Molecular identification and first description of the male of Neoechinorhynchus schmidti (Acanthocephala: Neoechinorhynchidae), a parasite of Trachemys scripta (Testudines) in México.

The morphology of the males of Neoechinorhynchus schmidti (Acanthocephala: Neoechinorhynchidae) is unknown, because this species was described based exclusively on females. However, recently we collected 2 common slider turtles Trachemys scripta in Centla swamps, Tabasco, Mexico, parasitized by 27 specimens of an acanthocephalan whose females were morphologically identical to N. schmidti. The domains D2 and D3 of the large subunit of the nuclear ribosomal RNA (LSU) of 3 males and 2 females of this material were sequenced. The sequences of both sexes were identical, and based on this result, we described for the first time the morphology of the males of N. schmidti. In addition, 6 sequences of a congeneric species, also parasite of turtles (Neoechinorhynchus emyditoides) were generated in the current research. The 11 sequences of these 2 species were aligned with 13 sequences of another 4 species of the same genus, producing a data set of 24 taxa with 674 nucleotides. The genetic divergence between N. schmidti and N. emyditoides was 4% and intraspecific differences ranged from 0.01 to 0.02%. Pairwise differences between either of these species and 4 other congeners parasitic in fresh and brackish water fishes (Neoechinorhynchus golvani, Neoechinorhynchus roseum, Neoechinorhynchus saginatus, and Neoechinorhynchus sp.) varied from 9.5 to 33%. Maximum likelihood and maximum parsimony analyses show that N. schmidti and N. emyditoides are sister taxa. Bootstrap analysis also indicates that the sister relationship is reliably supported.

DNA synthesis generates terminal duplications that seal end-to-end chromosome fusions.

End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. For more than 70 years, these duplications have been attributed solely to breakage-fusion-bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from Caenorhabditis elegans telomere replication mutants. Genome-level rearrangements revealed fused chromosome ends having interrupted terminal duplications accompanied by template-switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, noncontiguous segments of the genome occurs. Thus, a DNA synthesis-based process may create duplications that seal end-to-end fusions, in the absence of breakage-fusion-bridge cycles.

Caenorhabditis elegans as a model system for mtDNA replication defects.

Since the isolation and physical characterization of mammalian mitochondrial DNA (mtDNA) over 35 years ago, numerous studies have been conducted in order to understand its structure and properties, including mode of mtDNA replication and transcription. Even today, the mode of mtDNA replication is still a matter of intense debate. We believe that Caenorhabditis elegans holds the promise of identifying molecular mechanisms of mitochondrial replication. C. elegans is a simple and extremely powerful genetic and developmental model system. Their small size, rapid life cycle, the ability to self-fertilize and somatic tissues that consist of post-mitotic cells offer an efficient way to study mitochondrial metabolism. We have recently developed a number of methods in order to study mitochondrial DNA level and mtDNA maintenance during the development of C. elegans. We hope that the techniques described here can assist laboratories interested in understanding modes of mtDNA replication, distribution and mitochondrial morphology in C. elegans.

Differential timing of S phases, X chromosome replication, and meiotic prophase in the C. elegans germ line.

The replication of chromosomes in meiosis is an important first step for subsequent chromosomal interactions that promote accurate disjunction in the first of two segregation events to generate haploid gametes. We have developed an assay to monitor DNA replication in vivo in mitotic and meiotic germline nuclei of the nematode Caenorhabditis elegans. Using mutants that affect the mitosis/meiosis switch, we show that meiotic S phase is at least twice as long as mitotic S phase in C. elegans germ cell nuclei. Furthermore, our assay reveals that different regions of the genome replicate at different times, with the heterochromatic-like X chromosomes replicating at a distinct time from the autosomes. Finally, we have exploited S-phase labeling to monitor the timing of progression through meiotic prophase. Meiotic prophase for oocyte production in hermaphrodites lasts 54-60 h. Further, we find that the duration of the pachytene sub-stage is modulated by the presence of sperm. On the other hand, meiotic prophase for sperm production in males is completed by 20-24 h. Possible sources for the sex-specific differences in meiotic prophase kinetics are discussed.

MEL-47, a novel protein required for early cell divisions in the nematode Caenorhabditis elegans.

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50-80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo.

Herbimycin A suppresses mitotic activity and egg production of female Schistosoma mansoni.

