Dynamics of transcriptome evolution in the model eukaryote Neurospora.

Mounting evidence indicates that changes in the transcriptome contribute significantly to the phenotypic differentiation of closely related species. Nonetheless, further genome-wide studies, spanning a broad range of organisms, are needed to decipher the factors driving transcriptome evolution. The model Neurospora (Ascomycota) comprises a simple system for empirically studying the evolutionary dynamics of the transcriptome. Here, we studied the evolution of gene expression in Neurospora crassa and Neurospora tetrasperma and show that patterns of transcriptome evolution are connected to genome evolution, tissue type and sexual identity (mating types, mat A and mat a) in these eukaryotes. Based on the comparisons of inter- and intraspecies expression divergence, our data reveal that rapid expression divergence is more apt to occur in sexual/female (SF) than vegetative/male (VM) tissues. In addition, interspecies gene expression and protein sequence divergence were strongly correlated for SF, but not VM, tissue. A correlation between transcriptome and protein evolution parallels findings from certain animals, but not yeast, and add support for the theory that expression evolution differs fundamentally among multicellular and unicellular eukaryotes. Finally, we found that sexual identity in these hermaphroditic Neurospora species is connected to interspecies expression divergence in a tissue-dependent manner: rapid divergence occurred for mat A- and mat a-biased genes from SF and VM tissues, respectively. Based on these findings, it is hypothesized that rapid interspecies transcriptome evolution is shifting the mating types of Neurospora towards distinct female and male phenotypes, that is, sexual dimorphism.

Genome-wide selection on codon usage at the population level in the fungal model organism Neurospora crassa.

Many organisms exhibit biased codon usage in their genome, including the fungal model organism Neurospora crassa. The preferential use of subset of synonymous codons (optimal codons) at the macroevolutionary level is believed to result from a history of selection to promote translational efficiency. At present, few data are available about selection on optimal codons at the microevolutionary scale, that is, at the population level. Herein, we conducted a large-scale assessment of codon mutations at biallelic sites, spanning more than 5,100 genes, in 2 distinct populations of N. crassa: the Caribbean and Louisiana populations. Based on analysis of the frequency spectra of synonymous codon mutations at biallelic sites, we found that derived (nonancestral) optimal codon mutations segregate at a higher frequency than derived nonoptimal codon mutations in each population; this is consistent with natural selection favoring optimal codons. We also report that optimal codon variants were less frequent in longer genes and that the fixation of optimal codons was reduced in rapidly evolving long genes/proteins, trends suggestive of genetic hitchhiking (Hill-Robertson) altering codon usage variation. Notably, nonsynonymous codon mutations segregated at a lower frequency than synonymous nonoptimal codon mutations (which impair translational efficiency) in each N. crassa population, suggesting that changes in protein composition are more detrimental to fitness than mutations altering translation. Overall, the present data demonstrate that selection, and partly genetic interference, shapes codon variation across the genome in N. crassa populations.

Membranous expression of podocalyxin-like protein is an independent factor of poor prognosis in urothelial bladder cancer.

BACKGROUND: Membranous expression of the anti-adhesive glycoprotein podocalyxin-like (PODXL) has previously been found to correlate with poor prognosis in several major cancer forms. Here we examined the prognostic impact of PODXL expression in urothelial bladder cancer. METHODS: Immunohistochemical PODXL expression was examined in tissue microarrays with tumours from two independent cohorts of patients with urothelial bladder cancer: n=100 (Cohort I) and n=343 (Cohort II). The impact of PODXL expression on disease-specific survival (DSS; Cohort II), 5-year overall survival (OS; both cohorts) and 2-year progression-free survival (PFS; Cohort II) was assessed. RESULTS: Membranous PODXL expression was significantly associated with more advanced tumour (T) stage and high-grade tumours in both cohorts, and a significantly reduced 5-year OS (unadjusted HR=2.25 in Cohort I and 3.10 in Cohort II, adjusted HR=2.05 in Cohort I and 2.18 in Cohort II) and DSS (unadjusted HR=4.36, adjusted HR=2.70). In patients with Ta and T1 tumours, membranous PODXL expression was an independent predictor of a reduced 2-year PFS (unadjusted HR=6.19, adjusted HR=4.60) and DSS (unadjusted HR=8.34, adjusted HR=7.16). CONCLUSION: Membranous PODXL expression is an independent risk factor for progressive disease and death in patients with urothelial bladder cancer.

Degeneration in codon usage within the region of suppressed recombination in the mating-type chromosomes of Neurospora tetrasperma.

