Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

Ancient mitochondrial DNA and morphology elucidate an extinct island radiation of Indian Ocean giant tortoise (Cylindrapsis)

Ancient mitochondrial DNA sequences were used for investigating the evolution of an entire clade of extinct vertebrates, the endemic tortoises (Cylindraspis) of the Mascarene Islands in the Indian Ocean. Mitochondrial DNA corroborates morphological evidence that there were five species of tortoise with the following relationships: Cylindraspis triserrata ((Cylindraspis vosmaeri and Cylindraspis peltastes) (Cylindraspis inepta and Cylindraspis indica)). Phylogeny indicates that the ancestor of the group first colonized Mauritius where speciation produced C. triserrata and the ancestor of the other species including a second sympatric Mauritian form, C. inepta. A propagule derived from this lineage colonized Rodrigues 590 km to the east, where a second within-island speciation took place producing the sympatric C. vosmaeri and C. peltastes. A recent colonization of Réunion 150 km to the southwest produced C. indica. In the virtual absence of predators, the defensive features of the shells of Mascarene tortoises were largely dismantled, apparently in two stages. 'Saddlebacked' shells with high fronts evolved independently on all three islands. This and other features, such as a derived jaw structure and small body size, may be associated with niche differentiation in sympatric species and may represent a striking example of parallel differentiation in a large terrestrial vertebrate. The history of Mascarene tortoises contrasts with that of the Galápagos, where only a single species is present and surviving populations are genetically much more similar. However, they too show some reduction in anti-predator mechanisms and multiple development of populations with saddlebacked shells.

Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice

Type I diabetes is thought to occur as a result of the loss of insulin-producing pancreatic beta cells by an environmentally triggered autoimmune reaction. In rodent models of diabetes, streptozotocin (STZ), a genotoxic methylating agent that is targeted to the beta cells, is used to trigger the initial cell death. High single doses of STZ cause extensive beta -cell necrosis, while multiple low doses induce limited apoptosis, which elicits an autoimmune reaction that eliminates the remaining cells. We now show that in mice lacking the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG), beta -cell necrosis was markedly attenuated after a single dose of STZ. This is most probably due to the reduction in the frequency of base excision repair-induced strand breaks and the consequent activation of poly(ADP-ribose) polymerase (PARP), which results in catastrophic ATP depletion and cell necrosis. Indeed, PARP activity was not induced in A-PNG(-/-) islet cells following treatment with STZ in vitro. However, 48 h after STZ treatment, there was a peak of apoptosis in the beta cells of APNG(-/-) mice. Apoptosis was not observed in PARP-inhibited APNG(+/+) mice, suggesting that apoptotic pathways are activated in the absence of significant numbers of DNA strand breaks. Interestingly, STZ-treated APNG(-/-) mice succumbed to diabetes 8 months after treatment, in contrast to previous work with PARP inhibitors, where a high incidence of beta -cell tumors was observed. In the multiple-low-dose model, STZ induced diabetes in both APNG(-/-) and APNG(-/-) mice; however, the initial peak of apoptosis was 2.5-fold greater in the APNG(-/-) mice. We conclude that APNG substrates are diabetogenic but by different mechanisms according to the status of APNG activity.

Identifying the origin of single corneal cells by DNA fingerprinting - Part I - Implications for corneal limbal allografting

Purpose. To demonstrate that the combination of impression cytology and single cell DNA fingerprinting represents a powerful tool that is suitable for detecting transplanted cells after corneal limbal allografting. Methods, Fifty single cells were obtained by corneal impression cytology from 12 patients undergoing cataract surgery. Individual cells were isolated from samples by micromanipulation. Polymerase chain reaction and short tandem repeat profiling was used to obtain forensic standard DNA fingerprints from single cells. Blood samples taken at the time of impression cytology provided control fingerprints. Results, informative DNA fingerprints were obtained from all corneal samples and 66% (33 of 50 cells) of isolated single cells, Of all fingerprints obtained, most (91%, 30 of 33 fingerprints) corneal fingerprints matched corresponding blond sample fingerprints. At least one corneal fingerprint matched the corresponding blood sample fingerprint in 83% (10 of 12 patients) of the patients in the study, Conclusions. This extremely specific single cell DNA fingerprinting system permits accurate identification of individual corneal epithelial cells, allowing very reliable determination of their origin, which will enable host and donor cells to be distinguished from each other after keratolimbal allografting procedures. even if the host and donor are the same sex or siblings. These DNA fingerprinting methods allow assessment of quality and quantity of donor cell survival, as well as survival time. The extreme sensitivity and accuracy of the technique means that should contamination occur, it would be identified, thus ensuring meaningful results.

Replication of bovine papillomavirus type 1 (BPV-1) DNA in Saccharomyces cerevisiae following infection with BPV-1 virions

Saccharomyces cerevisiae protoplasts exposed to bovine papillomavirus type 1 (BPV-1) virions demonstrated uptake of virions on electron microscopy. S. cerevisiae cells looked larger after exposure to BPV-1 virions, and cell wall regeneration was delayed. Southern blot hybridization of Hirt DNA from cells exposed to BPV-1 virions demonstrated BPV-1 DNA, which could be detected over 80 days of culture and at least 13 rounds of division. Two-dimensional gel analysis of Hirt DNA showed replicative intermediates, confirming that the BPV-1 genome was replicating within S. cerevisiae. Nicked circle, linear, and supercoiled BPV-1 DNA species were observed in Hirt DNA preparations from S. cerevisiae cells infected for over 50 days, and restriction digestion showed fragments hybridizing to BPV-1 in accord with the predicted restriction map for circular BPV-1 episomes. These data suggest that BPV-1 can infect S. cerevisiae and that BPV-1 episomes can replicate in the infected S. cerevisiae cells.

