Phylogenetic relationships of Australian strongyloid nematodes inferred from ribosomal DNA sequence data

The sequence of the second internal transcribed spacer of the ribosomal DNA was determined for the following strongyloid nematodes: Cylicocyclus insignis, Chabertia ovina, Oesophagostomum venulosum, Cloacina communis, Cloacina hydriformis, Labiostrongylus labiostrongylus, Parazoniolaimus collaris, Macropostrongylus macropostrongylus, Macropostrongylus yorkei, Rugopharynx australis, Rugopharynx rosemariae, Macropostrongyloides baylisi, Oesophagostomoides longispicularis and Paramacropostrongylus toraliformis, and compared with published sequences for species of Strongylus and for Hypodontus macropi. The resultant phylogenetic trees supported current hypotheses based on morphological evidence for the separation of the families Strongylidae and Chabertiidae, but did not support the separation of the endemic Australian genera as a distinctive clade within the Chabertiidae. The implications of this finding for the phylogenetic origins of the Australian strongyloids are discussed.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

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.     

A comparison of the first internal transcribed spacer of ribosomal DNA in seven species of Trichostrongylus (Nematoda: Trichostrongylidae)

The first internal transcribed spacer (ITS-1) of the ribosomal DNA of seven species of Trichostrongylus was sequenced. The length of ITS-1 in the different species varied from 387 to 390 bases. The G + C content of the ITS-1 sequences were approximately 42%. Little or no intraspecific variation was detected in the three species. Trichostrongylus axei, Trichostrongylus colubriformis and Trichostrongylus vitrinus, for which multiple isolates from different geographical regions were sequenced. In contrast, the level of ITS-1 sequence differences between species ranged from 1.3% to 5.7%. The greatest sequence differences were detected between T. tenuis, the parasite species which infects birds and the six species found in mammals. Some of the nucleotide differences occurred at sites corresponding to recognition sites for restriction endonucleases. These results are compared with previous data obtained for the second internal transcribed spacer (ITS-2). The ITS-1 data indicate that this region of rDNA may also be useful for systematic studies in trichostrongylid nematodes.     

Systematic relationships among pocket gopher chewing lice (Phthiraptera: Trichodectidae) inferred from electrophoretic data

This study was designed to explore the efficacy of gonadotrophin-releasing hormone (GnRH) to antagonize the effect of gonadotrophin surge-inhibiting factor (GnSIF) on the timing of the induction by GnRH of the maximal self-priming effect on pituitary LH responsiveness. The GnSIF levels were increased by FSH treatment and reduced after gonadectomy. Female rats were injected s.c. with 10 IU FSH or saline (control) on three occasions during the 4-day cycle. Serial i.v. injections of GnRH (500 pmol/kg body weight) were administered to intact rats on the afternoon of pro-oestrus or 15-30 min after ovariectomy. Intact male rats were given 10 IU FSH and 500 or 2000 pmol GnRH/kg body weight on an equivalent time-schedule. Endogenous GnRH release was suppressed with phenobarbital. In intact female control rats, the timing of the maximally primed LH response was delayed as the GnRH pulse-interval increased. FSH treatment of female rats induced a suppression of the initial unprimed LH response and delayed the maximally primed LH response, which showed further delay as the GnRH pulse-interval was increased. When the pulsatile administration of GnRH was started 15-30 min after ovariectomy, the priming effect of GnRH did not change as the GnRH pulse-interval was increased in the saline-treated rats. However, FSH treatment caused a suppression of the unprimed LH response, a delay in the primed LH response and decreased the delay of the maximally primed LH response to GnRH when the GnRH pulse-interval was decreased. Increasing the interval between ovariectomy and the first GnRH pulse to 4 h diminished the efficacy of the FSH treatment: GnRH-induced priming was delayed by only one pulse instead of the two pulses in control rats. In intact males but not in orchidectomized rats, a self-priming effect was demonstrated during GnRH pulses which were 1 h apart. The effect of 2 nmol GnRH/kg body weight was the most pronounced. Compared with intact female rats, the timing of the maximally primed LH response was delayed by 1 h. FSH treatment did not affect the pituitary LH response to both dose levels of GnRH. It is concluded that FSH treatment increased the release of GnSIF by the ovary, then induced a state of low responsiveness of the pituitary gland to GnRH and subsequently delayed GnRH-induced maximal self-priming. The efficacy of GnRH to prime the pituitary gland was higher when GnSIF levels were decreasing after removal of the ovaries.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single-strand conformation polymorphism (SSCP)-based mutation scanning approaches to fingerprint sequence variation in ribosomal DNA of ascaridoid nematodes

In this study, we assessed single-strand conformation polymorphism (SSCP)-based approaches for their capacity to fingerprint sequence variation in ribosomal DNA (rDNA) of ascaridoid nematodes of veterinary and/or human health significance. The second internal transcribed spacer region (ITS-2) of rDNA was utilised as the target region because it is known to provide species-specific markers for this group of parasites. ITS-2 was amplified by PCR from genomic DNA derived from individual parasites and subjected to analysis. Direct SSCP analysis of amplicons from seven taxa (Toxocara vitulorum, Toxocara cati, Toxocara canis, Toxascaris leonina, Baylisascaris procyonis, Ascaris suum and Parascaris equorum) showed that the single-strand (ss) ITS-2 patterns produced allowed their unequivocal identification to species. While no variation in SSCP patterns was detected in the ITS-2 within four species for which multiple samples were available, the method allowed the direct display of four distinct sequence types of ITS-2 among individual worms of T. cati. Comparison of SSCP/sequencing with the methods of dideoxy fingerprinting (ddF) and restriction endonuclease fingerprinting (REF) revealed that also ddF allowed the definition of the four sequence types, whereas REF displayed three of four. The findings indicate the usefulness of the SSCP-based approaches for the identification of ascaridoid nematodes to species, the direct display of sequence variation in rDNA and the detection of population variation. The ability to fingerprint microheterogeneity in ITS-2 rDNA using such approaches also has implications for studying fundamental aspects relating to mutational change in rDNA.     

Cross-species amplification of soybean (Glycine max) simple sequence repeats (SSRs) within the genus and other legume genera: implications for the transferability of SSRs in plants

We investigated the transferability of 31 soybean (Glycine max) simple sequence repeat (SSR) loci to wild congeners and to other legume genera. Up to 65% of the soybean primer pairs amplified SSRs within Glycine, but frequently, the SSRs were short and interrupted compared with those of soybeans. Nevertheless, 85% of the loci were polymorphic within G. clandestina. Cross-species amplification outside of the genus was much lower (3%-13%), with polymorphism restricted to one primer pair, AG81. AG81 amplified loci in Glycine, Kennedia, and Vigna (Phaseoleae), Vicia (Vicieae), Trifolium (Trifolieae), and Lupinus (Genisteae) within the Papilionoideae, and in Albizia within the Mimosoideae. The primer conservation at AG81 may be explained by its apparent proximity to the seryl-tRNA synthetase gene. Interspecific differences in allele size at AG81 loci reflected repeat length variation within the SSR region and indels in the flanking region. Alleles of identical size with different underlying sequences (size homoplasy) were observed. Our findings and the emerging patterns in other plant studies suggest that in contrast to animals, successful cross-species amplification of SSRs in plants is largely restricted to congeners or closely related genera. Because mutations in both the SSR region and the flanking region contribute to variation in allele size among species, knowledge of DNA sequence is essential before SSR loci can be meaningfully used to address applied and evolutionary questions.     

The complete mitochondrial DNA sequence of the pig (Sus scrofa)

Familial adenomatous polyposis (FAP) is an autosomal dominant hereditary disorder caused by a germline inactivating mutation of the adenomatous polyposis coli (APC) gene. Patients with FAP sometimes develop various extracolonic manifestations including adrenocortical neoplasms. We present a 14-year-old boy with FAP who had an adrenocortical tumor with atypical histopathologic features, ie, sex-cord-like differentiation. Immunohistochemical studies of adrenal 4 binding protein (Ad4BP) and steroidogenic enzymes showed the capacity of these tumor cells to produce steroids. Genetic analysis of the tumor disclosed a two-hit mutation in APC: a germline 5-base pair deletion accompanied by a loss of the normal allele. Because there were no reports of genetic alterations in adrenocortical tumors developed in FAP patients, we examined 10 sporadic adrenal tumors (four carcinomas and six adenomas) for mutations in APC. However, no mutations were found in these 10 sporadic adrenal tumors. These results suggest that mutation of APC is also responsible for some fraction of the adrenocortical tumors: the tumor in this case is included.     