The origin and early evolution of sex chromosomes are currently poorly understood. The Neurospora tetrasperma mating-type (mat) chromosomes have recently emerged as a model system for the study of early sex chromosome evolution, since they contain a young (<6 million years ago [Mya]), large (>6.6-Mb) region of suppressed recombination. Here we examined preferred-codon usage in 290 genes (121,831 codon positions) in order to test for early signs of genomic degeneration in N. tetrasperma mat chromosomes. We report several key findings about codon usage in the region of recombination suppression, including the following: (i) this region has been subjected to marked and largely independent degeneration among gene alleles; (ii) the level of degeneration is magnified over longer periods of recombination suppression; and (iii) both mat a and mat A chromosomes have been subjected to deterioration. The frequency of shifts from preferred codons to nonpreferred codons is greater for shorter genes than for longer genes, suggesting that short genes play an especially significant role in early sex chromosome evolution. Furthermore, we show that these degenerative changes in codon usage are best explained by altered selection efficiency in the recombinationally suppressed region. These findings demonstrate that the fungus N. tetrasperma provides an effective system for the study of degenerative genomic changes in young regions of recombination suppression in sex-regulating chromosomes.

Consequences of reproductive mode on genome evolution in fungi.

An organism's reproductive mode is believed to be a major factor driving its genome evolution. In theory, sexual inbreeding and asexuality are associated with lower effective recombination levels and smaller effective population sizes than sexual outbreeding, giving rise to reduced selection efficiency and genetic hitchhiking. This, in turn, is predicted to result in the accumulation of deleterious mutations and other genomic changes, for example the accumulation of repetitive elements. Empirical data from plants and animals supporting/refuting these theories are sparse and have yielded few conclusive results. A growing body of data from the fungal kingdom, wherein reproductive behavior varies extensively within and among taxonomic groups, has provided new insights into the role of mating systems (e.g., homothallism, heterothallism, pseudohomothallism) and asexuality, on genome evolution. Herein, we briefly review the theoretical relationships between reproductive mode and genome evolution and give examples of empirical data on the topic derived to date from plants and animals. We subsequently focus on the available data from fungi, which suggest that reproductive mode alters the rates and patterns of genome evolution in these organisms, e.g., protein evolution, mutation rate, codon usage, frequency of genome rearrangements and repetitive elements, and variation in chromosome size.

Evidence of the accumulation of allele-specific non-synonymous substitutions in the young region of recombination suppression within the mating-type chromosomes of Neurospora tetrasperma.

Currently, little is known about the origin and early evolution of sex chromosomes. This is largely due to the fact that ancient non-recombining sex chromosomes are highly degenerated, and thus provide little information about the early genomic events in their evolution. The Neurospora tetrasperma mating-type (mat) chromosomes contain a young (<6 Mya) and large region (>6.6 Mb) of suppressed recombination, thereby providing a model system to study early stages of sex chromosome evolution. Here, we examined alleles of 207 genes located on the N. tetrasperma mat a and mat A chromosomes to test for signs of genomic alterations at the protein level in the young region of recombination suppression. We report that the N. tetrasperma mat a and mat A chromosomes have each independently accumulated allele-specific non-synonymous codon substitutions in a time-dependent, and gene-specific manner in the recombinationally suppressed region. In addition, examination of the ratio (ω) of non-synonymous substitutions (dN) to synonymous substitutions (dS) using maximum likelihood analyses, indicates that such changes are associated with relaxed purifying selection, a finding consistent with genomic degeneration. We also reveal that sex specific biases in mutation rates or selection pressures are not necessary for genomic alterations in sex chromosomes, and that recombination suppression in itself is sufficient to explain these results. The present findings extend our current understanding of genomic events associated within the young region of recombination suppression in these fungal sex-regulating chromosomes.

Phylogenetic and biological species diversity within the Neurospora tetrasperma complex.

The objective of this study was to explore the evolutionary history of the morphologically recognized filamentous ascomycete Neurospora tetrasperma, and to reveal the genetic and reproductive relationships among its individuals and populations. We applied both phylogenetic and biological species recognition to a collection of strains representing the geographic and genetic diversity of N. tetrasperma. First, we were able to confirm a monophyletic origin of N. tetrasperma. Furthermore, we found nine phylogenetic species within the morphospecies. When using the traditional broad biological species recognition all investigated strains of N. tetrasperma constituted a single biological species. In contrast, when using a quantitative measurement of the reproductive success, incorporating characters such as viability and fertility of offspring, we found a high congruence between the phylogenetic and biological species recognition. Taken together, phylogenetically and biologically defined groups of individuals exist in N. tetrasperma, and these should be taken into account in future studies of its life history traits.

