IgG autoantibodies to complement C1q in pediatric-onset systemic lupus erythematosus

The ribosomal internal transcribed spacer (ITS) region from individual Gyrodactylus specimens was amplified by polymerase chain reaction (PCR). The reaction amplified the entire ITS1-5.8S-ITS2 region of the ribosomal RNA gene cluster using primers that hybridize to the 3' terminus of the small subunit and the 5' terminus of the large subunit ribosomal RNA genes. The PCR products from Gyrodactylus salaris and Gyrodactylus thymalli were cloned and sequenced. The Gyrodactylus 5.8S gene was identified following comparative alignment of the G. salaris sequence and a Schistosoma 5.8S gene sequence. The ITS regions from G. salaris, G. thymalli, Gyrodactylus derjavini, and Gyrodactylus truttae were compared by restriction enzyme analysis and interspecific restriction fragment length polymorphisms were found. Gyrodactylus salaris and G. thymalli restriction fragment sizes were confirmed from sequence data. ITS amplification followed by Sau3AI digestion enables rapid and clear differentiation of G. salaris, G. derjavini, and G. truttae.     

Molecular characteristation of digenetic trematodes associated with Cyathura carinata (Crustacea: Isopoda) with a note on the utility of 18S ribosomal DNA for phylogenetic analysis in the Digenea (Platyhelminthes: Trematoda)

Analysis of 18S rDNA sequences isolated from Cyathura carinata (Crustacea: Isopoda) indicate that two different species of the Digenea (Platyhelminthes: Trematoda) parasitize this isopod species for which parasitic associations have previously been unknown. Phylogenetic analysis, based on the largest data set of digenetic 18S rDNA sequences published to date, also suggests that 18S rDNA is unsuitable for the reconstruction of phylogenetic relationships of the major digenetic lineages either due to rapid radiation of these lineages or the presence of homoplasy at this level.     

Multiple L1 progenitors in prosimian primates: phylogenetic evidence from ORF1 sequences

One of the uncertainties regarding the evolution of L1 elements is whether there are numerous progenitor genes. We present phylogenetic evidence from ORF1 sequences of slow loris (Nycticebus coucang) and galago (Galago crassicaudatus) that there were at least two distinct progenitors, active at the same time, in the ancestor of this family of prosimian primates. A maximum parsimony analysis that included representative L1s from human, rabbit, and rodents, along with the prosimian sequences, revealed that one of the galago L1s (Gc11) grouped very strongly with the slow loris sequences. The remaining galago elements formed their own unique and strongly supported clade. An analysis of replacement and silent site changes for each link of the most parsimonious tree indicated that during the descent of the Gc11 sequence approximately two times more synonymous than nonsynonymous substitutions had occurred, implying that the Gc11 founder was functional for some time after the split of galago and slow loris. Strong purifying selection was also evident on the galago branch of the tree. These data indicate that there were two distinct and contemporaneous L1 progenitors in the lorisoid ancestor, evolving under purifying selection, that were retained as functional L1s in the galago lineage (and presumably also in the slow loris). The prosimian ORF1 sequences could be further subdivided into subfamilies. ORF1 sequences from both the galago and slow loris have a premature termination codon near the 3' end, not shared by the other mammalian sequences, that shortens the open reading frame by 288 bp. An analysis of synonymous and nonsynonymous substitutions for the 5' and 3' portions, that included intra- and inter-subfamily comparisons, as well as comparisons among the other mammalian sequences, suggested that this premature stop codon is a prosimian acquisition that has rendered the 3' portion of ORF1 in these primates noncoding.     

Phytopathogenic filamentous (Ashbya, Eremothecium) and dimorphic fungi (Holleya, Nematospora) with needle-shaped ascospores as new members within the Saccharomycetaceae

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J Industr. Microbiol. 14, 523-530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle-shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542-549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianius, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution.     

The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.     

A DNA-based demonstration of a three-host life-cycle for the Bivesiculidae (Platyhelminthes: Digenea)

Immature bivesiculid trematodes collected from the intestine of Thlalassoma lunare (Labridae) are shown to be morphologically consistent with adults of Bivesicula claviformis from Epinephelus fasciatus (Serranidae). In addition, the immature bivesiculids have the same sequence for the second internal transcribed spacer of the ribosomal DNA. Comparison with three other species of Bivesiculidae showed differences of between 23% and 30%. These results show that bivesiculids may have three-host life-cycles in addition to the two-host life-cycles that have been demonstrated previously. The three-host life-cycle enables bivesiculids to infect large carnivorous fishes.     

Zea systematics: ribosomal ITS evidence

Ribosomal internal transcribed spacer (ITS) sequences were used to evaluate the phylogenetics of Zea and Tripsacum. Maximum likelihood and polymorphism parsimony were used for phylogenetic reconstructions. Zea ITS nucleotide diversity was high compared to other plant species, but approximately equivalent to other maize loci. Coalescence of ITS alleles was rapid relative to other nuclear loci; however, there was still much diversity within populations. Zea and Tripsacum form a clade clearly differentiated from all other Poaceae. Four Zea ITS pseudogenes were identified by phylogenetic position and nucleotide composition. The phylogenetic position of Z. mays ssp. huehuetenangensis was clearly established as basal to the other Z. mays. The ITS phylogeny disfavored a Z. luxurians and Z. diploperennis clade, which conflicted with some previous studies. The introgression of Z. mays alleles into Z. perennis and Z. diploperennis was also established. The ITS data indicated a near contemporary divergence of domesticated maize and its two closest wild relatives.     

