Delivery of nucleic acid into mammalian cells by anthrax toxin

Gene delivery vehicles based on receptor-mediated endocytosis offer an attractive long-term solution as they might overcome the limitations of toxicity and cargo capacity inherent to many viral gene delivery systems. The protective antigen component of anthrax toxin bind to specific receptors and deliver lethal factor or edema factor into the cytosol of mammalian cells. The N-terminal 254 amino acids of LF (LF(1-254)) binds to PA and, when fused to heterologous proteins, delivers such proteins into the cytosol. However, so far no attempt has been made to use the anthrax toxin system for the intracellular delivery of DNA. In the present study, LF(1-254) of anthrax toxin was fused to the DNA-binding domain of GAL4 protein. The fusion protein (LF(254)-GAL4DBD) showed both PA binding as well as DNA-binding activity in solution. The complex of fusion protein with plasmid DNA containing a reporter gene (luciferase or green fluorescent protein) along with PA delivered plasmid DNA into the cytosol of COS-1 cells. These results suggest that anthrax toxin components can be used as a non-viral system for the efficient delivery of DNA into the cytosol of mammalian cells.     

Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses

Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.     

Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum

We describe the first complete mitochondrial genome sequence from a representative of the insect order Coleoptera, the flour beetle Tribolium castaneum. The 15,881 bp long Tribolium mitochondrial genome encodes 13 putative proteins, two ribosomal RNAs and 22 tRNAs canonical for animal mitochondrial genomes. Their arrangement is identical to that in Drosophila melanogaster, which is considered ancestral for insects and crustaceans (Boore et al., 1998; Hwang, et al., 2001a). Nucleotide composition, amino acid composition, and codon usage fall within the range of values observed in other insect mitochondrial genomes. Most notable features are the use of TCT as tRNA(Ser(AGN)) anticodon instead of GCT, which is used in most other arthropod species, and the relative scarcity of special sequence motifs in the 1431 bp long control region. Phylogenetic analysis confirmed resolving power in the conserved regions of the mitochondrial proteome regarding diversification events, which predate the emergence of pterygote insects, while little resolution was obtained at the level of basal perygote diversification. The partition of faster evolving amino acid sites harbored strong support for joining Lepidoptera with Diptera, which is consistent with a monophyletic Mecopterida.     

A phylogeny of Packera (Senecioneae; asteraceae) based on internal transcribed spacer region sequence data and a broad sampling of outgroups

A phylogeny of Packera is presented based on sequence data from the internal transcribed spacer region of nuclear ribosomal DNA of 26 species (28 populations) of Packera and 23 outgroup taxa, including representatives of all three subtribes of the Senecioneae. The results support a Mexican origin for Packera, with its closest relatives found among Old World taxa in the subtribe Senecioninae, such as Senecio jacobaea and Pericallis. Packera species from the west coast of the United States, previously included in the section Bolanderi of Greenman, are part of a basal assemblage including species of Greenman's Mexican section Sanguisorboidei. The rest of Packera separates into two sister groups, one containing species from the Arizona-New Mexico region and the other containing more geographically diverse taxa. Among the outgroups, New World Senecio species are monophyletic and two Tussilaginoid assemblages are strongly supported; the Tephroseroid group (Tephroseris and Sinosenecio) plus Petasites combine with the Luina complex to form a clade of north temperate taxa, and the four Mexican genera (Psacalium, Robinsonecio, Barkleyanthus, and Pittocaulon) form a monophyletic group.     

Phylogenetic Studies on Didelphid Marsupials I. Introduction and Preliminary Results from Nuclear IRBP Gene Sequences

We report and analyze nucleotide sequence variation in the first exon (1158 bp) of the nuclear gene encoding the Interphotoreceptor Retinoid Binding Protein (IRBP) among 21 species representing all 15 currently recognized genera of living didelphids. Six previously published IRBP sequences representing five nondidelphimorph marsupial orders were also analyzed to test didelphid monophyly, and 12 published sequences representing ten placental orders were used as outgroups. No gaps (indels) are necessary to align didelphid sequences, but one short region (35 bp) is alignment-ambiguous among nondidelphids. Uncorrected pairwise sequence divergence ranges from 0.7 to 5.7% among nonconspecific didelphids, from 9.2 to 15.3% between didelphids and nondidelphid marsupials, and from 24.9 to 32.1% between marsupials and placentals. Neither transitions nor transversions exhibit saturation for any codon position at any level of taxonomic comparison. Parsimony analyses of these data provide strong support (bootstrap values >95%, Bremer values 7) for the monophyly of (1) Didelphidae ("caluromyines" + Didelphinae); (2) a group containing Caluromys and Caluromysiops; (3) Didelphinae; (4) a group of large opossums that includes Metachirus; (5) a group containing the remaining large opossums (with 2N = 22 chromosomes); (6) a group containing Marmosa and Micoureus; (7) a group containing Thylamys, Lestodelphys, and Gracilinanus; and (8) a group containing the last three genera plus a monophyletic Marmosops. In addition, we found moderate support (bootstrap values >80%, Bremer values 2) for the monophyly of Thylamys + Lestodelphys and for a sister-group relationship between Monodelphis and Marmosa + Micoureus. Sensitivity analysis suggests that all of these clades, together with their associated levels of bootstrap and Bremer support, are robust to alternative hypotheses of positional homology within the ambiguously alignable region. Although some of the relationships supported by IRBP are not consistent with the results of published morphological analyses, our reassessment of the morphological data suggests that many conflicts are more apparent than real.     