Dimethyl sulfoxide binding to globular proteins: a nuclear magnetic relaxation dispersion study.

The 2H magnetic relaxation dispersion (NMRD) technique was used to characterize interactions of dimethyl sulfoxide (DMSO) with globular proteins. A difference NMRD experiment involving the N-acetylglucosamine trisaccharide inhibitor, demonstrated that the DMSO 2H NMRD profile in lysozyme solution is due to a single DMSO molecule bound in the active cleft, with a molecular order parameter of 0.47 +/- 0.05 and a residence time in the range 10 ns to 5 ms. With the aid of transverse 2H relaxation data, the upper bound of the residence time was further reduced to 100 microns. A 1H shift titration experiment was also performed, yielding a binding constant of 2.3 +/- 0.3 M-1 at 27 degrees C. In contrast to lysozyme, no DMSO dispersion was observed for bovine pancreatic trypsin inhibitor (BPTI), indicating that a stable DMSO-protein complex requires a cleft of appropriate geometry in addition to hydrogen-bond and hydrophobic interactions.

The LAMB1 gene from the clubmoss, Lycopodium annotinum, is a divergent MADS-box gene, expressed specifically in sporogenic structures.

Transcription factors encoded by the large MADS-box gene family have important developmental functions in angiosperms, the flowering plants. Mutations in certain MADS-box genes are known to cause homeotic alterations in floral organ identity, and the establishment of floral organ identity is the most well-studied developmental process in which MADS-box genes are known to function. Our interest is in the potential connection between the duplication history of this gene family and the evolutionary origin of the structures that the different MADS-box genes developmentally regulate in plants. Previous studies have demonstrated that the origin of the MADS-box genes that control floral organ identity predate the evolutionary origin of the flower itself, since gymnosperms have genes that are orthologous to angiosperm floral homeotic MADS-box genes, whereas ferns appear to lack such genes. Here we report on the isolation of a MADS-box gene from Lycopodium annotinum, which belongs to the clubmosses, the phylogenetic sister group to other vascular plants. The gene, LAMB1, in the sporophyte is expressed exclusively in the reproductive structure, the strobilus, during sporogenesis. LAMB1 is similar to other plant MADS-box genes in that it contains a MADS-box as well as a second conserved element, a K-box. However, it differs in length and in exon/intron structure in the region between the MADS- and K-box, and also in the length and structure of the C-terminal region. A phylogenetic analysis indicates that LAMB1 is not closely related to other plant-type MADS-box genes, and may represent one of the basal branches in the phylogenetic tree of plant MADS-box genes.

Model-free analysis of stretched relaxation dispersions.

Nuclear magnetic relaxation dispersion (NMRD) measurements can provide valuable information about the dynamics and structure of macromolecular solutions and other complex fluids. A large number of 1H NMRD studies of water in concentrated protein solutions and in semisolid biological samples have been reported. The observed dispersion usually extends over a wide frequency range and then cannot be described by a Lorentzian spectral density function. We propose here a model-free approach for analyzing such stretched dispersion profiles. Unlike the traditional empirical fitting procedures, the model-free approach is based on rigorous theory and produces parameters with well-defined physical significance. The model-free approach is validated with the aid of synthetic relaxation data, showing that it is robust and accurate, and is then applied to new water 1H NMRD data from solutions of the protein bovine pancreatic trypsin inhibitor (BPTI). By separating the static and dynamic information content of the relaxation dispersion, the model-free analysis shows that the dramatic salt effect observed in BPTI solutions is due almost entirely to a slowing down of protein rotation with little change of protein structure. An analysis of the same data in terms of the empirical dispersion function used in most 1H NMRD studies leads to a qualitatively different picture. We demonstrate that this widely used dispersion function is unphysical and that its parameters do not have the physical meaning usually ascribed to them.

Evolution of synonymous codon usage in Neurospora tetrasperma and Neurospora discreta.