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57X FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells. (C) 2002 Elsevier Science (USA).

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer. (C) 2002 Elsevier Science Ltd. All rights reserved.

Rapid isolation of total RNA and genomic DNA from Hakea actities

Tissues of the Australian native plant species Hakea actities (Proteaceae) contain numerous metabolites and structural compounds that hinder the isolation of nucleic acids. Separate RNA and genomic DNA extraction procedures were developed to isolate high quality nucleic acids from H. actities. Total RNA was extracted from leaves, roots and cluster roots of H. actities grown in low nutrient levels. Cluster root formation in H. actities only occurs when the plants are grown in low nutrient concentrations. However, under these conditions, nucleic acid extraction becomes increasingly difficult. The new procedures are faster than many of the published nucleic acid extraction protocols, and avoid the use of hazardous chemicals. The RNA extraction method was used successfully on another Australian species and a crop species, suggesting that the procedure is useful for molecular studies of a broad range of plants.

Categorization and characterization of transcript-confirmed constitutively and alternatively spliced introns and exons from human

By spliced alignment of human DNA and transcript sequence data we constructed a data set of transcript-confirmed exons and introns from 2793 genes, 796 of which (28%) were seen to have multiple isoforms. We find that over one-third of human exons can translate in more than one frame, and that this is highly correlated with G+C content. Introns containing adenosine at donor site position +3 (A3), rather than guanosine (G3), are more common in low G+C regions, while the converse is true in high G+C regions. These two classes of introns are shown to have distinct lengths, consensus sequences and correlations among splice signals, leading to the hypothesis that A3 donor sites are associated with exon definition, and G3 donor sites with intron definition. Minor classes of introns, including GC-AG, U12-type GT-AG, weak, and putative AG-dependant introns are identified and characterized. Cassette exons are more prevalent in low G+C regions, while exon isoforms are more prevalent in high G+C regions. Cassette exon events outnumber other alternative events, while exon isoform events involve truncation twice as often as extension, and occur at acceptor sites twice as often as at donor sites. Alternative splicing is usually associated with weak splice signals, and in a majority of cases, preserves the coding frame. The reported characteristics of constitutive and alternative splice signals, and the hypotheses offered regarding alternative splicing and genome organization, have important implications for experimental research into RNA processing. The 'AltExtron' data sets are available at http://www.bit.uq.edu.au/altExtron/ and http://www.ebi.ac.uk/similar tothanaraj/altExtron/.

Cationic drug pharmacokinetics in diseased livers determined by fibrosis index, hepatic protein content, microsomal activity, and nature of drug

The disposition kinetics of six cationic drugs in perfused diseased and normal rat livers were determined by multiple indicator dilution and related to the drug physicochemical properties and liver histopathology. A carbon tetrachloride (CCl4)induced acute hepatocellular injury model had a higher fibrosis index (FI), determined by computer-assisted image analysis, than did an alcohol-induced chronic hepatocellular injury model. The alcohol-treated group had the highest hepatic alpha(1)- acid glycoprotein, microsomal protein (MP), and cytochrome P450 (P450) concentrations. Various pharmacokinetic parameters could be related to the octanol-water partition coefficient (log P-app) of the drug as a surrogate for plasma membrane partition coefficient and affinity for MP or P450, the dependence being lower in the CCl4-treated group and higher in the alcohol-treated group relative to controls. Stepwise regression analysis showed that hepatic extraction ratio, permeability-surface area product, tissue-binding constant, intrinsic clearance, partition ratio of influx (k(in)) and efflux rate constant (k(out)), and k(in)/k(out) were related to physicochemical properties of drug (log P-app or pK(a)) and liver histopathology (FI, MP, or P450). In addition, hepatocyte organelle ion trapping of cationic drugs was evident in all groups. It is concluded that fibrosis-inducing hepatic disease effects on cationic drug disposition in the liver may be predicted from drug properties and liver histopathology.

Molecular phylogenetics of Lentibulariaceae inferred from plastid rps16 Intron and trnL-F DNA sequences: implications for character evolution and biogeography

Phylogenetic relationships among 75 species of Lentibulariaceae, representing the three recognized genera, were assessed by cladistic analysis of DNA sequences from the plastid rps16 intron and the trnL-F region. Sequence data from the two loci were analyzed both separately and in combination. Consensus trees from all analyses are congruent, and parsimony jackknife results demonstrate strong support for relationships both between and within each of the three demonstrably monophyletic genera. The genus Pinguicula is sister to a Genlisea-Utricularia clade, the phylogenetic structure within this clade closely follows Taylor's recent sectional delimitations based on morphology. Three principal clades are shown within Utricularia, with the basal sections Polypoinpholyx and Pleiochasia together forming the sister lineage of the remaining Utricularia species. Of the fundamental morphological specializations, the stoloniferous growth form apparently arose independently within Genlisea and Utricularia three times, and within Utricularia itself, perhaps more than once. The epiphytic habit has evolved independently at least three times, in Pinguicula, in Utricularia section Phyllaria, and within the two sections Orchidioides and Iperua (in the latter as bromeliad tank-epiphytes). The suspended aquatic habit may have evolved independently within sections Utricularia and Vesiculina. Biogeographic optimization on the phylogeny demonstrates patterns commonly associated with the boreotropics hypothesis and limits the spatial origin of Lentibulariaceae to temperate Eurasia or tropical America.