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

Immature bivesiculid trematodes collected from the intestine of Thalassoma 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. (C) 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. All rights reserved.

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.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences; In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

Gene and genotypic diversity of Phytophthora cinnamomi in South Africa and Australia revealed by DNA polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D-m = 0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D-m South Africa = 0.020 and D-m Australia = 0.025 respectively), negative fixation indices, and significant deviations from Hardy-Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.

Molecular characterization of the toxic cyanobacterium Cylindrospermopsis raciborskii and design of a species-specific PCR

Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C, raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.

Activation of the human diaphragm during a repetitive postural task

1. The co-ordination between respiratory and postural functions of the diaphragm was investigated during repetitive upper Limb movement. It was hypothesised that diaphragm activity would occur either tonically or phasically in association with the forces from each movement and that this activity would combine with phasic respiratory activity. 2. Movements of the upper limb and ribcage were measured while standing subjects performed repetitive upper limb movements 'as fast as possible'. Electromyographic (EMG) recordings of the costal diaphragm were made using intramuscular electrodes in four subjects. Surface electrodes were placed over the deltoid and erector spinae muscles. 3. In contrast to standing at rest, diaphragm activity was present throughout expiration at 78 +/- 17% (mean +/- S.D.) of its peak inspiratory magnitude during repeated upper limb movement. 4. Bursts of deltoid and erector spinae EMG activity occurred at the Limb movement frequency (similar to 2.9 Hz). Although the majority of diaphragm EMG power was at the respiratory frequency (similar to 0.4 Hz), a peak was also present at the movement frequency. This finding was corroborated by averaged EMG activity triggered from upper limb movement. In addition, diaphragm EMG activity was coherent with ribcage motion at the respiratory frequency and with upper limb movement at the movement frequency. 5. The diaphragm response was similar when movement was performed while sitting. In addition, when subjects moved with increasing frequency the peak upper limb acceleration correlated with diaphragm EMG amplitude. These findings support the argument that diaphragm contraction is related to trunk control. 6. The results indicate that activity of human phrenic motoneurones is organised such that it contributes to both posture and respiration during a task which repetitively challenges trunk posture.

Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.

Chromosomal fragile site FRA16D and DNA instability in cancer

It has been proposed that common aphidicolin-inducible fragile sites, in general, predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Although this appears to be the case for the fragile site FRA3B and may be the case for FRA7G, it is not Set clear whether this association is a general property of this class of fragile site. The major aim of the present study was to determine whether the FRA16D chromosomal fragile site locus has a role to play in predisposing DNA sequences within and adjacent to the fragile site to DNA instability (such as deletion or translocation), which could lead to or be associated with neoplasia. We report the localization of FRA16D within a contig of cloned DNA and demonstrate that this fragile site coincides with a region of homozygous deletion in a gastric adenocarcinoma cell line and is bracketed by translocation breakpoints in multiple myeloma, as reported previously (Chesi, M., et al., Blood, 91: 4457-4463, 1998), Therefore, given similar findings at the FRA3B and FRA7G fragile sites, it is likely that common aphidicolin-inducible fragile sites exhibit the general property of localized DNA instability in cancer cells.

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-I DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP. (C) 2000 Elsevier Science B.V. All rights reserved.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.

Characterisation of Australian isolates of Fusarium oxysporum f. sp cubense by DNA fingerprinting analysis