Instability of quartet analyses of molecular sequence data by the maximum likelihood method: the Cetacea/Artiodactyla relationships

As laparoscopic techniques show definite advantages in terms of pain and postoperative pulmonary function, high-risk patients especially benefit from such procedures. Pathophysiological changes caused by the pneumoperitoneum can be managed with invasive monitoring and resulting therapy. The increase in systemic vascular resistance with reduction in cardiac output can be kept to a minimum using low intraabdominal pressures (8-10 mmHg) under adequate muscular relaxation. Additional peripheral vasodilators and positive inotropic medication may be necessary. As the adverse hemodynamic effects end almost instantaneously with the reduction of the intraabdominal pressure, it is allways possible to revert to an open procedure after a laparoscopic try. If ventilation can not be increased adequately to maintain isocapnia in a patient suffering from pulmonary disease, the resulting increase in paCO2 will usually be moderate. In some instances it may be necessary to prolong artificial ventilation postoperatively, until isocapnia is reached with minuteventilation as at the beginning of anaesthesia.     

Observations on the morphology, systematics, and biogeography of the genus Truttaedacnitis (Nematoda:Cucullanidae)

All species of Truttaedacnitis Petter, 1974, were examined for a phylogenetic analysis. Morphological studies showed that Truttaedacnitis truttae (Fabricius, 1794) from North America possessed a reduced button-shaped or nipple-shaped caudal mucron that is in contrast to the sharp spike-shaped mucron commonly reported in Eurasian specimens. Histological studies showed that the excretory pore in Truttaedacnitis sphaerocephala (Rudolphi, 1809) was situated near the level of the pseudobuccal capsule between the 2 parts of the ventral cephalic ridge. Cucullanus heterodonti Johnston and Mawson, 1943 is transferred to Truttaedacnitis based on the presence of prominent cephalic plates separated by sutures. An hypothesis regarding interrelationships among Truttaedacnitis spp. was developed using phylogenetic systematics. The anterior excretory pore is a synapomorphy supporting the monophyly of Truttaedacnitis clitellarius (Ward and Magath, 1917) and T. sphaerocephala parasitizing sturgeons. The arrangement of cephalic plates indicates that Truttaedacnitis pybusae Anderson, 1992 and T. truttae may form a monophyletic group and further supports the monophyly of T. clitellarius and T. sphaerocephala. Using unordered character states of different cephalic plates produced a cladogram that suggested that species in holarctic/circumboreal families (Acipenseridae, Salmonidae, Petromyzontidae) may be monophyletic. The systematics and biogeography of the various species of Truttaedacnitis and their host specificity indicate that associations in this parasite genus are very ancient and of a relictual nature, determined largely by past continental and oceanic configurations, and a combination of vicariance and dispersal.     

BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data

We propose an improved version of the neighbor-joining (NJ) algorithm of Saitou and Nei. This new algorithm, BIONJ, follows the same agglomerative scheme as NJ, which consists of iteratively picking a pair of taxa, creating a new mode which represents the cluster of these taxa, and reducing the distance matrix by replacing both taxa by this node. Moreover, BIONJ uses a simple first-order model of the variances and covariances of evolutionary distance estimates. This model is well adapted when these estimates are obtained from aligned sequences. At each step it permits the selection, from the class of admissible reductions, of the reduction which minimizes the variance of the new distance matrix. In this way, we obtain better estimates to choose the pair of taxa to be agglomerated during the next steps. Moreover, in comparison with NJ's estimates, these estimates become better and better as the algorithm proceeds. BIONJ retains the good properties of NJ--especially its low run time. Computer simulations have been performed with 12-taxon model trees to determine BIONJ's efficiency. When the substitution rates are low (maximum pairwise divergence approximately 0.1 substitutions per site) or when they are constant among lineages, BIONJ is only slightly better than NJ. When the substitution rates are higher and vary among lineages,BIONJ clearly has better topological accuracy. In the latter case, for the model trees and the conditions of evolution tested, the topological error reduction is on the average around 20%. With highly-varying-rate trees and with high substitution rates (maximum pairwise divergence approximately 1.0 substitutions per site), the error reduction may even rise above 50%, while the probability of finding the correct tree may be augmented by as much as 15%.     

Molecular phylogeny of the families Campulidae and Nasitrematidae (Trematoda) based on mtDNA sequence comparison

The heavy and light chains of IgG monoclonal antibodies (mAbs) can be shown to be heterogeneous, with respect to isoelectric points, when analyzed by two-dimensional electrophoresis (2-DE). The molecular basis for this charge heterogeneity has not been clearly defined but it has been suggested that it could be due, in part, to differences in glycosylation. To investigate this possibility we have compared the 2-DE pattern of glycosylated and aglycosylated forms of the mouse IgG1 mAb (1B7-11), produced in vitro in the presence and absence of tunicamycin. Charge heterogeneity was shown not to be a consequence of glycosylation status. Intracellular and secreted IgG mAbs were also analyzed to investigate the time course of change in charge properties of the heavy and light chains. The charge heterogeneity was found to be generated intracellularly, and alterations in charge properties could be induced during incubation under physiological conditions. Semilogarithmic plots of the density of the principal heavy and light chain spots against incubation time showed linear relationships, suggesting that the charge shifts result from a first-order reaction. The semilogarithmic plot for the light chain correlated well with the time after IgG synthesis. These results suggest that the charge heterogeneity of an IgG mAb is due to intra- and extracellular modifications of the polypeptide chains which reflect "aging" of antibody molecules.     

