Ecological differentiation and sympatry of cryptic species in the Sphagnum magellanicum complex (Bryophyta).

PREMISE: Sphagnum magellanicum (Sphagnaceae, Bryophyta) has been considered to be a single semi-cosmopolitan species, but recent molecular analyses have shown that it comprises a complex of at least seven reciprocally monophyletic groups, that are difficult or impossible to distinguish morphologically. METHODS: Newly developed barcode markers and RADseq analyses were used to identify species among 808 samples from 119 sites. Molecular approaches were used to assess the geographic ranges of four North American species, the frequency at which they occur sympatrically, and ecological differentiation among them. Microhabitats were classified with regard to hydrology and shade. Hierarchical modelling of species communities was used to assess climate variation among the species. Climate niches were projected back to 22,000 years BP to assess the likelihood that the North American species had sympatric ranges during the late Pleistocene. RESULTS: The species exhibited parallel morphological variation, making them extremely difficult to distinguish phenotypically. Two to three species frequently co-occurred within peatlands. They had broadly overlapping microhabitat and climate niches. Barcode- versus RADseq-based identifications were in conflict for 6% of the samples and always involved S. diabolicum vs. S. magniae. CONCLUSIONS: These species co-occur within peatlands at scales that could permit interbreeding, yet they remain largely distinct genetically and phylogenetically. The four cryptic species exhibited distinct geographic and ecological patterns. Conflicting identifications from barcode vs. RADseq analyses for S. diabolicum versus S. magniae could reflect incomplete speciation or hybridization. They comprise a valuable study system for additional work on climate adaptation.

Parallel patterns of genetic diversity and structure in circumboreal species of the Sphagnum capillifolium complex.

PREMISE: Shared geographical patterns of population genetic variation among related species is a powerful means to identify the historical events that drive diversification. The Sphagnum capillifolium complex is a group of closely related peat mosses within the Sphagnum subgenus Acutifolia and contains several circumboreal species whose ranges encompass both glaciated and unglaciated regions across the northern hemisphere. In this paper, we (1) inferred the phylogeny of subg. Acutifolia and (2) investigated patterns of population structure and genetic diversity among five circumboreal species within the S. capillifolium complex. METHODS: We generated RAD sequencing data from most species of the subg. Acutifolia and samples from across the distribution ranges of circumboreal species within the S. capillifolium complex. RESULTS: We resolved at least 14 phylogenetic clusters within the S. capillifolium complex. Five circumboreal species show some common patterns: One population system comprises plants in eastern North America and Europe, and another comprises plants in the Pacific Northwest or around the Beringian and Arctic regions. Alaska appears to be a hotspot for genetic admixture, genetic diversity, and sometimes endemic subclades. CONCLUSIONS: Our results support the hypothesis that populations of five circumboreal species within the S. capillifolium complex survived in multiple refugia during the last glacial maximum. Long-distance dispersal out of refugia, population bottlenecks, and possible adaptations to conditions unique to each refugium could have contributed to current geographic patterns. These results indicate the important role of historical events in shaping the complex population structure of plants with broad distribution ranges.

Divergent selection and climate adaptation fuel genomic differentiation between sister species of Sphagnum (peat moss).