Neurospora comprises a primary model system for the study of fungal genetics and biology. In spite of this, little is known about genome evolution in Neurospora. For example, the evolution of synonymous codon usage is largely unknown in this genus. In the present investigation, we conducted a comprehensive analysis of synonymous codon usage and its relationship to gene expression and gene length (GL) in Neurospora tetrasperma and Neurospora discreta. For our analysis, we examined codon usage among 2,079 genes per organism and assessed gene expression using large-scale expressed sequenced tag (EST) data sets (279,323 and 453,559 ESTs for N. tetrasperma and N. discreta, respectively). Data on relative synonymous codon usage revealed 24 codons (and two putative codons) that are more frequently used in genes with high than with low expression and thus were defined as optimal codons. Although codon-usage bias was highly correlated with gene expression, it was independent of selectively neutral base composition (introns); thus demonstrating that translational selection drives synonymous codon usage in these genomes. We also report that GL (coding sequences [CDS]) was inversely associated with optimal codon usage at each gene expression level, with highly expressed short genes having the greatest frequency of optimal codons. Optimal codon frequency was moderately higher in N. tetrasperma than in N. discreta, which might be due to variation in selective pressures and/or mating systems.

Phase-specific gene expression underlying morphological adaptations of the dimorphic human pathogenic fungus, Coccidioides posadasii.

Coccidioides posadasii is a dimorphic fungal pathogen that grows as a filamentous saprobe in the soil and as endosporulating spherules within the host. To identify genes specific to the pathogenic phase of Co. posadasii, we carried out a large-scale study of gene expression in two isolates of the species. From the sequenced Co. posadasii genome, we chose 1,000 open reading frames to construct a 70-mer microarray. RNA was recovered from both isolates at three life-cycle phases: hyphae, presegmented spherules, and spherules releasing endospores. Comparative hybridizations were conducted in a circuit design, permitting comparison between both isolates at all three life-cycle phases, and among all life-cycle phases for each isolate. By using this approach, we identified 92 genes that were differentially expressed between pathogenic and saprobic phases in both fungal isolates, and 43 genes with consistent differential expression between the two parasitic developmental phases. Genes with elevated expression in the pathogenic phases of both isolates included a number of genes that were involved in the response to environmental stress as well as in the metabolism of lipids. The latter observation is in agreement with previous studies demonstrating that spherules contain a higher proportion of lipids than saprobic phase tissue. Intriguingly, we discovered statistically significant and divergent levels of gene expression between the two isolates profiled for 64 genes. The results suggest that incorporating more than one isolate in the experimental design offers a means of categorizing the large collection of candidate genes that transcriptional profiling typically identifies into those that are strain-specific and those that characterize the entire species.

DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro.

Homeodomain-leucine zipper (HDZip) proteins constitute a large family of transcription factors apparently unique to plants. In this report we characterize the DNA-binding and dimerization preferences in vitro of class I HDZip proteins. Using gel-exclusion chromatography and in vitro protein binding assays we demonstrate that the HDZip class I protein ATHB5 forms a homodimeric complex in solution. Consistent with this finding we have demonstrated the sequence-specific interaction of ATHB5 with a 9 bp pseudopalindromic DNA sequence, CAATNATTG, composed of two half-sites overlapping at a central position, by use of a PCR-assisted binding-site selection assay and competitive EMSA experiments. A majority of other known members of HDZip class I interacted with similar DNA sequences, but differed in their preference for A/T versus G/C in the central position of the binding site. Selective heterodimerization in vitro was demonstrated between ATHB5 and different class I HDZip proteins. Heterodimer formation between class I HDZip proteins is of potential functional significance for the integration of information from different signalling pathways in the control of plant development.

Sugar-dependent alterations in cotyledon and leaf development in transgenic plants expressing the HDZhdip gene ATHB13.

ATHB13 is a new member of the homeodomain leucine zipper (HDZip) transcription factor family of Arabidopsis thaliana. Constitutive high-level expression of the ATHB13 cDNA in transgenic plants results in altered development of cotyledons and leaves, specifically in plants grown on media containing metabolizable sugars. Cotyledons and leaves of sugar-grown transgenic plants are more narrow and the junction between the petiole and the leaf blade less distinct, as compared to the wild type. High-level expression of ATHB13 affects cotyledon shape by inhibiting lateral expansion of epidermal cells in sugar-treated seedlings. Experiments with non-metabolizable sugars indicate that the alteration in leaf shape in the ATHB13 transgenics is mediated by sucrose sensing. ATHB13 further affects a subset of the gene expression responses of the wild-type plant to sugars. The expression of genes encoding beta-amylase and vegetative storage protein is induced to higher levels in response to sucrose in the transgenic plants as compared to the wild type. The expression of other sugar-regulated genes examined is unaffected by ATHB13. These data suggest that ATHB13 may be a component of the sucrose-signalling pathway, active close to the targets of the signal transduction.