The eggs of the endoparasite Schistosoma are the causative agent of schistosomiasis, an important disease of humans, which is endemic in (sub-) tropical regions. The absence of a vaccine with sufficient protective qualities and increasing resistance to approved and established drugs like praziquantel, justify the exploration of novel ways to fight schistosomes. Our strategy is based on interference with the sexual maturation of the female. Prerequisites for gonad development in adult females are a continuous pairing contact with the male and significantly increased mitotic activity. In this study we show that the male governs sexual maturation of the female, as the separation of couples causes a clear reduction of female mitotic activity and, consequently, egg production. We demonstrate that treatment of schistosomes with Herbimycin A, an inhibitor of protein tyrosine kinases (PTKs), mimics the separation of couples as the drug blocks mitotic activity and egg production of paired females. However, the synthesis of the eggshell precursor protein p14 is elevated. Furthermore, we show for the first time in invertebrates that Herbimycin A decreases tyrosine phosphorylation and PTK stability in schistosomes. Summarised, our data provide evidence that PTKs have key functions in regulating gonad development, eggshell gene expression and, consequently, egg production. Therefore, we suggest envisaging schistosome PTKs as novel targets for strategies to combat schistosomiasis.

Coordinates, DNA content and heterogeneity of cell nuclei and segments of the Caenorhabditis elegans intestine.

A specific protocol allowed to selectively stain and image the cell nuclei of the intestine of the nematode Caenorhabditis elegans. Digital processing of these images yielded quantitative information on the coordinates and the relative DNA content of these cell nuclei. With this technique, the exact locations (in relation to the length of worm or intestine) of the 20 intestinal cell nuclei of larval stage L1 and of the 34 intestinal cell nuclei of larval stages L2-4 and adults were determined. Tracking the DNA content of an individual intestinal cell nuclei allowed to study nuclear DNA endoreduplications during larval growth. The evaluation of the DNA content of all intestinal cell nuclei revealed a strong heterogeneity between the cell nuclei and intestinal segments with a maximum in segments int4-int5 in the L1 stage and int5-int6 in the L2-L4 stages. Minimum values were detected in the segments int2 and int8-int9 of all larval stages. Heterogeneity between the intestinal segments of C. elegans does not only concern the DNA content of cell nuclei, but also other cellular features like the quantity of intracellular vesicles or the absorption of particles out of the intestinal lumen.

Early post-larval development of the endoparasitic platyhelminth Mesocestoides corti: trypsin provokes reversible tegumental damage leading to serum-induced cell proliferation and growth.

Mesocestoides corti is a suitable in vitro model for studying the development of human endoparasitic platyhelminthes. Treatment with trypsin, supplemented with fetal bovine serum (FBS), induces M. corti development from larvae (tetrathyridia) to segmented adult worm; however, the role of this protease and of FBS in post-larval development induction remains unknown. To characterize the participation of trypsin enzymatic activity and of FBS in the induction of tetrathyridia growth and development, both stimuli were added to the larvae either together or sequentially. Additionally, specific inhibition of trypsin activity was also monitored. Finally, the effect of the enzyme on the parasite tegument as well as the proliferative activity and location of proliferating cells after induction of tetrathyridia development were also studied. We conclude that trypsin-induced tetrathyridia development to adult worm is FBS-dependent and that the effect of serum factors is dependent upon a previous trypsin-induced reversible damage to the larva tegument. In dividing and non-dividing tetrathyridia, proliferative activity of cells is mainly located within the apical massif in the anterior region and nerve cords of larvae, respectively. In tetrathyridia stimulated to develop to adult worms, an intense proliferative activity is evident along the nerve cords. Our results suggest that in natural infections the tetrathyridia tegument is temporally made permeable to growth factors by proteolytic enzyme activity in the intestine juice of the definitive host, thus leading to development to adult worms.

CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing.

To maintain genome stability, DNA replication is strictly regulated to occur only once per cell cycle. In eukaryotes, the presence of 'licensing proteins' at replication origins during the G1 cell-cycle phase allows the formation of the pre-replicative complex. The removal of licensing proteins from chromatin during the S phase ensures that origins fire only once per cell cycle. Here we show that the CUL-4 ubiquitin ligase temporally restricts DNA-replication licensing in Caenorhabditis elegans. Inactivation of CUL-4 causes massive DNA re-replication, producing cells with up to 100C DNA content. The C. elegans orthologue of the replication-licensing factor Cdt1 (refs 2, 3) is required for DNA replication. C. elegans CDT-1 is present in G1-phase nuclei but disappears as cells enter S phase. In cells lacking CUL-4, CDT-1 levels fail to decrease during S phase and instead remain constant in the re-replicating cells. Removal of one genomic copy of cdt-1 suppresses the cul-4 re-replication phenotype. We propose that CUL-4 prevents aberrant re-initiation of DNA replication, at least in part, by facilitating the degradation of CDT-1.

Dna2 requirement for normal reproduction of Caenorhabditis elegans is temperature-dependent.