BACKGROUND AND AIMS: New plant species can evolve through the reinforcement of reproductive isolation via local adaptation along habitat gradients. Peat mosses (Sphagnaceae) are an emerging model system for the study of evolutionary genomics and have well-documented niche differentiation among species. Recent molecular studies have demonstrated that the globally distributed species Sphagnum magellanicum is a complex of morphologically cryptic lineages that are phylogenetically and ecologically distinct. Here, we describe the architecture of genomic differentiation between two sister species in this complex known from eastern North America: the northern S. diabolicum and the largely southern S. magniae. METHODS: We sampled plant populations from across a latitudinal gradient in eastern North America and performed whole genome and restriction-site associated DNA sequencing. These sequencing data were then analyzed computationally. KEY RESULTS: Using sliding-window population genetic analyses we find that differentiation is concentrated within 'islands' of the genome spanning up to 400 kb that are characterized by elevated genetic divergence, suppressed recombination, reduced nucleotide diversity and increased rates of non-synonymous substitution. Sequence variants that are significantly associated with genetic structure and bioclimatic variables occur within genes that have functional enrichment for biological processes including abiotic stress response, photoperiodism and hormone-mediated signalling. Demographic modelling demonstrates that these two species diverged no more than 225 000 generations ago with secondary contact occurring where their ranges overlap. CONCLUSIONS: We suggest that this heterogeneity of genomic differentiation is a result of linked selection and reflects the role of local adaptation to contrasting climatic zones in driving speciation. This research provides insight into the process of speciation in a group of ecologically important plants and strengthens our predictive understanding of how plant populations will respond as Earth's climate rapidly changes.

Clonality, local population structure and gametophyte sex ratios in cryptic species of the Sphagnum magellanicum complex.

BACKGROUND AND AIMS: Sphagnum (peatmoss) comprises a moss (Bryophyta) clade with ~300-500 species. The genus has unparalleled ecological importance because Sphagnum-dominated peatlands store almost a third of the terrestrial carbon pool and peatmosses engineer the formation and microtopography of peatlands. Genomic resources for Sphagnum are being actively expanded, but many aspects of their biology are still poorly known. Among these are the degree to which Sphagnum species reproduce asexually, and the relative frequencies of male and female gametophytes in these haploid-dominant plants. We assess clonality and gametophyte sex ratios and test hypotheses about the local-scale distribution of clones and sexes in four North American species of the S. magellanicum complex. These four species are difficult to distinguish morphologically and are very closely related. We also assess microbial communities associated with Sphagnum host plant clones and sexes at two sites. METHODS: Four hundred and five samples of the four species, representing 57 populations, were subjected to restriction site-associated DNA sequencing (RADseq). Analyses of population structure and clonality based on the molecular data utilized both phylogenetic and phenetic approaches. Multi-locus genotypes (genets) were identified using the RADseq data. Sexes of sampled ramets were determined using a molecular approach that utilized coverage of loci on the sex chromosomes after the method was validated using a sample of plants that expressed sex phenotypically. Sex ratios were estimated for each species, and populations within species. Difference in fitness between genets was estimated as the numbers of ramets each genet comprised. Degrees of clonality [numbers of genets/numbers of ramets (samples)] within species, among sites, and between gametophyte sexes were estimated. Sex ratios were estimated for each species, and populations within species. Sphagnum-associated microbial communities were assessed at two sites in relation to Sphagnum clonality and sex. KEY RESULTS: All four species appear to engage in a mixture of sexual and asexual (clonal) reproduction. A single ramet represents most genets but two to eight ramets were dsumbers ansd text etected for some genets. Only one genet is represented by ramets in multiple populations; all other genets are restricted to a single population. Within populations ramets of individual genets are spatially clustered, suggesting limited dispersal even within peatlands. Sex ratios are male-biased in S. diabolicum but female-biased in the other three species, although significantly so only in S. divinum. Neither species nor males/females differ in levels of clonal propagation. At St Regis Lake (NY) and Franklin Bog (VT), microbial community composition is strongly differentiated between the sites, but differences between species, genets and sexes were not detected. Within S. divinum, however, female gametophytes harboured two to three times the number of microbial taxa as males. CONCLUSIONS: These four Sphagnum species all exhibit similar reproductive patterns that result from a mixture of sexual and asexual reproduction. The spatial patterns of clonally replicated ramets of genets suggest that these species fall between the so-called phalanx patterns, where genets abut one another but do not extensively mix because of limited ramet fragmentation, and the guerrilla patterns, where extensive genet fragmentation and dispersal result in greater mixing of different genets. Although sex ratios in bryophytes are most often female-biased, both male and female biases occur in this complex of closely related species. The association of far greater microbial diversity for female gametophytes in S. divinum, which has a female-biased sex ratio, suggests additional research to determine if levels of microbial diversity are consistently correlated with differing patterns of sex ratio biases.

