Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.

Marine fungi: their ecology and molecular diversity.

Fungi appear to be rare in marine environments. There are relatively few marine isolates in culture, and fungal small subunit ribosomal DNA (SSU rDNA) sequences are rarely recovered in marine clone library experiments (i.e., culture-independent sequence surveys of eukaryotic microbial diversity from environmental DNA samples). To explore the diversity of marine fungi, we took a broad selection of SSU rDNA data sets and calculated a summary phylogeny. Bringing these data together identified a diverse collection of marine fungi, including sequences branching close to chytrids (flagellated fungi), filamentous hypha-forming fungi, and multicellular fungi. However, the majority of the sequences branched with ascomycete and basidiomycete yeasts. We discuss evidence for 36 novel marine lineages, the majority and most divergent of which branch with the chytrids. We then investigate what these data mean for the evolutionary history of the Fungi and specifically marine-terrestrial transitions. Finally, we discuss the roles of fungi in marine ecosystems.

Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes.

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides, a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.

Discovery of novel intermediate forms redefines the fungal tree of life.

Fungi are the principal degraders of biomass in terrestrial ecosystems and establish important interactions with plants and animals. However, our current understanding of fungal evolutionary diversity is incomplete and is based upon species amenable to growth in culture. These culturable fungi are typically yeast or filamentous forms, bound by a rigid cell wall rich in chitin. Evolution of this body plan was thought critical for the success of the Fungi, enabling them to adapt to heterogeneous habitats and live by osmotrophy: extracellular digestion followed by nutrient uptake. Here we investigate the ecology and cell biology of a previously undescribed and highly diverse form of eukaryotic life that branches with the Fungi, using environmental DNA analyses combined with fluorescent detection via DNA probes. This clade is present in numerous ecosystems including soil, freshwater and aquatic sediments. Phylogenetic analyses using multiple ribosomal RNA genes place this clade with Rozella, the putative primary branch of the fungal kingdom. Tyramide signal amplification coupled with group-specific fluorescence in situ hybridization reveals that the target cells are small eukaryotes of 3-5 µm in length, capable of forming a microtubule-based flagellum. Co-staining with cell wall markers demonstrates that representatives from the clade do not produce a chitin-rich cell wall during any of the life cycle stages observed and therefore do not conform to the standard fungal body plan. We name this highly diverse clade the cryptomycota in anticipation of formal classification.

Newly identified and diverse plastid-bearing branch on the eukaryotic tree of life.

The use of molecular methods is altering our understanding of the microbial biosphere and the complexity of the tree of life. Here, we report a newly discovered uncultured plastid-bearing eukaryotic lineage named the rappemonads. Phylogenies using near-complete plastid ribosomal DNA (rDNA) operons demonstrate that this group represents an evolutionarily distinct lineage branching with haptophyte and cryptophyte algae. Environmental DNA sequencing revealed extensive diversity at North Atlantic, North Pacific, and European freshwater sites, suggesting a broad ecophysiology and wide habitat distribution. Quantitative PCR analyses demonstrate that the rappemonads are often rare but can form transient blooms in the Sargasso Sea, where high 16S rRNA gene copies mL(-1) were detected in late winter. This pattern is consistent with these microbes being a member of the rare biosphere, whose constituents have been proposed to play important roles under ecosystem change. Fluorescence in situ hybridization revealed that cells from this unique lineage were 6.6 ± 1.2 × 5.7 ± 1.0 µm, larger than numerically dominant open-ocean phytoplankton, and appear to contain two to four plastids. The rappemonads are unique, widespread, putatively photosynthetic algae that are absent from present-day ecosystem models and current versions of the tree of life.

Validation and justification of the phylum name Cryptomycota phyl. nov.

The recently proposed new phylum name Cryptomycota phyl. nov. is validly published in order to facilitate its use in future discussions of the ecology, biology, and phylogenetic relationships of the constituent organisms. This name is preferred over the previously tentatively proposed "Rozellida" as new data suggest that the life-style and morphology of Rozella is not representative of the large radiation to which it and other Cryptomycota belong. Furthermore, taxa at higher ranks such as phylum are considered better not based on individual names of included genera, but rather on some special characteristics - in this case the cryptic nature of this group and that they were initially revealed by molecular methods rather than morphological discovery. If the group were later viewed as a member of a different kingdom, the name should be retained to indicate its fungal affinities, as is the practice for other fungal-like protist groups.

