗

Methods for genome-wide analysis of gene expression changes in polyploids.

Polyploidy is an evolutionary innovation, providing extra sets of genetic material for phenotypic variation and adaptation. It is predicted that changes of gene expression by genetic and epigenetic mechanisms are responsible for novel variation in nascent and established polyploids (Liu and Wendel, 2002; Osborn et al., 2003; Pikaard, 2001). Studying gene expression changes in allopolyploids is more complicated than in autopolyploids, because allopolyploids contain more than two sets of genomes originating from divergent, but related, species. Here we describe two methods that are applicable to the genome-wide analysis of gene expression differences resulting from genome duplication in autopolyploids or interactions between homoeologous genomes in allopolyploids. First, we describe an amplified fragment length polymorphism (AFLP)--complementary DNA (cDNA) display method that allows the discrimination of homoeologous loci based on restriction polymorphisms between the progenitors. Second, we describe microarray analyses that can be used to compare gene expression differences between the allopolyploids and respective progenitors using appropriate experimental design and statistical analysis. We demonstrate the utility of these two complementary methods and discuss the pros and cons of using the methods to analyze gene expression changes in autopolyploids and allopolyploids. Furthermore, we describe these methods in general terms to be of wider applicability for comparative gene expression in a variety of evolutionary, genetic, biological, and physiological contexts.

Arabidopsis↗

Genetic factors accountable for line-specific DNA fingerprint bands in quail.

DNA fingerprints, prepared from mixes of DNA of individuals sampled from lines of Japanese quail selected for high or low 4-week body weight, were used to evaluate the relative contribution of several evolutionary forces to genetic diversity among populations. Comparisons between lines--two replicates of each selection direction and a control unselected line--were used to determine the frequency of line-specific DNA fingerprint bands produced by each of three major evolutionary forces: 1) mutation; 2) genetic drift; 3) selection. The latter force is expected to generate line-specific bands only if there is linkage disequilibrium between DNA fingerprint loci and quantitative loci (QTLs) controlling body weight. Using probes 33.6 and R18.1, an average of 48.4 DNA fingerprint bands in each line were analyzed. On average, 27.8 bands were found to be line-specific among the 96.8 (2 x 48.4) bands analyzed in an average comparison between pairs of lines. Based on the frequencies of line-specific bands in each particular comparison, it was calculated that 21% of the line-specific bands were due to mutation, 11% due to a single genetic drift event, 11% due to selection, 21% due to the combined effects of genetic drift and selection, 22% due to double independent events of genetic drift, and 14% due to undefined factors. Although evidence was found for a high frequency of genetic changes attributable to genetic drift, and a higher than expected frequency of linkage disequilibrium, the emphasis of this report is on the methodology suggested rather than on the particular results.

Animals↗

Lactone-ring-cleaving enzyme: genetic analysis, novel RNA editing, and evolutionary implications.

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1, 000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

Amino Acid Sequence↗

Life cycle patterns and their genetic control: an attempt to reconcile evolutionary and mechanistic speculation.

A model is proposed which implicates molecular recognition systems as the major controlling factors in life cycle expression. It is envisaged that such systems are important in immune functioning and catabolic, metabolic molecule recognition at both inter- and intra-cellular level. These recognition systems have the following characteristics: 1) Specific recognition molecules (recognisers), e.g. vertebrate antibodies, invertebrate agglutinins and plant agglutinins may recognise specific substances, e.g. antigens, catabolic and metabolic molecules. 2) The range of possible recognisable substances is very wide and variable. 3) The recognisers may themselves be recognised by other recognisers. 4) Recognisers are usually produced in large amounts only on presentation of the appropriate recognisable molecule. 5) The progressive introduction of new recognisable molecules increases the recogniser interaction, this interaction causing depression of some recogniser types (immune depression) and facilitation of other types among which may be recognisers specific for self components (e.g. auto-immunity). 6) Low juvenile viability is associated with a restricted range of available recognisers, high adult viability with increasing recogniser range and some auto-immunity/immune depression, senescence with a wide range of available recognisers and extensive auto-immunity/immune depression. Life cycle patterns and their control are discussed. It is suggested control mechanisms may include: 1) Dietary restriction and in some periods complete nutritional abstinence. 2) Specific recogniser depression, genes implicated here are the various antigens (species and polymorphic) found on cell surfaces, in the serum and in various body fluids of vertebrates, e.g. ABO, MNSs, P, Rh, Le and other blood groups, the ABO and Le secretor antigens and the HL-A antigens. In addition the immune response and mixed lymphocyte culture loci are implicated. Finally life cycle control is discussed with relation to sexual selection.