Phylogenomic structure and speciation in an emerging model: the Sphagnum magellanicum complex (Bryophyta).

Sphagnum magellanicum is one of two Sphagnum species for which a reference-quality genome exists to facilitate research in ecological genomics. Phylogenetic and comparative genomic analyses were conducted based on resequencing data from 48 samples and RADseq analyses based on 187 samples. We report herein that there are four clades/species within the S. magellanicum complex in eastern North America and that the reference genome belongs to Sphagnum divinum. The species exhibit tens of thousands (RADseq) to millions (resequencing) of fixed nucleotide differences. Two species, however, referred to informally as S. diabolicum and S. magni because they have not been formally described, are differentiated by only 100 (RADseq) to 1000 (resequencing) of differences. Introgression among species in the complex is demonstrated using D-statistics and f4 ratios. One ecologically important functional trait, tissue decomposability, which underlies peat (carbon) accumulation, does not differ between segregates in the S. magellanicum complex, although previous research showed that many closely related Sphagnum species have evolved differences in decomposability/carbon sequestration. Phylogenetic resolution and more accurate species delimitation in the S. magellanicum complex substantially increase the value of this group for studying the early evolutionary stages of climate adaptation and ecological evolution more broadly.

Phylogenetic structure in the Sphagnum recurvum complex (Bryophyta) in relation to taxonomy and geography.

PREMISE: The Sphagnum recurvum complex comprises a group of closely related peat mosses that are dominant components of many northern wetland ecosystems. Taxonomic hypotheses for the group range from interpreting the whole complex as one polymorphic species to distinguishing 6-10 species. The complex occurs throughout the Northern Hemisphere, and some of the putative species have intercontinental ranges. Our goals were to delimit the complex and assess its phylogenetic structure in relation to morphologically defined species and intercontinental geography. METHODS: RADseq analyses were applied to a sample of 384 collections from Europe, North America, and Asia. The data were subjected to maximum likelihood phylogenetic analyses and analyses of genetic structure using the software STRUCTURE and multivariate ordination approaches. RESULTS: The S. recurvum complex includes S. angustifolium, S. fallax, S. flexuosum, S. pacificum, and S. recurvum as clades with little evidence of admixture. We also resolved an unnamed clade that is referred to here as S. "pseudopacificum." We confirm that S. balticum and S. obtusum are nested within the complex. Species with bluntly acute to obtuse stem leaf apices are sister to those with acute to apiculate leaves. Most of the species exhibit some differentiation between intraspecific population systems disjunct on different continents. CONCLUSIONS: We recognize seven species in the amended S. recurvum complex, including S. balticum and S. obtusum, in addition to the informal clade S. "pseudopacificum." Although we detected some geographically correlated phylogenetic structure within widespread morphospecies, our RADseq data support the interpretation that these species have intercontinental geographic ranges.

Robust production and passaging of infectious HPV in squamous epithelium of primary human keratinocytes.

Using Cre-loxP-mediated recombination, we established a highly efficient and reproducible system that generates autonomous HPV-18 genomes in primary human keratinocytes (PHKs), the organotypic raft cultures of which recapitulated a robust productive program. While E7 promoted S-phase re-entry in numerous suprabasal differentiated cells, HPV DNA unexpectedly amplified following a prolonged G2 arrest in mid- and upper spinous cells. As viral DNA levels intensified, E7 activity diminished and then extinguished. These cells then exited the cell cycle to undergo virion morphogenesis. High titers of progeny virus generated an indistinguishable productive infection in naïve PHK raft cultures as before, never before achieved until now. An immortalization-defective HPV-18 E6 mutant genome was also characterized for the first time. Numerous cells accumulated p53 protein, without inducing apoptosis, but the productive program was severely curtailed. Complementation of mutant genomes by E6-expressing retrovirus restored proper degradation of p53 as well as viral DNA amplification and L1 production. This system will be invaluable for HPV genetic dissection and serves as a faithful ex vivo model for investigating infections and interventions.