To determine the function of Dna2 in a multicellular organism, the Caenorhabditis elegans Dna2 expression was probed and deletion mutant phenotypes were analyzed. Dna2 was localized to the nuclei of C. elegans oocytes and early embryos by immunostaining or green fluorescent protein-tagging. A homozygous dna2 deletion mutant showed a reduced brood size and embryonic lethality, and the phenotypes greatly depended on growth temperature and aggravated in the succeeding generation. The mutant embryos also showed delayed cell divisions, which together with temperature-dependence of the mutant phenotypes supported the well-conserved role of Dna2 in DNA replication.

New telomere formation during the process of chromatin diminution in Ascaris suum.

Chromatin diminution in the parasitic nematode Ascaris suum represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. At the molecular level, it is a rather complex event including chromosome breakage, new telomere formation and DNA degradation. Analysis of a cloned somatic telomere (pTel1) revealed that it has been newly created during the process of chromatin diminution by the addition of telomeric repeats (TTAGGC)n to a chromosomal breakage site (Müller et al., 1991). However, telomere addition does not occur at a single chromosomal locus, but at many different sites within a short chromosomal region, termed CBR1 (chromosomal breakage region 1). Here we present the cloning and the analysis of 83 different PCR amplified telomere addition sites from the region of CBR1. The lack of any obvious sequence homology shared among them argues for a telomerase-mediated healing process, rather than for a recombinational event. This hypothesis is strongly supported by the existence of 1-6 nucleotides corresponding to and being in frame with the newly added telomeric repeats at almost all of the telomere addition sites. Furthermore, we show that telomeres are not only added to the ends of the retained chromosomal portions, but also to the eliminated part of the chromosomes, which later on become degraded in the cytoplasm. This result suggests that de novo telomere formation during the process of chromatin diminution represents a non-specific process which can heal any broken DNA end.

MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans.

The germ line is an immortal cell lineage that is passed indefinitely from one generation to the next. To identify the genes that are required for germline immortality, we isolated Caenorhabditis elegans mutants with mortal germ lines--worms that can reproduce for several healthy generations but eventually become sterile. One of these mortal germline (mrt) mutants, mrt-2, exhibits progressive telomere shortening and accumulates end-to-end chromosome fusions in later generations, indicating that the MRT-2 protein is required for telomere replication. In addition, the germ line of mrt-2 is hypersensitive to X-rays and to transposon activity. Therefore, mrt-2 has defects in responding both to damaged DNA and to normal double-strand breaks present at telomeres. mrt-2 encodes a homologue of a checkpoint gene that is required to sense DNA damage in yeast. These results indicate that telomeres may be identified as a type of DNA damage and then repaired by the telomere-replication enzyme telomerase.

Fasciola hepatica: stage-specific expression of novel gene sequences as identified by differential display.

Differences in gene expression between adult and immature Fasciola hepatica (liver fluke) parasites isolated from the mammalian host were investigated using the technique of differential display. For any given primer combination used to produce these displays there were, on average, 22% apparently adult-specific and 14% apparently immature-specific cDNA products able to be identified, consistent with a high degree of differential gene expression between these two parasite developmental stages. Several cDNA fragments specific to immature parasite RNA were isolated and cloned. An abundant 400- to 500-bp RNA species was identified on a Northern blot by hybridization to the cloned DD2 cDNA fragment and was determined to be expressed at levels at least 10-fold higher in immature parasites relative to adult parasites. mRNA transcripts corresponding to the remaining cDNA fragments (DD14, DD16, DISP10, and DISP2) were apparently expressed at levels below the sensitivity limits of Northern analysis, although differential expression of these transcripts was confirmed by reverse transcriptase PCR (RT-PCR). The identities or functional significance of each of the five differentially expressed cDNAs identified in this study is still unclear due to the lack of any significant sequence similarity to the entries currently held within sequence databases.

Ordered differential display: a simple method for systematic comparison of gene expression profiles.

A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .

Endoreplication in the ovary, testis, and intestine of Strongyloides stercoralis.

During development of the free-living adults of the human parasitic nematode Stronglyoides stercoralis, cells in certain tissues grow by endoreplication in which rounds of DNA replication occur without cell or nuclear division. The DNA content of individual nuclei was measured by microdensitometry of Feulgen-stained preparations. In females, some ovarian cells have up to 800 times the haploid DNA content (800C). In males, some cells of the testis have up to 100C. Intestinal cells in both sexes have up to 16C, whereas most other somatic cells have 2C.

Endoreplication in the ovary, testis, and intestine of strongyloides sterocalis

During development of the free-living adults of the human parasitic nematode Strongyloides stercoralis, cells in certain tissues grow by endoreplication in which rounds of DNA replication occur without cell or nuclear division. The DNA content of individual nuclei was measured by microdensitometry of Feulgenstained preparations. In females, some ovarian cells have up to 800 times the haploid DNA content (800C). In males, some cells of the testis have up to 100C. Intestinal cells in both sexes have up to 16C, whereas most other somatic cells have 2C (AU)