Descriptions and phylogenetic systematics of Myxobolus spp. from cyprinids in Algonquin Park, Ontario

Eight species of Myxobolus were collected from four species of cyprinids in Algonquin Park, Ontario. On the basis of spore morphology, five of these species are described as new and two are redescribed. The evolutionary relationships among these eight species were studied using partial small subunit ribosomal DNA (ssu-rDNA) sequence data. The resulting cladograms, which were highly resolved and with strongly supported relationships, allowed for the evaluation of spore morphology, host specificity, and tissue tropism, criteria traditionally used in species identification. These criteria, recently criticized for creating artificial rather than natural taxonomic groupings, were evaluated for their reliability in the systematics of the species examined. The data showed that distantly related species often infect the same host and tissue, and that closely related species often occur in different hosts. Morphologically similar species are more closely related to each other and the taxonomy based on spore morphology is consistent with the relationships depicted in the phylogenies. These results suggest that spore morphology is better than host specificity and tissue tropism as a species character, as well as for determining evolutionary relationships among the species of Myxobolus examined.     

Distribution and Geographic Variation in the Western Woolly Lemur (Avahi occidentalis) with Description of a New Species (A. unicolor

The western woolly lemur is distributed through central western, northwestern and possibly northern Madagascar and is traditionally viewed as a monotypic species (Avahi occidentalis) or as a subspecies of A. laniger. We present new data on body weights of Avahi which, together with previously available body weights, provide additional evidence for recognizing eastern and western woolly lemurs as two distinct species. We then reexamine the distributions of, and chromatic variation within and between, populations of western woolly lemurs. A comparative study of wild subjects and museum skins reveals that several geographically discrete morphs exist in western Avahi, which is clearly polytypic. Marked differences between populations in fur coloration suggest that 3 distinct taxa should be recognized. The populations differ considerably in their habitat structure. In addition, field observations suggest clear differences in population density between 2 of the populations. We describe and name one new species.     

Characterization of 10 KDA prolamin genes in Phyllostachys aurea (Bambusoideae, Poaceae)

Prolamin is the dominant class of seed storage protein in grasses (Poaceae). Information on the 10 kDa multigene family coding for prolamins characteristic of the bambusoid-oryzoid grasses is limited. Two genes encoding 10 kDa prolamin were cloned and sequenced in the bambusoid species Phyllostachys aurea to assess the sequence diversity of this gene family in the oryzoid-bambusoid grasses. The genes, ~417 bp in length, were 96% similar at the DNA sequence level, differing in 12 base substitutions dispersed throughout the sequence. Eight of these mutations were nonsynonymous, leading to amino acid substitutions in the coding region, and one was nonsense, producing an amber stop codon. One gene had an open reading frame (ORF) of 139 amino acids, while the other gene had a shorter ORF (106 amino acids) due to the presence of a stop codon in the coding region and, thus, represents a pseudogene. Deduced proteins showed amino acid composition similar to that of rice. The study underscores the overall conserved nature of this multigene family and reflects considerable sequence divergence at the DNA and amino acid levels between the Oryza and the Phyllostachys genes. The systematic implication of the data is discussed in light of the inconsistent placement of Oryza in the Bambusoideae or Oryzoideae.     

Candida arabinofermentans, a new L-arabinose fermenting yeast

Candida arabinofermentans (type strain NRRL YB-2248, CBS 8468), a new yeast that ferments the pentose L-arabinose, is described. The three known strains of this new species were isolated from insect frass of pine and larch trees in the U.S. Phylogenetic analysis of nucleotide sequences from the D1/D2 domain of large subunit (26S) ribosomal DNA places C. arabinofermentans among the methanol-assimilating yeasts and most closely related to Candida ovalis. Strains of the new species produce 0.7-1.9 g/l ethanol from L-arabinose.     

EFFECTS OF POSTGLACIAL RANGE EXPANSION ON ALLOZYME AND QUANTITATIVE GENETIC VARIATION OF THE PITCHER-PLANT MOSQUITO,WYEOMYIA SMITHII

We determined allozyme variability of 34 populations of the pitcher-plant mosquito, Wyeomyia smithii, from Florida (30°N) to northern Manitoba (54°N) and compared allozyme variability with the additive genetic variance for preadult development time and photoperiodic response determined previously for six populations over a similar range (30–50°N). Phylogenetic analysis of allozymes shows a well-defined split between Gulf Coast and lowland North Carolina populations, similar to previously observed phylogeographic patterns in a wide variety of taxa. A deeper split in the phylogeny of W. smithii coincides with the location of the maximum extent of the Laurentide Ice Sheet. Furthermore, both average heterozygosity and patterns of isolation-by-distance decline in populations north of the former glacial border. It is likely that northern populations are the result of a range expansion that occurred subsequent to the late-Wisconsin retreat of the Laurentide Ice Sheet and that these populations have not yet reached a drift-migration equilibrium. The northern decline in allozyme heterozygosity contrasts sharply with the northern increase in additive genetic variance of development time and photoperiodic response found in previous studies. These previous studies also showed that the genetic divergence of populations has involved stochastic variation in the contribution of dominance and epistasis to the genetic architecture underlying demographic traits, including preadult development time, and photoperiodic response. When taken together, the present and prior studies identify the genetic processes underlying the lack of concordance between geographic patterns of allozyme and quantitative genetic variation in natural populations of W. smithii. In the presence of nonadditive genetic variation, isolation and drift can result in opposite patterns of genetic variation for structural genes and quantitative traits.