Phylogenetic relationships among tick subfamilies (Ixodida: Ixodidae: Argasidae) based on the 18S nuclear rDNA gene

A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.     

Molecular phylogeny of forest and field mice of the genus Apodemus (Muridae, Rodentia) based on the data on restriction analysis of total nuclear DNA

Based on restriction-fragment length polymorphism (RFLP) of total nuclear DNA (nDNA), analyses of phylogenetic relations and genetic similarity were performed in nine species of forest and field mice of the genus Apodemus. Genetic distances calculated for different species pairs ranged from 0.24 to 12.53%; i.e., the differences were 50-fold. The estimated evolutionary age of the genus Apodemus is approximately 12 million years. In general, the obtained data on genetic similarity and phylogenetic relationship allow us to differentiate at least three groups of species: (1) southern Paleoarctic (A. argenteus), (2) eastern (A. peninsulae, A. speciosus, and A. agrarius), and (3) western (A. sylvaticus, A. flavicollis, A. ponticus, A. uralensis, and A. fulvipectus) ones. The latter two groups are related to the northern Paleoarctic. Such a division into groups corresponds to characteristic features of karyotype organization and segmentation of satellite DNA (satDNA) of these species, as well as the nature of variation in isozymes and in a fragment of the enzyme-encoding sequence of cytochrome b gene isolated from the mitochondrial genome. Species groups (1) and (3) exhibited a high probability of a monophyletic origin (70 and 99%, respectively). Group (2) is unlikely to be monophyletic, and the genetic distances in it are significantly greater than those in group 3. A. argenteus is the most diverged, both phenogenetically and phylogenetically. The data are consistent with a new zoological classification, which assumes the division of the unified genus Apodemus into two taxa of generic rank and suggest that the southern Paleoarctic forest mouse should be regarded as a separate taxon of at least subgeneric rank.     

A highly repetitive sequence isolated from genomic DNA of the medaka (Oryzias latipes)

In order to investigate the relationship between various temporomandibular joint (TMJ) pain levels and the detection of high signal intensity (joint effusion) on T2 weighted magnetic resonance imaging (MRI), 19 consecutive patients who complained of unilateral painful TMJ hypomobility (closed locking) were involved in this study. All patients were clinically examined in a routine manner, and all patients rated their pain levels by a visual analogue scale and eight pain questionnaire prior MRI study. T1 and T2 weighted MRI was taken in sagittal section at unilateral affected joint side. The presence or absence of a high signal intensity spot within the TMJ compartment were judged by three examiners. The high signal intensity was detected in 10 joints, but not in 9 joints. In between these two groups, the pain ratio was calculated and compared. The data showed that there was no significant statistical correlation between pain levels and the presence of high signals. This study disclosed that the MRI detection of high signal intensity in the closed locking TMJ did not directly relate to the presence of TMJ pain nor the increased pain level. These indicate the need of further larger studies.     

Allozyme and DNA sequence data support speciation of northern and southern populations of silver catfish, Schilbe intermedius (Rüppel, 1832)

Patterns of genetic variation in Schilbe intermedius were investigated due to morphological differences and taxonomic uncertainties regarding the Southern African schilbeids. A total of three populations, two Southern populations representing the former Eutropius depressirostris and a Northern population representing S. mystus, were electrophoretically analysed to determine the extent of genetic differentiation among these populations. The Northern and Southern populations were fixed for different alleles at the G3PDH-2 protein coding locus and allozyme differentiation between populations, using the 0.95 criterion, were also encountered at the PGDH-2 locus. Genetic distance values indicate greater genetic differentiation between the Northern and Southern populations compared to the two Southern populations. DNA sequence analysis of 900-1000 nucleotides of the cytochrome b gene revealed distances of 3.2-3.5% between the Schilbe/Eutropius complex. This finding, together with ingroup and outgroup analysis of evolutionary relationships, is congruent with the results from the electrophoretic analysis of the taxa. Sufficient differentiation exist between the Northern and Southern populations to regard them as distinct species.