Newly identified sex chromosomes in the Sphagnum (peat moss) genome alter carbon sequestration and ecosystem dynamics.

Peatlands are crucial sinks for atmospheric carbon but are critically threatened due to warming climates. Sphagnum (peat moss) species are keystone members of peatland communities where they actively engineer hyperacidic conditions, which improves their competitive advantage and accelerates ecosystem-level carbon sequestration. To dissect the molecular and physiological sources of this unique biology, we generated chromosome-scale genomes of two Sphagnum species: S. divinum and S. angustifolium. Sphagnum genomes show no gene colinearity with any other reference genome to date, demonstrating that Sphagnum represents an unsampled lineage of land plant evolution. The genomes also revealed an average recombination rate an order of magnitude higher than vascular land plants and short putative U/V sex chromosomes. These newly described sex chromosomes interact with autosomal loci that significantly impact growth across diverse pH conditions. This discovery demonstrates that the ability of Sphagnum to sequester carbon in acidic peat bogs is mediated by interactions between sex, autosomes and environment.

Demographic history and gene flow in the peatmosses Sphagnum recurvum and Sphagnum flexuosum (Bryophyta: Sphagnaceae).

Population size changes and gene flow are processes that can have significant impacts on evolution. The aim of this study was to investigate the relationship of geography to patterns of gene flow and population size changes in a pair of closely related Sphagnum (peatmoss) species: S. recurvum and S. flexuosum. Both species occur in eastern North America, and S. flexuosum also occurs in Europe. Genetic data from restriction-site-associated DNA sequencing (RAD-seq) were used in this study. Analyses of gene flow were accomplished using coalescent simulations of site frequency spectra (SFSs). Signatures of gene flow were confirmed by f 4 statistics. For S. flexuosum, genetic diversity of plants in glaciated areas appeared to be lower than that in unglaciated areas, suggesting that glaciation can have an impact on effective population sizes. There is asymmetric gene flow from eastern North America to Europe, suggesting that Europe might have been colonized by plants from eastern North America after the last glacial maximum. The rate of gene flow between S. flexuosum and S. recurvum is lower than that between geographically disjunct S. flexuosum populations. The rate of gene flow between species is higher among sympatric plants of the two species than between currently allopatric S. flexuosum populations. There was also gene flow from S. recurvum to the ancestor S. flexuosum on both continents which occurred through secondary contact. These results illustrate a complex history of interspecific gene flow between S. flexuosum and S. recurvum, which occurred in at least two phases: between ancestral populations after secondary contact and between currently sympatric plants.

The evolution of chloroplast genes and genomes in ferns.

Most of the publicly available data on chloroplast (plastid) genes and genomes come from seed plants, with relatively little information from their sister group, the ferns. Here we describe several broad evolutionary patterns and processes in fern plastid genomes (plastomes), and we include some new plastome sequence data. We review what we know about the evolutionary history of plastome structure across the fern phylogeny and we compare plastome organization and patterns of evolution in ferns to those in seed plants. A large clade of ferns is characterized by a plastome that has been reorganized with respect to the ancestral gene order (a similar order that is ancestral in seed plants). We review the sequence of inversions that gave rise to this organization. We also explore global nucleotide substitution patterns in ferns versus those found in seed plants across plastid genes, and we review the high levels of RNA editing observed in fern plastomes.

Complete plastome sequences of Equisetum arvense and Isoetes flaccida: implications for phylogeny and plastid genome evolution of early land plant lineages.