Multiple marker parallel tag environmental DNA sequencing reveals a highly complex eukaryotic community in marine anoxic water.

Sequencing of ribosomal DNA clone libraries amplified from environmental DNA has revolutionized our understanding of microbial eukaryote diversity and ecology. The results of these analyses have shown that protist groups are far more genetically heterogeneous than their morphological diversity suggests. However, the clone library approach is labour-intensive, relatively expensive, and methodologically biased. Therefore, even the most intensive rDNA library analyses have recovered only small samples of much larger assemblages, indicating that global environments harbour a vast array of unexplored biodiversity. High-throughput parallel tag 454 sequencing offers an unprecedented scale of sampling for molecular detection of microbial diversity. Here, we report a 454 protocol for sampling and characterizing assemblages of eukaryote microbes. We use this approach to sequence two SSU rDNA diversity markers-the variable V4 and V9 regions-from 10 L of anoxic Norwegian fjord water. We identified 38 116 V4 and 15 156 V9 unique sequences. Both markers detect a wide range of taxonomic groups but in both cases the diversity detected was dominated by dinoflagellates and close relatives. Long-tailed rank abundance curves suggest that the 454 sequencing approach provides improved access to rare genotypes. Most tags detected represent genotypes not currently in GenBank, although many are similar to database sequences. We suggest that current understanding of the ecological complexity of protist communities, genetic diversity, and global species richness are severely limited by the sequence data hitherto available, and we discuss the biological significance of this high amplicon diversity.

Stable marker gene transfer into human bone marrow stromal cells and their progenitors using novel herpesvirus saimiri-based vectors.

We have evaluated the ability of new herpesvirus saimiri (HVS)-based vectors to deliver a marker gene green fluorescent protein (GFP) into human bone marrow (BM) stromal cells and their progenitors. Stromal cells expanded from adherent layers of long-term BM cultures (LTC) were susceptible to HVS-based infection in a dose-dependent manner, and the efficiency of 94.8 +/- 2.0% was achieved using single exposure with HVS/EGFP vector at multiplicity of infection (moi) of approximately 50. Colony-forming unit-fibroblast (CFU-F) assay established the ability of HVS-based vectors to infect progenitors for bone marrow stroma fibroblasts and transfer the marker gene over multiple cell divisions in the absence of selective pressure. HVS was not toxic for stromal cells and progenitors and no viral replication was detected upon growth of modified stroma. On the basis these data, we believe that HVS-based constructs can offer a new opportunity for selective gene delivery into bone marrow stromal cells and progenitors. The ability of HVS to infect nondividing cells can be considered advantageous in the development of both ex vivo and in vivo strategies.

A herpesvirus saimiri-based gene therapy vector with potential for use in cancer immunotherapy.

The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.

Herpesvirus saimiri-based gene delivery vectors maintain heterologous expression throughout mouse embryonic stem cell differentiation in vitro.

In order to achieve a high efficiency of gene delivery into rare cell types like stem cells the use of viral vectors is presently without alternative. An ideal stem cell gene therapy vector would be able to infect primitive progenitor cells and sustain or activate gene expression in differentiated progeny. However, many viral vectors are inactivated when introduced in developing systems where cell differentiation occurs. To this end, we have developed a mouse in vitro model for testing herpesvirus saimiri (HVS)-based gene therapy vectors. We demonstrate here for the first time that HVS is able to infect totipotent mouse embryonic stem (ES) cells with high efficiency. We have transduced ES cells with a recombinant virus carrying the enhanced green fluorescent protein (EGFP) gene and the neomycin resistance gene (NeoR) driven by a CMV promoter and the SV40 promoter, respectively. ES cells maintain the viral episomal genome and can be terminally differentiated into mature haematopoietic cells. Moreover, heterologous gene expression is maintained throughout in vitro differentiation. Besides its obvious use in gene therapy, this unique expression system has wide ranging applications in studies aimed at understanding gene function and expression in cell differentiation and development.

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

Three-dimensional structure of herpes simplex virus type 1 glycoprotein D at 2.4-nanometer resolution.

