Evidence of a spotted fever-like rickettsia and a potential new vector from northeastern Australia

A spotted fever-like rickettsia was identified in a Hemaphysalis tick by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA, ompA, and ompB genes. A comparison of these nucleotide sequences with those of other spotted fever group (SFG) rickettsiae revealed that the Hemaphysalis tick rickettsia was distinct from other previously reported strains. Phylogenetic analysis based on both ompA and ompB also indicates that the strain’s closest relatives are the agents of Thai tick typhus (Rickettsia honei strain TT-118) and Flinders Island spotted fever (R. honei). This study represents the first report of an R. honei-like agent from a Hemaphysalis tick in Australia and of a spotted fever group rickettsia from Cape York Peninsula, Queensland.

Molecular phylogeny of Wolbachia endosymbionts in southeast asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the α-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.

wsp Gene sequences from the Wolbachia of filarial nematodes

Wolbachia endosymbiotic bacteria are widespread in arthropods and are also present in filarial nematodes. Almost all filarial species so far examined have been found to harbor these endosymbionts. The sequences of only three genes have been published for nematode Wolbachia (i.e., the genes coding for the proteins FtsZ and catalase and for 16S rRNA). Here we present the sequences of the genes coding for the Wolbachia surface protein (WSP) from the endosymbionts of eight species of filaria. Complete gene sequences were obtained from the endosymbionts of two different species, Dirofilaria immitis and Brugia malayi. These sequences allowed us to design general primers for amplification of the wsp gene from the Wolbachia of all filarial species examined. For these species, partial WSP sequences (about 600 base pairs) were obtained with these primers. Phylogenetic analysis groups these nematode wsp sequences into a coherent cluster. Within the nematode cluster, wsp-based Wolbachia phylogeny matches a previous phylogeny obtained with ftsZ gene sequences, with a good consistency of the phylogeny of hosts (nematodes) and symbionts (Wolbachia). In addition, different individuals of the same host species (Dirofilaria immitis and Wuchereria bancrofti) show identical wsp gene sequences.

A two-phase strategy for detecting recombination in nucleotide sequences

Genetic recombination can produce heterogeneous phylogenetic histories within a set of homologous genes. Delineating recombination events is important in the study of molecular evolution, as inference of such events provides a clearer picture of the phylogenetic relationships among different gene sequences or genomes. Nevertheless, detecting recombination events can be a daunting task, as the performance of different recombination-detecting approaches can vary, depending on evolutionary events that take place after recombination. We previously evaluated the effects of post-recombination events on the prediction accuracy of recombination-detecting approaches using simulated nucleotide sequence data. The main conclusion, supported by other studies, is that one should not depend on a single method when searching for recombination events. In this paper, we introduce a two-phase strategy, applying three statistical measures to detect the occurrence of recombination events, and a Bayesian phylogenetic approach to delineate breakpoints of such events in nucleotide sequences. We evaluate the performance of these approaches using simulated data, and demonstrate the applicability of this strategy to empirical data. The two-phase strategy proves to be time-efficient when applied to large datasets, and yields high-confidence results.

Bellerophon: a program to detect chimeric sequences in multiple sequence alignments

Bellerophon is a program for detecting chimeric sequences in multiple sequence datasets by an adaption of partial treeing analysis. Bellerophon was specifically developed to detect 16S rRNA gene chimeras in PCR-clone libraries of environmental samples but can be applied to other nucleotide sequence alignments.

Specific PCR based detection of Phytophthora medicaginis using the intergenic spacer region of the ribosomal DNA

A technique based on the polymerase chain reaction (PCR) for the specific detection of Phytophthora medicaginis was developed using nucleotide sequence information of the ribosomal DNA (rDNA) regions. The complete IGS 2 region between the 5 S gene of one rDNA repeat and the small subunit of the adjacent repeat was sequenced for P. medicaginis and related species. The entire nucleotide sequence length of the IGS 2 of P. medicaginis was 3566 bp. A pair of oligonucleotide primers (PPED04 and PPED05), which allowed amplification of a specific fragment (364 bp) within the IGS 2 of P. medicaginis using the PCR, was designed. Specific amplification of this fragment from P. medicaginis was highly sensitive, detecting template DNA as low as 4 ng and in a host-pathogen DNA ratio of 1000000:1. Specific PCR amplification using PPED04 and PPED05 was successful in detecting P. medicaginis in lucerne stems infected under glasshouse conditions and field infected lucerne roots. The procedures developed in this work have application to improved identification and detection of a wide range of Phytophthora spp. in plants and soil.