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.     

Ribosomal DNA ITS-1 and ITS-2 sequence comparisons as a tool for predicting genetic relatedness

The determination of the secondary structure of the internal transcribed spacer (ITS) regions separating nuclear ribosomal RNA genes of Chlorophytes has improved the fidelity of alignment of nuclear ribosomal ITS sequences from related organisms. Application of this information to sequences from green algae and plants suggested that a subset of the ITS-2 positions is relatively conserved. Organisms that can mate are identical at all of these 116 positions, or differ by at most, one nucleotide change. Here we sequenced and compared the ITS-1 and ITS-2 of 40 green flagellates in search of the nearest relative to Chlamydomonas reinhardtii. The analysis clearly revealed one unique candidate, C. incerta. Several ancillary benefits of the analysis included the identification of mislabelled cultures, the resolution of confusion concerning C. smithii, the discovery of misidentified sequences in GenBank derived from a green algal contaminant, and an overview of evolutionary relationships among the Volvocales, which is congruent with that derived from rDNA gene sequence comparisons but improves upon its resolution. The study further delineates the taxonomic level at which ITS sequences, in comparison to ribosomal gene sequences, are most useful in systematic and other studies.     

Phylogenetic analyses of Chlamydia psittaci strains from birds based on 16S rRNA gene sequence

The nucleotide sequences of 16S ribosomal DNA (rDNA) were determined for 39 strains of Chlamydia psittaci (34 from birds and 5 from mammals) and for 4 Chlamydia pecorum strains. The sequences were compared phylogenetically with the gene sequences of nine Chlamydia strains (covering four species of the genus) retrieved from nucleotide databases. In the neighbor-joining tree, C. psittaci strains were more closely related to each other than to the other Chlamydia species, although a feline pneumonitis strain was distinct (983 to 98.6% similarity to other strains) and appeared to form the deepest subline within the species of C. psittaci (bootstrap value, 99%). The other strains of C. psittaci exhibiting similarity values of more than 99% were branched into several subgroups. Two pigeon strains and one turkey strain formed a distinct clade recovered in 97% of the bootstrapped trees. The other pigeon strains seemed to be distinct from the strains from psittacine birds, with 88% of bootstrap value. In the cluster of psittacine strains, three parakeet strains and an ovine abortion strain exhibited a specific association (level of sequence similarity, 99.9% or more; bootstrap value, 95%). These suggest that at least four groups of strains exist within the species C. psittaci. The 16S rDNA sequence is a valuable phylogenetic marker for the taxonomy of chlamydiae, and its analysis is a reliable tool for identification of the organisms.     

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.     

Preparation of a novel streptokinase mutant with improved stability

The recent finding of the 37-collar-spined Echinostoma revolutum in North America prompted rDNA nucleotide sequence comparisons between this worm and the sympatric Echinostoma trivolvis. Three isolates of E. revolutum from distinct sites and 2 isolates of E. trivolvis collected from a single site were used in this analysis. Sequence data were compared to those from previously sequenced members of the 37-collar-spine group. The 3 North American isolates of E. revolutum were found to be identical, but they differed from Eurasian isolates of E. revolutum at 9 of the 1,006 sites sequenced. Further, 1 of the E. trivolvis isolates studied herein was identical to the published sequence for this species, but 6 nucleotide changes were observed in the second E. trivolvis isolate. Restriction fragment length polymorphisms at this locus support the nucleotide differences found between the E. trivolvis isolates. The degree of intraspecific variation detected raises questions regarding the utility of the internal-transcribed spacer regions of the ribosomal DNA repeat for taxonomic diagnosis and in phylogenetic studies for poorly differentiated groups, such as the 37-collar-spined congeners.     

Comparisons of two polymorphic species of Ostertagia and phylogenetic relationships within the Ostertagiinae (Nematoda: Trichostrongyloidea) inferred from ribosomal DNA repeat and mitochondrial DNA sequences