Herpes simplex virus type 1 glycoprotein D (gD) is essential for virus infectivity and is responsible for binding to cellular membrane proteins and subsequently promoting fusion between the virus envelope and the cell. No structural data are available for gD or for any other herpesvirus envelope protein. Here we present a three-dimensional model for the baculovirus-expressed truncated protein gD1(306t) based on electron microscopic data. We demonstrate that gD1(306t) appears as a homotetramer containing a pronounced pocket in the center of the molecule. Monoclonal antibody binding demonstrates that the molecule is oriented such that the pocket protrudes away from the virus envelope.

The gene product encoded by ORF 57 of herpesvirus saimiri regulates the redistribution of the splicing factor SC-35.

The herpesvirus saimiri (HVS) gene product encoded by ORF 57 shares limited C-terminal similarity with herpes simplex virus 1 ICP27, a protein that has been demonstrated to be involved in the inhibition of host-cell splicing and is responsible for the redistribution of components of the spliceosome. It has previously been shown that ORF 57 can either activate or repress viral gene expression by a post-transcriptional mechanism. Furthermore, repression of gene expression by ORF 57 is dependent on the presence of an intron within the target gene coding region. In this report, it is shown that HVS infection results in the redistribution of the SC-35 splicing factor in the infected cell nucleus. Furthermore, the redistributed SC-35 colocalized with the ORF 57 protein product and expression of the protein alone was sufficient to cause the redistribution of the spliceosome components. These results suggest that the mechanism by which ORF 57 down-regulates expression of intron-containing genes involves the redistribution of the spliceosome complex.

Assessment of Herpesvirus saimiri as a potential human gene therapy vector.

Herpesvirus saimiri has characteristics that make it amenable to development as a gene therapy vector. The viral genome is thought to be capable of accommodating large quantities of heterologous DNA while the virus itself can infect many different cell types. Virus infection has been shown in many cases to be persistent by virtue of episomal maintenance in the target cell. In this article we examine the ability of nonselectable recombinant viruses expressing the beta-galactosidase gene product to infect a variety of human cells and demonstrate that this virus could be developed as an alternative hematopoietic stem cell gene therapy vector. In contrast to earlier observations, we demonstrate by a number of methods that the virus has the ability to replicate in many human cell types, suggesting the need for the development of a disabled virus for use as a gene therapy vector.

DNA mismatch repair genes and their association with colorectal cancer (Review).

Mismatch repair genes are involved in increasing the fidelity of replication by specific repair of DNA polymerase incorporation errors. In Escherichia coli, the best studied mismatch repair (MMR) pathway is the methyl-directed long patch repair system which is mediated by three gene products; MutS, MutL and MutH. These are conserved in higher eukaryotes. Mutations in human homologues of these proteins have been shown to be implicated in hereditary non-polyposis colorectal cancer (HNPCC). Alterations in the coding regions of MMR genes result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair.

Differences in the intracellular localization and fate of herpes simplex virus tegument proteins early in the infection of Vero cells.

The fate of herpes simplex virus 1 (HSV-1) tegument proteins during infection in Vero cells was investigated immunochemically. Input virion-associated VP13/14 and VP16 localized to the nucleus early in infection, while VP1/2 localized to the nuclear envelope of the cell and VP22 could not be detected using monoclonal antibody P43. Western blotting suggested that virion-associated VP13/14, VP16 and VP22 were stable in infected cells whereas VP1/2 appeared to be processed or modified. Further studies showed that P43 recognized a phosphorylation-sensitive epitope in VP22 and suggested that virion-associated VP22 was phosphorylated upon entry to the cell. VP13/14 and VP16 were easily extracted from cells early in infection whereas VP22 was largely insoluble. Phosphatase treatment of soluble extracts caused a shift in the molecular mass of VP16 showing it was phosphorylated. As infection progressed VP16 was observed in discrete nuclear compartments where it co-localized with ICP8 and the capsid-associated protein VP22a. VP13/14 was also observed in the nucleus. P43 immunostaining appeared around 6 h post-infection as punctate nuclear foci which often localized to the edge of VP16-immunoreactive areas. Punctate P43 cytoplasmic staining appeared around 12 h post-infection. By 18 h the nuclear pattern had disappeared and an extensive cytoplasmic stain was observed which closely overlapped that of other tegument proteins. On the basis of these data we suggest that virion-associated VP22 is phosphorylated upon entry of the virus into the cell and that unphosphorylated VP22, which is preferentially recognized by P43, becomes available later in infection, initially in the nucleus, for packaging into virions.