Genetic variation among Australian isolates of the fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt in banana, was examined using DNA amplification fingerprinting (DAF). Ninety-four isolates which represented Races 1, 2, 3, and 4, and vegetative compatibility groups (VCGs) 0120, 0124, 0125, 0128, 0129, 01211, 01213/16, and 01220 were analysed. The genetic relatedness among isolates within each VCG, and between the 8 different VCGs of Foc present in Australia was determined. The DNA fingerprint patterns were VCG-specific, with each VCG representing a unique genotype. The genetic similarity among isolates within each VCG ranged from 97% to 100%. Among the different VCGs of Foc, 3 major clusters were distinguished which corresponded with race. All Race 1 and 2 isolates (VCGs 0124, 0125, 0128, and 01220) were closely related and clustered together, the Race 3 isolates from Heliconia clustered separately, and all Race 4 isolates (VCGs 0120, 0129, 01211, and 01213/16) clustered together. Fifteen isolates from Alstonville, NSW, were characterised because although they were classified as Race 2 based on their recovery from cooking banana cultivars, they belonged in VCG 0124, which had previously contained only Race 1 isolates. The occurrence of more than one race within a VCG means that vegetative compatibility grouping cannot be used to assign pathotype to pathogenic race as previously thought. It was possible to distinguish the Race 1 and Race 2 isolates within VCG 0124 using DNA fingerprinting, as each race produced a unique DNA fingerprint pattern. Among the Australian isolates, DNA fingerprinting analysis identified 9 different VCGs and genotypes of Foc.

Polyinosinic acid and polycationic liposomes attenuate the hepatic clearance of circulating plasmid DNA

DNA that enters the circulation is rapidly cleared both by tissue uptake and by DNase-mediated degradation. In this study, we have examined the uptake of linear plasmid DNA in an isolated perfused liver model and following intra-arterial administration to rats. We found that the DNA was rapidly taken up by the isolated perfused liver without degradation. The single-pass extraction ratio was 0.76 +/- 0.05, the mean transit time was 15.3 +/- 3.6 s, and the volume of distribution was 0.29 +/- 0.07 ml/g. Hepatic uptake was saturable and was inhibited by polyinosinic acid or polycationic liposomes but not by condensation of the DNA with polylysine. When the linear plasmid DNA was administered in vivo, plasma half-life was 3.1 +/- 0.2 min, volume of distribution was 670 +/- 85 ml/kg, and clearance was 32 +/- 4 min. Coadministration of cationic liposomes decreased the volume of distribution to 180 +/- 28 ml/kg as well as the half-life (2.6 +/- 0.2 min). By contrast, polyinosinic acid significantly increased the circulating half-life (7.7 +/- 0.5 min), decreased the volume of distribution (95 +/- 17 ml/kg), and partially inhibited DNA degradation. When administered along with the liposomes and the polyinosinic acid, the distribution of plasmid-derived radioactivity decreased in the liver and increased in most other peripheral tissues. This study shows that pharmacological manipulation of the uptake and degradation of DNA can alter its distribution and clearance in vivo. These results may be useful in optimizing gene delivery procedures for in vivo gene therapy.

A preliminary phylogeny of the scale insects (Hemiptera : Sternorrhyncha : Coccoidea) based on nuclear small-subunit ribosomal DNA

Scale insects (Hemiptera: Sternorrhyncha: Coccoidea) are a speciose and morphologically specialized group of plant-feeding bugs in which evolutionary relationships and thus higher classification are controversial. Sequences derived from nuclear small-subunit ribosomal DNA were used to generate a preliminary molecular phylogeny for the Coccoidea based on 39 species representing 14 putative families. Monophyly of the archaeococcoids (comprising Ortheziidae, Margarodidae sensu lato, and Phenacoleachia) was equivocal, whereas monophyly of the neococcoids was supported. Putoidae, represented by Puto yuccae, was found to be outside the remainder of the neococcoid clade. These data are consistent with a single origin (in the ancestor of the neococcoid clade) of a chromosome system involving paternal genome elimination in males. Pseudococcidae (mealybugs) appear to be sister to the rest of the neococcoids and there are indications that Coccidae (soft scales) and Kerriidae (lac scales) are sister taxa. The Eriococcidae (felt scales) was not recovered as a monophyletic group and the eriococcid genus Eriococcus sensu lato was polyphyletic. (C) 2002 Elsevier Science (USA). All rights reserved.