Ontogeny and phylogeny of a Scutellospora heterogama mutant, with implications for morphological recognition of species in Glomeromycota.

A putative mutant of Scutellospora heterogama has been maintained for 19 pot culture generations over 15y. The mutant differed phenotypically from the wild-type parental lineage in characteristics of the spore wall: albino versus pigmented red-brown color, high plasticity in acidic mutants versus rigid and finely laminate, deep red-purple reaction versus no reaction in Melzer's reagent, respectively. This variation was equivalent to or greater than that between any two species in Scutellospora or any other genus in Glomeromycota. Comparison of spore ontogenesis revealed that the terminal (mature) state of the albino mutant was similar to a transient intermediate stage in the wild-type. The albino phenotype, therefore, did not result from emergence of a unique morphological innovation. Rather, it arose from a mutation that led to premature termination of spore ontogenesis so that a unique transient juvenile stage became permanent in mature spores. Because this mutation was homogeneous in all progeny populations, it is hypothesized to be a recessive trait expressed only after the allele was distributed in all nuclei of the fungal thallus. Sampling of the genomes of the putative mutant and wild-type isolates by microsatellite-primed PCR suggested a local mutation. The profile of the mutant was identical to that of the wild-type parent and was 60-97 % similar to those of four other S. heterogama isolates. Phylogenetic analysis of the D1-D2 domains of the 25S rRNA gene and a ß-tubulin gene with and without three variable introns placed the albino mutant solidly within the S. heterogama clade. These results suggest that stability of morphological traits is not a suitable criterion by itself to recognize species. The albino phenotype was a discrete and heritable mutation that became fixed in a population and was stable through time and space. In the absence of negative selection, this mutation could persist, disperse and then be misinterpreted as a new species in nature. Genetic markers expose this mutation as a population-level variant and therefore of no macroevolutionary significance. Assessment of genetic divergence amongst multiple isolates is important in ascertaining the contribution of morphological characters toward recognition of species in glomeromycotan clades.

Phylogenies from genetic and morphological characters do not support a revision of Gigasporaceae (Glomeromycota) into four families and five genera.

The family Gigasporaceae consisted of the two genera Gigaspora and Scutellospora when first erected. In a recent revision of this classification, Scutellospora was divided into three families and four genera based on two main lines of evidence: (1) phylogenetic patterns of coevolving small and large rRNA genes and (2) morphology of spore germination shields. The rRNA trees were assumed to accurately reflect species evolution, and shield characters were selected because they correlated with gene trees. These characters then were used selectively to support gene trees and validate the classification. To test this new classification, a phylogenetic tree was reconstructed from concatenated 25S rRNA and ß-tubulin gene sequences using 35% of known species in Gigasporaceae. A tree also was reconstructed from 23 morphological characters represented in 71% of known species. Results from both datasets showed that the revised classification was untenable. The classification also failed to accurately represent sister group relationships amongst higher taxa. Only two clades were fully resolved and congruent among datasets: Gigaspora and Racocetra (a clade consisting of species with spores having one inner germinal wall). Other clades were unresolved, which was attributed in part to undersampling of species. Topology of the morphology-based phylogeny was incongruent with gene evolution. Five shield characters were reduced to three, of which two were phylogenetically uninformative because they were homoplastic. Therefore, most taxa erected in the new classification are rejected. The classification is revised to restore the family Gigasporaceae, within which are the three genera Gigaspora, Racocetra, and Scutellospora. This classification does not reflect strict topology of either gene or morphological evolution. Further revisions must await sampling of additional characters and taxa to better ascertain congruence between datasets and infer a more accurate phylogeny of this important group of fungi.

DNA double-strand breaks by asbestos, silica, and titanium dioxide: possible biomarker of carcinogenic potential?

DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to radiation, exposure to toxins, or other environmental stresses. A complex DNA-damage-response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DNA DSBs is the phosphorylation of a histone, H2AX, at serine 139 (gamma-H2AX), which can be detected by a fluorescent antibody. A study was undertaken to compare the induction of DNA DSBs in normal (small airway epithelial) cells and cancer cells (A549) after exposure to asbestos (crocidolite), a proven carcinogen, silica, a suspected carcinogen, and titanium dioxide (TiO(2)), an inert particle recently reported to be carcinogenic in animals. The results indicate that crocidolite induced greater DNA DSBs than silica and TiO(2), regardless of cell type. DNA DSBs caused by crocidolite were higher in normal cells than in cancer cells. Silica and TiO(2) induced higher DNA DSBs in cancer cells than in normal cells. The production of reactive oxygen species was found to be highest in cells exposed to crocidolite, followed, in potency, by silica and TiO(2). The generation of reactive oxygen species was higher in normal cells than in cancer cells. Cell viability assay indicated that crocidolite caused the greatest cytotoxicity in both cell types. Apoptosis, measured by caspase 3/7 and poly (ADP-Ribose) polymerase activation, was highest in crocidolite-exposed cells, followed by TiO(2) and silica. The results of this study indicate that crocidolite has a greater carcinogenic potential than silica and TiO(2), judged by its ability to cause sustained genomic instability in normal lung cells.

Isolation and sequence analysis of a beta-tubulin gene from arbuscular mycorrhizal fungi.

A full-length beta-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in beta-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5' and 3' splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two beta-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.

Phylogenetic analysis of the Glomeromycota by partial beta-tubulin gene sequences.

The 3' end of the beta-tubulin gene was amplified from 50 isolates of 45 species in Glomeromycota. The analyses included a representative selection of all families except Pacisporaceae and Geosiphonaceae. Phylogenetic analyses excluded three intron regions at the same relative positions in all species due to sequence and length polymorphisms. The beta-tubulin gene phylogeny was similar to the 18S rRNA gene phylogeny at the family and species level, but it was not concordant at the order level. Species in Gigasporaceae and Glomeraceae grouped together but without statistical support. Paralogous sequences in Glomus species likely contributed to phylogenetic ambiguity. Trees generated using different fungal phyla as out-groups yielded a concordant topology. Family relationships within the Glomeromycota did not change regardless if the third codon position was included or excluded from the analysis. Multiple clones from three isolates of Scutellospora heterogama yielded divergent sequences. However, phylogenetic patterns suggested that only a single copy of the beta-tubulin gene was present, with variation attributed to intraspecific sequence divergence.

Confirmation of gene expression-based prediction of survival in non-small cell lung cancer.

PURPOSE: It is a critical challenge to determine the risk of recurrence in early stage non-small cell lung cancer (NSCLC) patients. Accurate gene expression signatures are needed to classify patients into high- and low-risk groups to improve the selection of patients for adjuvant therapy. EXPERIMENTAL DESIGN: Multiple published microarray data sets were used to evaluate our previously identified lung cancer prognostic gene signature. Expression of the signature genes was further validated with real-time reverse transcription-PCR and Western blot assays of snap-frozen lung cancer tumor tissues. RESULTS: Our previously identified 35-gene signature stratified 264 patients with NSCLC into high- and low-risk groups with distinct overall survival rates (P < 0.05, Kaplan-Meier analysis, log-rank tests). The 35-gene signature further stratified patients with clinical stage 1A diseases into poor prognostic and good prognostic subgroups (P = 0.0007, Kaplan-Meier analysis, log-rank tests). This signature is independent of other prognostic factors for NSCLC, including age, sex, tumor differentiation, tumor grade, and tumor stage. The expression of the signature genes was validated with real-time reverse transcription-PCR analysis of lung cancer tumor specimens. Protein expression of two signature genes, TAL2 and ILF3, was confirmed in lung adenocarcinoma tumors by using Western blot analysis. These two biomarkers showed correlated mRNA and protein overexpression in lung cancer development and progression. CONCLUSIONS: The results indicate that the identified 35-gene signature is an accurate predictor of survival in NSCLC. It provides independent prognostic information in addition to traditional clinicopathologic criteria.