Phosphorylation of structural components promotes dissociation of the herpes simplex virus type 1 tegument.

The role of phosphorylation in the dissociation of structural components of the herpes simplex virus type 1 (HSV-1) tegument was investigated, using an in vitro assay. Addition of physiological concentrations of ATP and magnesium to wild-type virions in the presence of detergent promoted the release of VP13/14 and VP22. VP1/2 and the UL13 protein kinase were not significantly solubilized. However, using a virus with an inactivated UL13 protein, we found that the release of VP22 was severely impaired. Addition of casein kinase II (CKII) to UL13 mutant virions promoted VP22 release. Heat inactivation of virions or addition of phosphatase inhibited the release of both proteins. Incorporation of radiolabeled ATP into the assay demonstrated the phosphorylation of VP1/2, VP13/14, VP16, and VP22. Incubation of detergent-purified, heat-inactivated capsid-tegument with recombinant kinases showed VP1/2 phosphorylation by CKII, VP13/14 phosphorylation by CKII, protein kinase A (PKA), and PKC, VP16 phosphorylation by PKA, and VP22 phosphorylation by CKII and PKC. Proteolytic mapping and phosphoamino acid analysis of phosphorylated VP22 correlated with previously published work. The phosphorylation of virion-associated VP13/14, VP16, and VP22 was demonstrated in cells infected in the presence of cycloheximide. Use of equine herpesvirus 1 in the in vitro release assay resulted in the enhanced release of VP10, the homolog of HSV-1 VP13/14. These results suggest that the dissociation of major tegument proteins from alphaherpesvirus virions in infected cells may be initiated by phosphorylation events mediated by both virion-associated and cellular kinases.

The open reading frame (ORF) 50a gene product regulates ORF 57 gene expression in herpesvirus saimiri.

We have previously demonstrated that open reading frame (ORF) 50 and ORF 57 encode transcriptional regulating genes in herpesvirus saimiri. ORF 50, a homolog of Epstein-Barr virus R protein, is a sequence-specific transactivator, whereas ORF 57 acts posttranscriptionally. In this report, we demonstrate that the ORF 57 gene is regulated by the ORF 50a gene product. We show that the ORF 57 gene is expressed at basal levels early in the virus replication cycle and that thereafter it is transactivated by the ORF 50a gene product, due to an increase in RNA levels. As it has been shown that the ORF 57 gene product downregulates ORF 50a due to the presence of its intron, these combined observations identify a feedback mechanism modulating gene expression in herpesvirus saimiri, whereby ORF 50a transcription is downregulated by the ORF 57 gene product, a gene which it specifically transactivates. Furthermore, we propose that the intron-containing ORF 57 gene downregulates itself by the same mechanism as that for ORF 50a, as both genes are downregulated at similar times during the replication cycle.

The immediate-early gene product encoded by open reading frame 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level.

The herpesvirus saimiri (HVS) immediate-early gene product encoded by open reading frame (ORF) 57 shares limited amino acid homology with HSV-1 ICP27 and Epstein-Barr virus BMLF1, both regulatory proteins. The ORF 57 gene has been proposed to be spliced based on the genome sequence, and here we confirm the intron-exon structure of the gene. We also demonstrate that a cDNA construct of the ORF 57 gene product represses the transactivating capability of the ORF 50a gene product (which is produced from a spliced transcript), but activates that of ORF 50b (an unspliced transcript). Further analyses with cotransfection experiments show that ORF 57 can either activate or repress expression from a range of both early and late HVS promoters, depending on the target gene. These results indicate that repression of gene expression mediated by the ORF 57 gene product is dependent on the presence of an intron within the target gene encoding region. Furthermore, Northern blot analysis demonstrates that the levels of mRNA transcribed from genes not containing an intron are not significantly affected in the presence of the ORF 57 gene product. This suggests that it regulates gene expression through a posttranscriptional mechanism.

EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.