The first internal transcribed spacer DNA (ITS-1) (rDNA) and the mitochondrial (mt) DNA-derived cytochrome oxidase I gene (COX-1) were enzymatically amplified, cloned and sequenced from 6 nominal species of Ostertagiinae as well as Haemonchus contortus and Haemonchus placei. The portion of the COX-1 gene analyzed was 393 base pairs (bp) in length and contained 33 within species polymorphic base changes at 28 synonymous sites. The ITS-1 rDNA consensus sequences ranged from 392 bp (Ostertagia ostertagi/Ostertagia lyrata, Teladorsagia circumcincta) to 404 bp (H. contortus, H. placei). These data were used both in a distance analysis to assess the concept of polymorphic species within the genus Ostertagia and in parsimony analysis to assess phylogenetic relationships within a limited group of Ostertagiinae. Pairwise similarity scores of both ITS-1 and COX-1 data showed the highest number of conserved sites between the proposed dimorphic species of Ostertagia. The level of similarity was lower in the COX-1 data due to the high number of synonymous base changes. Analysis by maximum parsimony of the same data did not refute O. ostertagi/O. lyrata and Ostertagia mossil/Ostertagia dikmansi as dimorphic species and supported monophyly of these ostertagiines relative to representatives of the haemonchine outgroup. In the single most parsimonious tree from ITS-1 rDNA data, a subclade of Ostertagia spp. included forms possessing parallel synlophes and long esophageal valves that typically occur in cervid hosts.     

Intracranial abnormalities in infants treated with extracorporeal membrane oxygenation: update on sonographic and CT findings

The phylogenetic relationship of the asexual mycorrhizal fungus Cenococcum geophilam Fr. among sexual ascomycetes was examined by phylogenetic analysis of nucleotide sequence data from the nuclear small subunit (18S) ribosomal RNA genie region. A specific focus of this study was to test the hypothesis that the genus Elaphomyces is the closest sexual relative of C. geophilum. Thus nucleotide sequence data of five C. geophilum isolates, three Elaphomyces species, and 44 additional genera of ascomycetes were included in the phylogenetic analyses. The percentage of similarity among the 18S rDNA sequences of the C. geophilum isolates examined was 99.8 to 100%, indicating that C. geophilum is monophyletic. Percent similarity of nucleotide sequence among the three Elaphomyces species was also high and ranged from 99.4 to 99.5%. DNA parsimony and distance analysis of the sequence data separated these 2 genera on distant clades when sequence from 44 additional genera of ascomycetes was included. Parsimony and distance analyses positioned C. geophilum as a basal, intermediate lineage between the two Loculoascomycete orders, the Pleosporales and the Dothidiales, and strongly supported Elaphomyces to be of Plectomycete origin. Among the sexual Ascomycetes examined, which included representative taxa from four classes of filamentous Ascomycetes (Plectomycetes, Pyrenomycetes, Discomycetes, and Loculoascomycetes), no close sexual relative to C. geophilum was identified. At least four independent lineages of mycorrhizal fungi were identified among the ascomycetes examined.     

Soluble complement receptor 1 is increased in patients with leukemia and after administration of granulocyte colony-stimulating factor

The morphologically uniform species Gonium pectorale is a colonial green flagellate of worldwide distribution. The affinities of 25 isolates from 18 sites on five continents were assessed by both DNA sequence comparisons and sexual compatibility. Complete sequences were obtained (i) for the internal transcribed spacer ITS-1 and ITS-2 regions of ribosomal DNA and (ii) for each of three single-copy spliceosomal introns, two in a small G protein and one in the actin gene. ITS sequences appeared to homogenize sufficiently rapidly to behave as a single copy gene. Intron sequence differences between isolates in this species reached nucleotide substitution saturation, while ITS sequences did not. Parsimony and evolutionary distance analysis of the two types of DNA data gave essentially the same tree conformation. By all these criteria, the group of G. pectorale isolates fell into two main clades, A and B. Clade A, with isolates from four continents, was comprised of four subclades of quite closely related isolates, plus one strain of ambiguous affinity. Clade B was comprised of two subclades represented by South African and South American isolates, respectively; thus, only subclades of clade B showed geographical localization. With respect to mating, all isolates except one homothallic strain and one apparently sterile strain fell into either one or the other of two mating types. Pairings in all possible combinations revealed that isolates from the same site formed abundant zygotes, which germinated to produce new, sexually active organisms. Zygotes were also formed in many pairings of other combinations, including crosses of clade A with clade B organisms, but none of the latter produced viable germlings. The ability to mate and produce viable progeny that were themselves capable of sexual reproduction was restricted to members of subclades established on the basis of DNA sequence similarities. Thus, the grades of difference in both nuclear intron sequences and rDNA ITS sequences paralleled those observed in the sexual analysis.     

Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae

Phylogenetic relationships of different members of the family Cucurbitaceae were estimated from sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the nuclear ribosomal RNA genes. Twenty-six species of different genera belonging to different tribes and several subtribes were analyzed. The whole ITS regions were amplified by PCR technique and cloned, and three to five different clones of each species were sequenced; for some species PCR products were sequenced directly. ITS1 and ITS2 regions are slightly variable in length, with each length appearing genus-specific. A substitution rate of 3.62 x 10(-9) substitutions per site per year was calculated assuming 40 MYA separation time. Phylogenetic relationships inferred from ITS sequences of some species is in agreement with morphological data, but deviations to the taxonomic classification were also observed. A polyphyletic origin of the New World species must be considered. In the genus Cucurbita different "types" of ITS sequences within one species exist, possibly due to the high frequency of introgression during domestication or due to polyploidization events; in contrast, low intraspecific variability was detectable in the genus Cucumis, indicating different stages of speciation.