Aging↗

Reinforcement of genetic coherence: a single-locus model.

Genetic coherence and genetic separation are the outcome of evolutionary mechanisms which maintain genetic variation within populations through recombination on the one hand, and which divide this variation via speciation between reproductively (recombinatorically) more or less isolated populations on the other. While mechanisms of speciation have received considerable attention in biology, their counterpart, mechanisms of genetic coherence, are addressed only implicitly, if at all. Usually, genetic coherence is intuitively associated with the forces maintaining genetic polymorphisms and thus potential for flexible adaptational reaction of populations. However, so far no models seem to exist which explain the evolution of genetic coherence as the natural counterpart of genetic separation or speciation. In this paper a single-locus model is analyzed, in which a mutant allele is introduced into a resident stable diallelic polymorphism, and where this allele is equivalent to one of the resident alleles in all respects with the exception of mating relations. The conditions for replacement of the resident allele by its selectively equivalent mutant are obtained with reference to the associated mating relations. It turned out that for heterozygote advantage the mutant replaces the selectively equivalent resident allele if it increases the mating preferences for carriers of other alleles. The evolution of lower such preferences requires heterozygote inferiority, which confirms the Wallace effect of speciation (by reinforcement). It is argued that this observation suggests that non-selective constituents of the mating system form the section of the genetic system that is responsible for moderating the genetic load implied by adapting selection while simultaneously securing the adaptational potential embodied in the resident allelic variation. Mating systems thus serve the preservation of adaptability.

Adaptation, Physiological↗

Amount and structure of intra- and interspecific genetic variation in the moss genus Polytrichum.

Allozyme electrophoresis was used to determine amount and structure of genetic variation within and between five congeneric haploid moss species: Polytrichum formosum, P. commune, P. uliginosum, P. piliferum and P. juniperinum. For the different species, gene diversity within populations (HS) ranged from very low (0.025) to moderate values (0.127), being, on average, lower than those observed for vascular plants and many other moss species. Polytrichum piliferum and P. juniperinum showed significantly higher levels of HS than the other species examined, which possibly might be explained by sexual reproduction being more prevalent in these two species, that often live in more dynamic habitats, where turnover of individuals is more frequent. Genetic variability was observed to be structured in contrasting ways at different levels. At the intraspecific level genetic differentiation among populations of most Polytrichum species was low, FST </= 0.1, indicating a considerable degree of gene flow by wind-dispersed spores over substantial distances. At the interspecific level strong divergence, genetic identities being on average I=0.222, was observed between most species studied, despite low levels of divergence at the morphological level. These I-values are significantly lower than observed for congeneric vascular plant species and most congeneric bryophyte species. This suggests that the morphological conservatism is not due to lack of genetic variability and evolutionary potential.

Genetic Variation↗

Localization and characterization of X chromosome inversion breakpoints separating Drosophila mojavensis and Drosophila arizonae.

Ectopic exchange between transposable elements or other repetitive sequences along a chromosome can produce chromosomal inversions. As a result, genome sequence studies typically find sequence similarity between corresponding inversion breakpoint regions. Here, we identify and investigate the breakpoint regions of the X chromosome inversion distinguishing Drosophila mojavensis and Drosophila arizonae. We localize one inversion breakpoint to 13.7 kb and localize the other to a 1-Mb interval. Using this localization and assuming microsynteny between Drosophila melanogaster and D. arizonae, we pinpoint likely positions of the inversion breakpoints to windows of less than 3000 bp. These breakpoints define the size of the inversion to approximately 11 Mb. However, in contrast to many other studies, we fail to find significant sequence similarity between the 2 breakpoint regions. The localization of these inversion breakpoints will facilitate future genetic and molecular evolutionary studies in this species group, an emerging model system for ecological genetics.

Animals↗

Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Base Sequence↗

Genetic organization of the mecA region in methicillin-susceptible and methicillin-resistant strains of Staphylococcus sciuri.