BACKGROUND: Despite considerable progress in our understanding of land plant phylogeny, several nodes in the green tree of life remain poorly resolved. Furthermore, the bulk of currently available data come from only a subset of major land plant clades. Here we examine early land plant evolution using complete plastome sequences including two previously unexamined and phylogenetically critical lineages. To better understand the evolution of land plants and their plastomes, we examined aligned nucleotide sequences, indels, gene and nucleotide composition, inversions, and gene order at the boundaries of the inverted repeats. RESULTS: We present the plastome sequences of Equisetum arvense, a horsetail, and of Isoetes flaccida, a heterosporous lycophyte. Phylogenetic analysis of aligned nucleotides from 49 plastome genes from 43 taxa supported monophyly for the following clades: embryophytes (land plants), lycophytes, monilophytes (leptosporangiate ferns + Angiopteris evecta + Psilotum nudum + Equisetum arvense), and seed plants. Resolution among the four monilophyte lineages remained moderate, although nucleotide analyses suggested that P. nudum and E. arvense form a clade sister to A. evecta + leptosporangiate ferns. Results from phylogenetic analyses of nucleotides were consistent with the distribution of plastome gene rearrangements and with analysis of sequence gaps resulting from insertions and deletions (indels). We found one new indel and an inversion of a block of genes that unites the monilophytes. CONCLUSIONS: Monophyly of monilophytes has been disputed on the basis of morphological and fossil evidence. In the context of a broad sampling of land plant data we find several new pieces of evidence for monilophyte monophyly. Results from this study demonstrate resolution among the four monilophytes lineages, albeit with moderate support; we posit a clade consisting of Equisetaceae and Psilotaceae that is sister to the "true ferns," including Marattiaceae.

Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes.

BACKGROUND: Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. RESULTS: The Tortula chloroplast genome is approximately 123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the approximately 71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. CONCLUSIONS: Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations.

The evolution of chloroplast genome structure in ferns.

The plastid genome (plastome) is a rich source of phylogenetic and other comparative data in plants. Most land plants possess a plastome of similar structure. However, in a major group of plants, the ferns, a unique plastome structure has evolved. The gene order in ferns has been explained by a series of genomic inversions relative to the plastome organization of seed plants. Here, we examine for the first time the structure of the plastome across fern phylogeny. We used a PCR-based strategy to map and partially sequence plastomes. We found that a pair of partially overlapping inversions in the region of the inverted repeat occurred in the common ancestor of most ferns. However, the ancestral (seed plant) structure is still found in early diverging branches leading to the osmundoid and filmy fern lineages. We found that a second pair of overlapping inversions occurred on a branch leading to the core leptosporangiates. We also found that the unique placement of the gene matK in ferns (lacking a flanking intron) is not a result of a large-scale inversion, as previously thought. This is because the intron loss maps to an earlier point on the phylogeny than the nearby inversion. We speculate on why inversions may occur in pairs and what this may mean for the dynamics of plastome evolution.

Transcriptional regulation of the AT1 receptor gene in immortalized human trophoblast cells.

Studies investigating the mechanisms that govern the expression of the human angiotensin II (Ang II) type 1 receptor (hAT1R) gene have progressed slowly due to the lack of human cell lines that express the AT1R. Recently, however, an immortalized human trophoblast cell line (HTR-8/SVNeo) was demonstrated to respond to Ang II. Therefore, we utilized this cell line to characterize the AT1R expressed on the cell surface and to investigate the mechanisms by which the hAT1R gene is regulated in these cells. HTR-8/SVNeo cells were shown to express functional high affinity AT1Rs having a Bmax value of 114+/-11 fmol/mg protein and a Kd value of 0.14+/-0.1 nM. Additionally, Ang II-induced IP3 production was mediated via the AT1R. Deletional analysis of the hAT1R promoter localized a major basal regulatory sequence within the -105 to -79 bp region, relative to the transcription start site, in HTR-8/SVNeo cells. Electrophoretic mobility shift assay (EMSA) and Chromatin Immunoprecipitation (ChIP) assay demonstrated that the transcription factors, Sp1 and Sp3, interact with this region of the hAT1R promoter in vitro and in vivo. Taken together, our data demonstrate that HTR-8/SVNeo cells express functional AT1Rs and that basal level expression of this gene is regulated, in part, by Sp1 and Sp3 in this cell line.

