Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

Detection and validation of single gene inversions.

MOTIVATION: The biologically meaningful algorithmic study of genome rearrangement should take into account the distribution of sizes of the rearranged genomic fragments. In particular, it is important to know the prevalence of short inversions in order to understand the patterns of gene order disruption observed in comparative genomics. RESULTS: We find a large excess of short inversions, especially those involving a single gene, in comparison with a random inversion model. This is demonstrated through comparison of four pairs of bacterial genomes, using a specially-designed implementation of the Hannenhalli-Pevzner theory, and validated through experimentation on pairs of random genomes matched to the real pairs.

Replication orientation affects the rate and direction of bacterial gene evolution.

In many bacterial genomes, the leading and lagging strands have different skews in base composition; for example, an excess of guanosine compared to cytosine on the leading strand. We find that Chlamydia genes that have switched their orientation relative to the direction of replication, for example by inversion, acquire the skew of their new "host" strand. In contrast to most evolutionary processes, which have unpredictable effects on the sequence of a gene, replication-related skews reflect a directional evolutionary force that causes predictable changes in the base composition of switched genes, resulting in increased DNA and amino acid sequence divergence.

Genome rearrangement by replication-directed translocation.

Gene order in bacteria is poorly conserved during evolution. For example, although many homologous genes are shared by the proteobacteria Escherichia coli, Haemophilus influenzae and Helicobacter pylori, their relative positions are very different in each genome, except local functional clusters such as operons. The complete sequences of the more closely related bacterial genomes, such as pairs of Chlamydia, H. pylori and Mycobacterium species, now allow identification of the processes and mechanisms involved in genome evolution. Here we provide evidence that a substantial proportion of rearrangements in gene order results from recombination sites that are determined by the positions of the replication forks. Our observations suggest that replication has a major role in directing genome evolution.

The contributions of replication orientation, gene direction, and signal sequences to base-composition asymmetries in bacterial genomes.

Asymmetries in base composition between the leading and the lagging strands have been observed previously in many prokaryotic genomes. Since a majority of genes is encoded on the leading strand in these genomes, previous analyses have not been able to determine the relative contribution to the base composition skews of replication processes and transcriptional and/or translational forces. Using qualitative graphical presentations and quantitative statistical analyses (analysis of variance), we have found that a significant proportion of the GC and AT skews can be attributed to replication orientation, i.e., the sequence of a gene is influenced by whether it is encoded on the leading or lagging strand. This effect of replication orientation on skews is independent of, and can be opposite in sign to, the effects of transcriptional or translational processes, such as selection for codon usage, amino acid preferences, expression levels (inferred from codon adaptation index), or potential short signal sequences (e.g., chi sequences). Mutational differences between the leading and the lagging strands are the most likely explanation for a significant proportion of the base composition skew in these bacterial genomes. The finding that base composition skews due to replication orientation are independent of those due to selection for function of the encoded protein may complicate the interpretation of phylogenetic relationships, conserved positions in nucleotide or amino acid sequence alignments, and codon usage patterns.

High apparent rate of simultaneous compensatory base-pair substitutions in ribosomal RNA.

We present a model for the evolution of paired bases in RNA sequences. The new model allows for the instantaneous rate of substitution of both members of a base pair in a compensatory substitution (e.g., A-U-->G-C) and expands our previous work by allowing for unpaired bases or noncanonical pairs. We implemented the model with distance and maximum likelihood methods to estimate the rates of simultaneous substitution of both bases, alphad, vs. rates of substitution of individual bases, alphas in rRNA. In the rapidly evolving D2 expansion segments of Drosophila large subunit rRNA, we estimate a low ratio of alphad/alphas, indicating that most compensatory substitutions involve a G-U intermediate. In contrast, we find a surprisingly high ratio of alphad/alphas in the core small subunit rRNA, indicating that the evolution of the slowly evolving rRNA sequences is modeled much more accurately if simultaneous substitution of both members of a base pair is allowed to occur approximately as often as substitution of individual bases. Using simulations, we have ruled out several potential sources of error in the estimation of alphad/alphas. We conclude that in the core rRNA sequences compensatory substitutions can be fixed so rapidly as to appear to be instantaneous.

Evolution of mobile group I introns: recognition of intron sequences by an intron-encoded endonuclease.

Mobile group I introns are hypothesized to have arisen after invasion by endonuclease-encoding open reading frames (ORFs), which mediate their mobility. Consistent with an endonuclease-ORF invasion event, we report similarity between exon junction sequences (the recognition site for the mobility endonuclease) and intron sequences flanking the endonuclease ORF in the sunY gene of phage T4. Furthermore, we have demonstrated the ability of the intron-encoded endonuclease to recognize and cleave these intron sequences when present in fused form in synthetic constructs. These observations and accompanying splicing data are consistent with models in which the invading endonuclease ORF is provided safe haven within a splicing element. In turn the intron is afforded immunity to the endonuclease product, which imparts mobility to the intron.

Nucleotide sequence requirements for self-cleavage of Neurospora VS RNA.

We have used several complementary approaches to investigate the minimal contiguous sequence required for the in vitro self cleavage reaction performed by Neurospora VS RNA. Deletion analysis and site-directed mutagenesis revealed that only a single nucleotide is required upstream of the self-cleavage site, and that the identity of this nucleotide is not critical. This distinguishes VS RNA from all currently known ribozymes except hepatitis delta virus RNA. The shortest contiguous sequence capable of cleavage contains 153 nt downstream of the cleavage site. Linker insertion mutagenesis suggests that much of this downstream sequence is important for self-cleavage. Comparative sequence analysis of the VS plasmid from six natural isolates supports the importance in vivo of the minimal region determined by in vitro methods. Also, phylogenetic analysis raises the possibility of a recent horizontal transfer of the VS plasmid from Neurospora intermedia to Neurospora sitophila.

A sampling theory of selectively neutral alleles in a subdivided population.

Ewens' sampling distribution is investigated for a structured population. Samples are assumed to be taken from a single subpopulation that exchanges migrants with other subpopulations. A complete description of the probability distribution for such samples is not a practical possibility but an equilibrium approximation can be found. This approximation extracts the information necessary for constructing a continuous approximation to the complete distribution using known values of the distribution and its derivatives in randomly mating populations. It is shown that this approximation is as complete a description of a single biologically realistic subpopulation as is possible given standard uncertainties about the actual size of the migration rates, relative sizes of each of the subpopulations and other factors that might affect the genetic structure of a subpopulation. Any further information must be gained at the expense of generality. This approximation is used to investigate the effect of population subdivision on Watterson's test of neutrality. It is known that the infinite allele, sample distribution is independent of mutation rate when made conditional on the number of alleles in the sample. It is shown that the conditional, infinite allele, sample distribution from this approximation is also independent of population structure and hence Watterson's test is still approximately valid for subdivided populations.