Expressed sequenced tags and new genes from the cattle tick, Boophilus microplus

We used the expressed sequenced tags (ESTs) approach to study the genome of the cattle tick Boophilus microplus. One hundred and forty-two of our 234 unique ESTs were from genes not previously identified from ticks, mites or any other arachnids. The largest class of identified ESTs (29%) was from genes involved in transcription and translation. Ninety-one ESTs (39% of all ESTs) did not match any sequences in international databases; some of these may be specific to ticks. Thirteen percent of our ESTs were from ribosomal proteins and two ESTs were for genes implicated in resistance to pesticides. (C) 1998 Chapman & Hall Ltd.

PCR bias toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Distinguishing species and populations of Rhipicephaline ticks with its 2 ribosomal RNA

Most populations and some species of ticks of the genera Boophilus (5 spp.) and Rhipicephalus (ca. 75 spp.) cannot be distinguished phenotypically. Moreover, there is doubt about the validity of species in these genera. I studied the entire second internal transcribed spacer (ITS 2) rRNA of 16 populations of rhipicephaline ticks to address these problems: Boophilus,microplus from Australia, Kenya, South Africa and Brazil (4 populations); Boophilus decoloratus from Kenya; Rhipicephalus appendiculatus from Kenya, Zimbabwe and Zambia (7 populations); Rhipicephalus zambesiensis from Zimbabwe (3 populations); and Rhipicephalus evertsi from Kenya. Each of the 16 populations had a unique ITS 2, but most of the nucleotide variation occurred among species and genera. ITS 2 rRNA can be used to distinguish the populations and species of Boophilus and Rhipicephalus studied here. Little support was found for the hypothesis that B. microplus from Australia and South Africa are different species. ITS 2 appears useful for phylogenetic inference in the Rhipicephalinae because in genetic distance, maximum likelihood, and maximum parsimony analyses, most branches leading to species had >95% bootstrap support. Rhipicephalus appendiculatus and R, zambeziensis are closely related, yet their ITS 2 sequences could be distinguished unambiguously. This lends weight to a previous proposal that Rhipicephalus sanguineus and Rhipicephalus turanicus, and Rhipicephalus pumlilio and Rhipicephalus camicasi, respectively, are conspecific, because each of these pairs of species had identical sequences for ca. 250 bp of ITS 2 rRNA.

Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.

Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins which contain classical monopartite and bipartite nuclear localization sequences (NLSs), and facilitates their transport into the nucleus. To determine the structural basis of the recognition of the two classes of NLSs by mammalian importin-alpha, we co-crystallized an N-terminally truncated mouse receptor protein with peptides corresponding to the monopartite NLS from the simian virus 40 (SV40) large T-antigen, and the bipartite NLS from nucleoplasmin. We show that the monopartite SV40 large T-antigen NLS binds to two binding sites on the receptor, similar to what was observed in yeast importin-alpha. The nucleoplasmin NLS-importin-alpha complex shows, for the first time, the mode of binding of bipartite NLSs to the receptor. The two basic clusters in the NLS occupy the two binding sites used by the monopartite NLS, while the sequence linking the two basic clusters is poorly ordered, consistent with its tolerance to mutations. The structures explain the structural basis for binding of diverse NLSs to the sole receptor protein. (C) 2000 Academic Press.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

Cryptococcus statzelliae sp nov and three novel strains of Cryptococcus victoriae, yeasts isolated from Antarctic soils

A morphological and physiological characterization of yeast strains CBS 8908, CBS 8915, CBS 8920, CBS 8925(T) and CBS 8926, isolated from Antarctic soils, was performed. Phylogenetic analyses of the sequences of the D1/D2 regions and the adjacent internal transcribed spacer (ITS) regions of the large-subunit rDNA of these strains placed them into the Tremellales clade of the Hymenomycetes. The sequence data identified strains CBS 8908, CBS 8915 and CBS 8920 as belonging to the species Cryptococcus victoriae. Strains CBS 8925(T) and CBS 8926 were found to represent an unique clade within the Hymenomycetes, with Dioszegia crocea CBS 6714(T) being their closest phylogenetic relative. Fatty acid composition and proteome fingerprint data for these novel strains were also obtained. No sexual state was observed. A novel basidiomycetous species, Cryptococcus statzelliae, is proposed for strains CBS 8925(T) and CBS 8926.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae, The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented, A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria, Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group, Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species, A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.