Translation of the human angiotensin II type 1 receptor mRNA is mediated by a highly efficient internal ribosome entry site.

Activation of the angiotensin II type 1 receptor (AT1R) is closely involved in the pathogenesis of cardiovascular disease. The human AT1R (hAT1R) mRNA splice variants have long 5'-untranslated regions (5'-UTRs) ranging from 272 to 414 bp that have the potential to form stable secondary structures. In this study, we show that the 5'-UTR of hAT(1)R mRNAs contains an internal ribosome entry site (IRES) located within the first 40 bp of the proximal end of exon 1. Experiments utilizing the hAT1R 5'-UTR as a molecular decoy demonstrate a reduction in IRES activity of approximately 50%. This inhibition is most efficient for the hAT1R IRES suggesting that a defined set of trans-factors are required to initiate translation through this cis-element. Translation initiation from the hAT1R IRES appears to be physiologically relevant since IRES activity was maintained during serum starvation, a cellular stress known to inhibit cap-dependent translation. These results suggest that cap-independent translation initiation by internal ribosome entry may represent an important mechanism for the regulation of hAT1R expression.

Genotyping-by-sequencing for Populus population genomics: an assessment of genome sampling patterns and filtering approaches.

Continuing advances in nucleotide sequencing technology are inspiring a suite of genomic approaches in studies of natural populations. Researchers are faced with data management and analytical scales that are increasing by orders of magnitude. With such dramatic advances comes a need to understand biases and error rates, which can be propagated and magnified in large-scale data acquisition and processing. Here we assess genomic sampling biases and the effects of various population-level data filtering strategies in a genotyping-by-sequencing (GBS) protocol. We focus on data from two species of Populus, because this genus has a relatively small genome and is emerging as a target for population genomic studies. We estimate the proportions and patterns of genomic sampling by examining the Populus trichocarpa genome (Nisqually-1), and demonstrate a pronounced bias towards coding regions when using the methylation-sensitive ApeKI restriction enzyme in this species. Using population-level data from a closely related species (P. tremuloides), we also investigate various approaches for filtering GBS data to retain high-depth, informative SNPs that can be used for population genetic analyses. We find a data filter that includes the designation of ambiguous alleles resulted in metrics of population structure and Hardy-Weinberg equilibrium that were most consistent with previous studies of the same populations based on other genetic markers. Analyses of the filtered data (27,910 SNPs) also resulted in patterns of heterozygosity and population structure similar to a previous study using microsatellites. Our application demonstrates that technically and analytically simple approaches can readily be developed for population genomics of natural populations.

Between two fern genomes.

Ferns are the only major lineage of vascular plants not represented by a sequenced nuclear genome. This lack of genome sequence information significantly impedes our ability to understand and reconstruct genome evolution not only in ferns, but across all land plants. Azolla and Ceratopteris are ideal and complementary candidates to be the first ferns to have their nuclear genomes sequenced. They differ dramatically in genome size, life history, and habit, and thus represent the immense diversity of extant ferns. Together, this pair of genomes will facilitate myriad large-scale comparative analyses across ferns and all land plants. Here we review the unique biological characteristics of ferns and describe a number of outstanding questions in plant biology that will benefit from the addition of ferns to the set of taxa with sequenced nuclear genomes. We explain why the fern clade is pivotal for understanding genome evolution across land plants, and we provide a rationale for how knowledge of fern genomes will enable progress in research beyond the ferns themselves.

Promotion of hepatocarcinogenesis by perfluoroalkyl acids in rainbow trout.