A homolog of the Staphylococcus aureus methicillin resistance gene mecA was recently shown to be ubiquitous in independent isolates of the animal species Staphylococcus sciuri. The mecA gene homolog and regions flanking it were cloned and sequenced from four strains of S. sciuri: strain K1 (ATCC 29062), a representative of S. sciuri subsp. sciuri; two strains (K3 and K8) representing S. sciuri subsp. rodentius; and strain K11, a representative of S. sciuri subsp. carnaticum. Strains K1 and K11 were susceptible to methicillin, while strains K3 and K8 showed heterogeneous resistance. The mecA genes of strains K1 and K11 and one of the two copies of mecA (mecA1) present in strain K3 had virtually identical DNA sequences in the mecA gene and were similar in genetic organization in the flanking regions. In contrast, the single copy of mecA in strain K8 and the second copy of mecA (mecA2) in strain K3 had mecA DNA sequences identical to that of S. aureus mecA, and the mecA region in these two strains was also similar to that of the same region in the S. aureus strain used for comparison. Interestingly, an open reading frame defining an N-terminal truncated polypeptide, NTORF101, with a high degree of homology to a DNA segment in the hypervariable region of methicillin-resistant S. aureus (and also similar to the Escherichia coli gene ugpQ) was also identified downstream of the mecA homolog of strain K11, representing S. sciuri subsp. carnaticum. The ugpQ-like gene is not present in methicillin-susceptible strains of S. aureus. The presence of such a ugpQ-like gene together with the homolog of mecA in strain K11 supports the speculation that these genetic elements may be evolutionary relatives and/or precursors of the genetic determinant of methicillin resistance in S. aureus.

Amino Acid Sequence↗

Nutrition and ontogenetic development of the intestine.

Fetal, suckling, and postweaning vertebrates have distinct dietary inputs that impose different functional demands on the developing intestine. Differences between species and life-history stages in intestinal structure and functions are set by genetic determinants that reflect evolutionary diets. Dietary inputs interacting with genetic determinants influence intestinal functions by triggering the production of new enterocyte populations and (or) by reprogramming existing enterocytes. In addition to nutrients, nonnutritive components of amniotic fluid and milk, such as growth factors and hormones, are important mediators of intestinal development and in humans can exert influences at as early as 10 weeks of gestation, when fetuses begin swallowing amniotic fluid. Changes in diet composition during suckling elicit limited and apparently nonspecific responses in intestinal structure and activities of brush-border hydrolases and transporters. The ability to adaptively modulate intestinal brush-border functions develops at weaning, when diet composition begins to vary unpredictably. Hydrolytic and transport capacities of the developing intestine are matched to age-related increases in dietary inputs, are not in great excess, and may be growth limiting. Although not as extensively studied, postnatal diet also influences development of intestinal endocrine and immune functions and has a complex, poorly understood interaction with the developing microflora.

Animal Nutritional Physiological Phenomena↗

Stochastic information processing biological systems.

We propose a simple, biochemically-based model for stochastic information processing in brain, genetic, and, consequently, evolutionary modelling. The essential features of reaction-diffusion processes are realized by intrinsically stochastic probabilistic automata (Shannon and Weaver, 1948; see also Ashby, 1958, von Neumann, 1966; Burks, 1970; Paz, 1971) whose definition extends that of classical automata. (Classical automata are deterministic; earlier work on probabilistic automata focused on error correction and at least approximating deterministic behavior.) We call these probabilistic automata biochemical to emphasize the role of intrinsically stochastic process in biological information processing. Our model yields descriptions of gradualism (Conrad, 1974), learning, and apparent inefficiencies in the brain, and partially resolves the near impossibility of simultaneous point mutations (Conrad, 1972, 1978) in genetics. The genetic model implies an evolutionary dynamics of punctuated equilibria (Gould and Eldredge, 1977).

Animals↗

Genetic Tools in the Nakaseomyces clade for Evolutionary Comparisons of Signal Transduction Pathways.

The genus Nakaseomyces provides four species that are closely related but have different characteristics. For example, N. glabratus (formerly known as Candida glabrata) is a common human pathogen, whereas N. bracarensis and N. nivariensis have been isolated in clinical settings but are not common human pathogens. N. delphensis was isolated from fruit and there is no evidence it is pathogenic. Given the differences, we developed the clade as a molecular genetic system where we could introduce plasmids and assess transcriptional output from cloned promoters. We engineered a CRISPR/Cas9 plasmid that allows for rapid Gibson cloning of gRNAs, generated auxotrophic strains for amino acids and nucleotides, and introduced plasmids into each species. We used promoter-YFP plasmids to determine that while there are differences between the species, each species likely has intact thiamine and phosphate (THI and PHO) signal transduction pathways, and that gene expression in N. glabratus and N. bracarensis is more similar to one another than to the other two species. Finally, we determine that N. glabratus, N. bracarensis, and N. nivariensis persist in a murine macrophage for 24&#x2009;h, whereas N. delphensis does not. This work describes new molecular tools for genetic manipulation in the Nakaseomyces clade and allows for evolutionary questions to be explored.

Signal Transduction↗