Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences

Homobasidiomycete fungi display many complex fruiting body morphologies, including mushrooms and puffballs, but their anatomical simplicity has confounded efforts to understand the evolution of these forms. We performed a comprehensive phylogenetic analysis of homobasidiomycetes, using sequences from nuclear and mitochondrial ribosomal DNA, with an emphasis on understanding evolutionary relationships of gilled mushrooms and puffballs. Parsimony-based optimization of character states on our phylogenetic trees suggested that strikingly similar gilled mushrooms evolved at least six times, from morphologically diverse precursors. Approximately 87% of gilled mushrooms are in a single lineage, which we call the “euagarics.” Recently discovered 90 million-year-old fossil mushrooms are probably euagarics, suggesting that (i) the origin of this clade must have occurred no later than the mid-Cretaceous and (ii) the gilled mushroom morphology has been maintained in certain lineages for tens of millions of years. Puffballs and other forms with enclosed spore-bearing structures (Gasteromycetes) evolved at least four times. Derivation of Gasteromycetes from forms with exposed spore-bearing structures (Hymenomycetes) is correlated with repeated loss of forcible spore discharge (ballistospory). Diverse fruiting body forms and spore dispersal mechanisms have evolved among Gasteromycetes. Nevertheless, it appears that Hymenomycetes have never been secondarily derived from Gasteromycetes, which suggests that the loss of ballistospory has constrained evolution in these lineages.

Evolution of plastid gene rps2 in a lineage of hemiparasitic and holoparasitic plants: Many losses of photosynthesis and complex patterns of rate variation

The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35s-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

Ecological factors rather than temporal factors dominate the evolution of vesicular stomatitis virus

Vesicular stomatitis New Jersey virus (VSV-NJ) is a rhabdovirus that causes economically important disease in cattle and other domestic animals in endemic areas from southeastern United States to northern South America. Its negatively stranded RNA genome is capable of undergoing rapid evolution, which allows phylogenetic analysis and molecular epidemiology studies to be performed. Previous epidemiological studies in Costa Rica showed the existence of at least two distinct ecological zones of high VSV-NJ activity, one located in the highlands (premontane tropical moist forest) and the other in the lowlands (tropical dry forest). We wanted to test the hypothesis that the viruses circulating in these ecological zones were genetically distinct. For this purpose, we sequenced the hypervariable region of the phosphoprotein gene for 50 VSV-NJ isolates from these areas. Phylogenetic analysis showed that viruses from each ecological zone had distinct genotypes. These genotypes were maintained in each area for periods of up to 8 years. This evolutionary pattern of VSV-NJ suggests an adaptation to ecological factors that could exert selective pressure on the virus. As previous data indicated an absence of virus adaptation to factors related to the bovine host (including immunological pressure), it appears that VSV genetic divergence represents positive selection to adapt to specific vectors and/or reservoirs at each ecological zone.

Chloroplast small heat shock proteins: Evidence for atypical evolution of an organelle-localized protein

Knowledge of the origin and evolution of gene families is critical to our understanding of the evolution of protein function. To gain a detailed understanding of the evolution of the small heat shock proteins (sHSPs) in plants, we have examined the evolutionary history of the chloroplast (CP)-localized sHSPs. Previously, these nuclear-encoded CP proteins had been identified only from angiosperms. This study reveals the presence of the CP sHSPs in a moss, Funaria hygrometrica. Two clones for CP sHSPs were isolated from a F. hygrometrica heat shock cDNA library that represent two distinct CP sHSP genes. Our analysis of the CP sHSPs reveals unexpected evolutionary relationships and patterns of sequence conservation. Phylogenetic analysis of the CP sHSPs with other plant CP sHSPs and eukaryotic, archaeal, and bacterial sHSPs shows that the CP sHSPs are not closely related to the cyanobacterial sHSPs. Thus, they most likely evolved via gene duplication from a nuclear-encoded cytosolic sHSP and not via gene transfer from the CP endosymbiont. Previous sequence analysis had shown that all angiosperm CP sHSPs possess a methionine-rich region in the N-terminal domain. The primary sequence of this region is not highly conserved in the F. hygrometrica CP sHSPs. This lack of sequence conservation indicates that sometime in land plant evolution, after the divergence of mosses from the common ancestor of angiosperms but before the monocot–dicot divergence, there was a change in the selective constraints acting on the CP sHSPs.

Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

Interferon-induced human MxA GTPase blocks nuclear import of Thogoto virus nucleocapsids

Interferon-induced human MxA protein belongs to the dynamin superfamily of large GTPases. It exhibits antiviral activity against a variety of RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Here, we report that MxA blocks the transport of Thogoto virus nucleocapsids into the nucleus, thereby preventing transcription of the viral genome. This interaction can be abolished by a mAb that neutralizes the antiviral activity of MxA. Our results reveal an antiviral mechanism whereby an interferon-induced protein traps the incoming virus and interferes with proper transport of the viral genome to its ultimate target compartment within the infected cell.

Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3′-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study

Rheumatoid arthritis (RA), the most common autoimmune disease, is associated in families with other autoimmune diseases, including insulin-dependent diabetes mellitus (IDDM). Its genetic component has been suggested by familial aggregation (λs = 5), twin studies, and segregation analysis. HLA, which is the only susceptibility locus known, has been estimated to account for one-third of this component. The aim of this paper was to identify new RA loci. A genome scan was performed with 114 European Caucasian RA sib pairs from 97 nuclear families. Linkage was significant only for HLA (P < 2.5⋅10−5) and nominal for 19 markers in 14 other regions (P < 0.05). Four of the loci implicated in IDDM potentially overlap with these regions: the putative IDDM6, IDDM9, IDDM13, and DXS998 loci. The first two of these candidate regions, defined in the RA genome scan by the markers D18S68-D18S61-D18S469 (18q22–23) and D3S1267 (3q13), respectively, were studied in 194 additional RA sib pairs from 164 nuclear families. Support for linkage to chromosome 3 only was extended significantly (P = 0.002). The analysis of all 261 families provided a linkage evidence of P = 0.001 and suggested an interaction between this putative RA locus and HLA. This locus could account for 16% of the genetic component of RA. Candidate genes include those coding for CD80 and CD86, molecules involved in antigen-specific T cell recognition. In conclusion, this first genome scan in RA Caucasian families revealed 14 candidate regions, one of which was supported further by the study of a second set of families.

Evolutionary specialization of the nuclear targeting apparatus

The α- and β-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All α-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various α-Kaps fall into two groups, α1 and α2. Whereas animals encode both types, the yeast genome encodes only an α1-Kap. The β-Kaps are characterized by 14–15 tandemly arranged HEAT motifs. We show that the Arm repeats of α-Kaps and the HEAT motifs of β-Kaps are similar, suggesting that the α-Kaps and β-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of β-Kaps, consistent with their proposed cargo specificities. We present a model in which an α-independent β-Kap progenitor gave rise to the α-dependent β-Kaps and the α-Kaps.

Coiled Bodies Preferentially Associate with U4, U11, and U12 Small Nuclear RNA Genes in Interphase HeLa Cells but Not with U6 and U7 Genes

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle–regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for χ2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Identification of a sequence element directing a protein to nuclear speckles

SF3b155 is an essential spliceosomal protein, highly conserved during evolution. It has been identified as a subunit of splicing factor SF3b, which, together with a second multimeric complex termed SF3a, interacts specifically with the 12S U2 snRNP and converts it into the active 17S form. The protein displays a characteristic intranuclear localization. It is diffusely distributed in the nucleoplasm but highly concentrated in defined intranuclear structures termed “speckles,” a subnuclear compartment enriched in small ribonucleoprotein particles and various splicing factors. The primary sequence of SF3b155 suggests a multidomain structure, different from those of other nuclear speckles components. To identify which part of SF3b155 determines its specific intranuclear localization, we have constructed expression vectors encoding a series of epitope-tagged SF3b155 deletion mutants as well as chimeric combinations of SF3b155 sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified (i) a functional nuclear localization signal of the monopartite type (KRKRR, amino acids 196–200) and (ii) a molecular segment with multiple threonine-proline repeats (amino acids 208–513), which is essential and sufficient to confer a specific accumulation in nuclear speckles. This latter sequence element, in particular amino acids 208–440, is required for correct subcellular localization of SF3b155 and is also sufficient to target a reporter protein to nuclear speckles. Moreover, this “speckle-targeting sequence” transfers the capacity for interaction with other U2 snRNP components.

Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts

Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.

Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation

To assess whether heterozygosity of the donor cell genome was a general parameter crucial for long-term survival of cloned animals, we tested the ability of embryonic stem (ES) cells with either an inbred or F1 genetic background to generate cloned mice by nuclear transfer. Most clones derived from five F1 ES cell lines survived to adulthood. In contrast, clones from three inbred ES cell lines invariably died shortly after birth due to respiratory failure. Comparison of mice derived from nuclear cloning, in which a complete blastocyst is derived from a single ES cell, and tetraploid blastocyst complementation, in which only the inner cell mass is formed from a few injected ES cells, allows us to determine which phenotypes depend on the technique or on the characteristics of the ES cell line. Neonatal lethality also has been reported in mice entirely derived from inbred ES cells that had been injected into tetraploid blastocysts (ES cell-tetraploids). Like inbred clones, ES cell-tetraploid pups derived from inbred ES cell lines died shortly after delivery with signs of respiratory distress. In contrast, most ES cell-tetraploid neonates, derived from six F1 ES cell lines, developed into fertile adults. Cloned pups obtained from both inbred and F1 ES cell nuclei frequently displayed increased placental and birth weights whereas ES cell-tetraploid pups were of normal weight. The potency of F1 ES cells to generate live, fertile adults was not lost after either long-term in vitro culture or serial gene targeting events. We conclude that genetic heterozygosity is a crucial parameter for postnatal survival of mice that are entirely derived from ES cells by either nuclear cloning or tetraploid embryo complementation. In addition, our results demonstrate that tetraploid embryo complementation using F1 ES cells represents a simple, efficient procedure for deriving animals with complex genetic alterations without the need for a chimeric intermediate.