Previously, we reported that perfluorooctanoic acid (PFOA) promotes liver cancer in a manner similar to that of 17ß-estradiol (E2) in rainbow trout. Also, other perfluoroalkyl acids (PFAAs) are weakly estrogenic in trout and bind the trout liver estrogen receptor. The primary objective of this study was to determine whether multiple PFAAs enhance hepatic tumorigenesis in trout, an animal model that represents human insensitivity to peroxisome proliferation. A two-stage chemical carcinogenesis model was employed in trout to evaluate PFOA, perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorooctane sulfonate (PFOS), and 8:2 fluorotelomer alcohol (8:2FtOH) as complete carcinogens or promoters of aflatoxin B(1) (AFB(1))- and/or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced liver cancer. A custom trout DNA microarray was used to assess hepatic transcriptional response to these dietary treatments in comparison with E2 and the classic peroxisome proliferator, clofibrate (CLOF). Incidence, multiplicity, and size of liver tumors in trout fed diets containing E2, PFOA, PFNA, and PFDA were significantly higher compared with AFB(1)-initiated animals fed control diet, whereas PFOS caused a minor increase in liver tumor incidence. E2 and PFOA also enhanced MNNG-initiated hepatocarcinogenesis. Pearson correlation analyses, unsupervised hierarchical clustering, and principal components analyses showed that the hepatic gene expression profiles for E2 and PFOA, PFNA, PFDA, and PFOS were overall highly similar, though distinct patterns of gene expression were evident for each treatment, particularly for PFNA. Overall, these data suggest that multiple PFAAs can promote liver cancer and that the mechanism of promotion may be similar to that of E2.

A highly efficient system to produce infectious human papillomavirus: Elucidation of natural virus-host interactions.

A simple, efficient system has been developed to produce high titers of infectious human papillomavirus type 18 (HPV-18) in organotypic raft cultures of primary human keratinocytes (PHKs). Molecular characterization elucidated key early and late events in the reproductive program. The system obviates the need for immortalized cells and allows the analyses of mutant HPV genomes not previously possible. An E6 deletion mutant incapable of causing p53 degradation is defective in viral DNA amplification and capsid protein production. The high levels of p53 protein which accumulated in numerous cells did not lead to apoptosis over a prolonged duration. Time course and metabolic labeling experiments revealed novel interactions with the host. Notably, post-mitotic, differentiated cells are induced by HPV E7 expression to reenter S phase, whereupon host chromosomes replicate, but HPV DNA does not amplify until the cells have progressed to and are arrested in G(2) phase. Here, we present data that strongly suggest that the abundant cytoplasmic viral E1;E4 protein is not responsible for this G(2) arrest, as described in the literature upon ectopic expression in cell lines. We provide additional insights into the viral life cycle and contrast them to conclusions derived from experiments in cell lines.

Conservation of selection on matK following an ancient loss of its flanking intron.

The chloroplast gene trnK and its associated group II intron appear to be absent in a large and ancient clade that includes nearly 90% of fern species. However, the maturase protein encoded within the intron (matK) is still present and located on the boundary of a large-scale inversion. We surveyed the chloroplast genome sequence of clade-member Adiantum capillus-veneris for evidence of a still present but fragmented trnK intron. Lack of signature structural domains and sequence motifs in the genome indicate loss of the trnK intron through degradation in an ancestor of the clade. In plants, matK preferentially catalyzes splicing of the trnK intron, but may also have a generalist function, splicing other group II introns in the chloroplast genome. We therefore tested whether a shift in selective constraint has occurred after loss of the trnK intron. Using previously unavailable sequences for several ferns, we compared matK sequences of the intron-less fern clade to sequences from seed plants and ferns with the intron and found no significant differences in selection among lineages using multiple methods. We conclude that matK in ferns has maintained its apparently ancient and generalized function in chloroplasts, even after the loss of its co-evolved group II intron. Finally, we also present primers that will allow amplification and nucleotide sequencing of the phylogenetically useful matK gene in additional fern taxa.