ALBERT

Description: CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa , we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus , evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and loss rather than recombination. Since the evolution of spacer content is characterized by a rapid turnover, it is likely that recombination is not beneficial for improving phage resistance in the strains under study, or that it cannot be detected in the resolution of intraspecies comparisons.

Description: Background The CRISPR/Cas system is known to act as an adaptive and heritable immune system in Eubacteria and Archaea. Immunity is encoded in an array of spacer sequences. Each spacer can provide specific immunity to invasive elements that carry the same or a similar sequence. Even in closely related strains, spacer content is very dynamic and evolves quickly. Standard models of nucleotide evolutioncannot be applied to quantify its rate of change since processes other than single nucleotide changes determine its evolution.Methods We present probabilistic models that are specific for spacer content evolution. They account for the different processes of insertion and deletion. Insertions can be constrained to occur on one end only or are allowed to occur throughout the array. One deletion event can affect one spacer or a whole fragment of adjacent spacers. Parameters of the underlying models are estimated for a pair of arrays by maximum likelihood using explicit ancestor enumeration.Results Simulations show that parameters are well estimated on average under the models presented here. There is a bias in the rate estimation when including fragment deletions. The models also estimate times between pairs of strains. But with increasing time, spacer overlap goes to zero, and thus there is an upper bound on the distance that can be estimated. Spacer content similarities are displayed in a distance based phylogeny using the estimated times.We use the presented models to analyze different Yersinia pestis data sets and find that the results among them are largely congruent. The models also capture the variation in diversity of spacers among the data sets. A comparison of spacer-based phylogenies and Cas gene phylogenies shows that they resolve very different time scales for this data set.Conclusions The simulations and data analyses show that the presented models are useful for quantifying spacer content evolution and for displaying spacer content similarities of closely related strains in a phylogeny. This allows for comparisons of different CRISPR arrays or for comparisons between CRISPR arrays and nucleotide substitution rates.

Description: The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data—a common practice in phylogenomic analyses—introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.

Description: Background: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts. Results: We generated a HGMEC SAGE library consisting of 68,987 transcript tags. Then taking advantage of large public databases and advanced bioinformatics we compared the HGMEC SAGE library with a SAGE library of non-cultured ex vivo human glomeruli (44,334 tags) which contained endothelial cells. The 823 tags common to both which would have the potential to be expressed in vivo were subsequently checked against 822,008 tags from 16 non-glomerular endothelial SAGE libraries. This resulted in 268 transcript tags differentially overexpressed in HGMEC compared to non-glomerular endothelia. These tags were filtered using a set of criteria: never before shown in kidney or any type of endothelial cell, absent in all nephron regions except the glomerulus, more highly expressed than statistically expected in HGMEC. Neurogranin, a direct target of thyroid hormone action which had been thought to be brain specific and never shown in endothelial cells before, fulfilled these criteria. Its expression in glomerular endothelium in vitro and in vivo was then verified by real-time-PCR, sequencing and immunohistochemistry. Conclusions: Our results represent an extensive molecular characterization of HGMEC beyond a mere database, underline the endothelial heterogeneity, and propose neurogranin as a potential link in the kidney-thyroid axis.

Description: Background: CRISPR is a microbial immune system likely to be involved in host-parasite coevolution. Itfunctions using target sequences encoded by the bacterial genome, which interfere with invadingnucleic acids using a homology-dependent system. The system also requires protospacer associatedmotifs (PAMs), short motifs close to the target sequence that are required for interference in CRISPRtypes I and II. Here, we investigate whether PAMs are depleted in phage genomes due to selectionpressure to escape recognition. Results: To this end, we analyzed two data sets. Phages infecting all bacterial hosts were analyzed first,followed by a detailed analysis of phages infecting the genus Streptococcus, where PAMs are bestunderstood. We use two different measures of motif underrepresentation that control for codon biasand the frequency of submotifs. We compare phages infecting species with a particular CRISPR typeto those infecting species without that type. Since only known PAMs were investigated, the analysisis restricted to CRISPR types I-C and I-E and in Streptococcus to types I-C and II. We foundevidence for PAM depletion in Streptococcus phages infecting hosts with CRISPR type I-C, in Vibriophages infecting hosts with CRISPR type I-E and in Streptococcus thermopilus phages infectinghosts with type II-A, known as CRISPR3. Conclusions: The observed motif depletion in phages with hosts having CRISPR can be attributed to selectionrather than to mutational bias, as mutational bias should affect the phages of all hosts. Thisobservation implies that the CRISPR system has been efficient in the groups discussed here.