Recursive partitioning for tumor classification with gene expression microarray data

Precise classification of tumors is critically important for cancer diagnosis and treatment. It is also a scientifically challenging task. Recently, efforts have been made to use gene expression profiles to improve the precision of classification, with limited success. Using a published data set for purposes of comparison, we introduce a methodology based on classification trees and demonstrate that it is significantly more accurate for discriminating among distinct colon cancer tissues than other statistical approaches used heretofore. In addition, competing classification trees are displayed, which suggest that different genes may coregulate colon cancers.

Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: Induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal

We have recently shown that VEGF functions as a survival factor for newly formed vessels during developmental neovascularization, but is not required for maintenance of mature vessels. Reasoning that expanding tumors contain a significant fraction of newly formed and remodeling vessels, we examined whether abrupt withdrawal of VEGF will result in regression of preformed tumor vessels. Using a tetracycline-regulated VEGF expression system in xenografted C6 glioma cells, we showed that shutting off VEGF production leads to detachment of endothelial cells from the walls of preformed vessels and their subsequent death by apoptosis. Vascular collapse then leads to hemorrhages and extensive tumor necrosis. These results suggest that enforced withdrawal of vascular survival factors can be applied to target preformed tumor vasculature in established tumors. The system was also used to examine phenotypes resulting from over-expression of VEGF. When expression of the transfected VEGF cDNA was continuously “on,” tumors became hyper-vascularized with abnormally large vessels, presumably arising from excessive fusions. Tumors were significantly less necrotic, suggesting that necrosis in these tumors is the result of insufficient angiogenesis.

Matchings and phylogenetic trees

This paper presents a natural coordinate system for phylogenetic trees using a correspondence with the set of perfect matchings in the complete graph. This correspondence produces a distance between phylogenetic trees, and a way of enumerating all trees in a minimal step order. It is useful in randomized algorithms because it enables moves on the space of trees that make random optimization strategies “mix” quickly. It also promises a generalization to intermediary trees when data are not decisive as to their choice of tree, and a new way of constructing Bayesian priors on tree space.

Life-span, T-cell responses, and incidence of lymphomas in congenic mice.

Survival, T-cell functions, and postmortem histopathology were studied in H-2 congenic strains of mice bearing H-2b, H-2k, and H-2d haplotypes. Males lived longer than females in all homozygous and heterozygous combinations except for H-2d homozygotes, which showed no differences between males and females. Association of heterozygosity with longer survival was observed only with H-2b/H-2b and H-2b/H-2d mice. Analysis using classification and regression trees (CART) showed that both males and females of H-2b homozygous and H-2k/H-2b mice had the shortest life-span of the strains studied. In histopathological analyses, lymphomas were noted to be more frequent in females, while hemangiosarcomas and hepatomas were more frequent in males. Lymphomas appeared earlier than hepatomas or hemangiosarcomas. The incidence of lymphomas was associated with the H-2 haplotype--e.g., H-2b homozygous mice had more lymphomas than did mice of the H-2d haplotype. More vigorous T-cell function was maintained with age (27 months) in H-2d, H-2b/H-2d, and H-2d/H-2k mice as compared with H-2b, H-2k, and H-2b/H-2k mice, which showed a decline of T-cell responses with age.

Origin and evolution of the slime molds (Mycetozoa)

The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid), and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. Ribosomal RNA trees show the myxogastrid and dictyostelid slime molds as unrelated early branching lineages, but actin and β-tubulin trees place them together as a single coherent (monophyletic) group, closely related to the animal–fungal clade. We have sequenced the elongation factor-1α genes from one member of each division of the Mycetozoa, including Dictyostelium discoideum, for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, with the myxogastrid and dictyostelid slime molds most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals + fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four that place these taxa within the “crown” of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and we emphasize the importance of developing multiple sequence data sets.

Point mutations and sequence variability in proteins: Redistributions of preexisting populations

Here we study the effect of point mutations in proteins on the redistributions of the conformational substates. We show that regardless of the location of a mutation in the protein structure and of its type, the observed movements of the backbone recur largely at the same positions in the structures. Despite the different interactions that are disrupted and formed by the residue substitution, not only are the conformations very similar, but the regions that move are also the same, regardless of their sequential or spatial distance from the mutation. This observation leads us to conclude that, apart from some extreme cases, the details of the interactions are not critically important in determining the protein conformation or in specifying which parts of the protein would be more prone to take on different local conformations in response to changes in the sequence. This finding further illustrates why proteins manifest a robustness toward many mutational events. This nonuniform distribution of the conformer population is consistently observed in a variety of protein structural types. Topology is critically important in determining folding pathways, kinetics, building block cutting, and anatomy trees. Here we show that topology is also very important in determining which regions of the protein structure will respond to sequence changes, regardless of the sequential or spatial location of the mutation.

Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

The optimization principle in phylogenetic analysis tends to give incorrect topologies when the number of nucleotides or amino acids used is small

In the maximum parsimony (MP) and minimum evolution (ME) methods of phylogenetic inference, evolutionary trees are constructed by searching for the topology that shows the minimum number of mutational changes required (M) and the smallest sum of branch lengths (S), respectively, whereas in the maximum likelihood (ML) method the topology showing the highest maximum likelihood (A) of observing a given data set is chosen. However, the theoretical basis of the optimization principle remains unclear. We therefore examined the relationships of M, S, and A for the MP, ME, and ML trees with those for the true tree by using computer simulation. The results show that M and S are generally greater for the true tree than for the MP and ME trees when the number of nucleotides examined (n) is relatively small, whereas A is generally lower for the true tree than for the ML tree. This finding indicates that the optimization principle tends to give incorrect topologies when n is small. To deal with this disturbing property of the optimization principle, we suggest that more attention should be given to testing the statistical reliability of an estimated tree rather than to finding the optimal tree with excessive efforts. When a reliability test is conducted, simplified MP, ME, and ML algorithms such as the neighbor-joining method generally give conclusions about phylogenetic inference very similar to those obtained by the more extensive tree search algorithms.

Phylogeny of rice genomes with emphasis on origins of allotetraploid species

The rice genus, Oryza, which comprises 23 species and 9 recognized genome types, represents an enormous gene pool for genetic improvement of rice cultivars. Clarification of phylogenetic relationships of rice genomes is critical for effective utilization of the wild rice germ plasm. By generating and comparing two nuclear gene (Adh1 and Adh2) trees and a chloroplast gene (matK) tree of all rice species, phylogenetic relationships among the rice genomes were inferred. Origins of the allotetraploid species, which constitute more than one-third of rice species diversity, were reconstructed based on the Adh gene phylogenies. Genome types of the maternal parents of allotetraploid species were determined based on the matK gene tree. The phylogenetic reconstruction largely supports the previous recognition of rice genomes. It further revealed that the EE genome species is most closely related to the DD genome progenitor that gave rise to the CCDD genome. Three species of the CCDD genome may have originated through a single hybridization event, and their maternal parent had the CC genome. The BBCC genome species had different origins, and their maternal parents had either a BB or CC genome. An additional genome type, HHKK, was recognized for Oryza schlechteri and Porteresia coarctata, suggesting that P. coarctata is an Oryza species. The AA genome lineage, which contains cultivated rice, is a recently diverged and rapidly radiated lineage within the rice genus.

PlantsP: a functional genomics database for plant phosphorylation

The PlantsP database is a curated database that combines information derived from sequences with experimental functional genomics information. PlantsP focuses on plant protein kinases and protein phosphatases. The database will specifically provide a resource for information on a collection of T-DNA insertion mutants (knockouts) in each protein kinase and phosphatase in Arabidopsis thaliana. PlantsP also provides a curated view of each protein that includes a comprehensive annotation of functionally related sequence motifs, sequence family definitions, alignments and phylogenetic trees, and descriptive information drawn directly from the literature. PlantsP is available at http://PlantsP.sdsc.edu.

Nitrate-Ammonium Synergism in Rice. A Subcellular Flux Analysis1

Many reports have shown that plant growth and yield is superior on mixtures of NO3− and NH4+ compared with provision of either N source alone. Despite its clear practical importance, the nature of this N-source synergism at the cellular level is poorly understood. In the present study we have used the technique of compartmental analysis by efflux and the radiotracer 13N to measure cellular turnover kinetics, patterns of flux partitioning, and cytosolic pool sizes of both NO3− and NH4+ in seedling roots of rice (Oryza sativa L. cv IR72), supplied simultaneously with the two N sources. We show that plasma membrane fluxes for NH4+, cytosolic NH4+ accumulation, and NH4+ metabolism are enhanced by the presence of NO3−, whereas NO3− fluxes, accumulation, and metabolism are strongly repressed by NH4+. However, net N acquisition and N translocation to the shoot with dual N-source provision are substantially larger than when NO3− or NH4+ is provided alone at identical N concentrations.

Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica.

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA.