Chromosome diminution in Ascaris suum: Chromatin structure

Ascaris suum loses 56% of its nuclear DNA during chromosome diminution. Measured values of histones per nucleus are relatively constant, resulting in an approximate doubling of histone: DNA ratios during this process. Experiments were performed in an effort directed towards ascertaining the location of the increased histones. Repeat lengths of micrococcal nuclease protected pre- and post-diminution DNA were determined. Nuclear sap proteins from post-diminution nuclei were also examined in order to test the possibility of nuclear pools of free histones.     

Chromatin diminution in Ascaris suum: nucleotide sequence of the eliminated satellite DNA.

We have determined the prototype sequence of the DNA which is eliminated in the course of chromatin diminution in Ascaris suum. This DNA which is virtually absent from somatic cells but retained in the germ line consists predominantly of highly repetitive sequences which are variants of an AT rich 123 base pair repeat unit. Both major and minor variants have been sequenced. The overall structure of this germ line limited DNA corresponds to the segmental organization characteristic of satellite DNAs. Possible correlations between the mechanism of chromatin diminution and some properties of the satellite sequence are discussed.     

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.     

Chromatin diminution in early embryogenesis of Ascaris lumbricoides L. var. suum

The occurrence of chromatin diminution in early Ascaris lumbricoides L. embryos has been studied in detail, and it is shown that it is possible to preselect three characteristic types of mitoses: pre-diminution, diminution, and post-diminution mitosis. The first three embryonic mitotic divisions are of the pre-diminution type. Chromatin diminution occurs after the third mitosis, but there is a variation from embryo to embryo as to whether or not chromosomal diminution occurs during the fourth, fifth, and six divisions. However, the seventh embryonic division, which gives rise to an eight-cell embryo, always exhibits chromatin diminution. Subsequent mitoses of somatic cells already in the diminished state are of the post-diminution type of mitosis.     

Developmentally regulated telomerase activity is correlated with chromosomal healing during chromatin diminution in Ascaris suum

Telomerase is the ribonucleoprotein complex responsible for the maintenance of the physical ends, or telomeres, of most eukaryotic chromosomes. In this study, telomerase activity has been identified in cell extracts from the nematode Ascaris suum. This parasitic nematode is particularly suited as a model system for the study of telomerase, because it shows the phenomenon of chromatin diminution, consisting of developmentally programmed chromosomal breakage, DNA elimination, and new telomere formation. In vitro, the A. suum telomerase is capable of efficiently recognizing and elongating nontelomeric primers with nematode-specific telomere repeats by using limited homology at the 3' end of the DNA to anneal with the putative telomerase RNA template. The activity of this enzyme is developmentally regulated, and it correlates temporally with the phenomenon of chromatin diminution. It is up-regulated during the first two rounds of embryonic cell divisions, to reach a peak in 4-cell-stage embryos, when three presomatic blastomeres prepare for chromatin diminution. The activity remains high until the beginning of gastrulation, when the last of the presomatic cells undergoes chromatin diminution, and then constantly decreases during further development. In summary, our data strongly argue for a role of this enzyme in chromosome healing during the process of chromatin diminution.     

Synthesis of nucleosomal histone variants during wheat grain development

The synthesis and distribution of histone subfractions (variants) were investigated during early grain development and in mature tissues of wheat (Tritium aestivum L.). Histones were extracted from purified chromatin and separated by two-dimensional polyacrylamide gel electrophoresis. There were no detectable differences in the patterns of histone variants from immature grain (3–16 days after fertilization), from mature embryos, from coleoptiles and roots of 4-day-old, etiolated seedlings and from leaves of 10-day-old, light-grown seedlings. Wheat H2 histones are composed of families of closely related variants. H2A consists of three major variants, and H2B consists of two major and four minor variants. The synthesis of these variants during early grain formation was determined by calculating the specific activities of the [³H]lysinelabeled proteins synthesized between 3 and 10 days after fertilization. The rate of synthesis of the nucleosomal histones closely parallels the declining rate of cell division in developing grains. Our results indicate that all the recognized wheat histone variants are present in developing wheat grains from the earliest time investigated (3 days after fertilization) and persist with no detectable changes in relative quantities throughout grain development and in several mature tissues.     

Changes in nucleosomal core histone variants during chicken development and maturation

The nucleosomal core histones H2A, H2B, and H3 of the chicken can be resolved by polyacrylamide gel electrophoresis in the presence of nonionic detergents into two primary structure variants each, which occur in different relative amounts in various adult tissues. Quantitative analysis of the histone components throughout embryonic development and posthatching maturation of the chicken revealed that the proportions of the three pairs of variants change independently. Thus, the two H2A variants occur in similar proportions throughout embryonic development and in all adult tissues. In contrast, only one variant each of H2B and H3 is detectable at the earliest stages (primitive streak). The second variant of these histones becomes detectable and increases gradually during somite formation (2-12 days of incubation) to reach a plateau at a level of about 3 and 10% of total H2B and H3 histones, respectively. After hatching, the relative amounts of the minor H2B and H3 variants remain at embryonic levels in those tissues which maintain a high mitotic activity such as blood-forming tissues, but increase with different kinetics in tissues which essentially stop cell division in adults (e.g., liver, kidney, etc.). However, while H2B.2 remains a very minor component in all tissues, H3.3 increases at a relatively high rate for more than a year to become the predominant H3 variant in the liver and kidney of older chickens. The changes in chicken core histone variant proportions appear to be related to changes in growth rate rather than cell differentiation. The extensive change of H3 variant proportions in nondividing adult tissues is most likely due to replication-independent incorporation of H3.3 into nucleosomes.     

Catalase activity during the development of the parasitic nematode, Ascaris suum

1. Catalase activity was partially purified from body wall muscle of the parasitic nematode, Ascaris suum, and was similar to catalases isolated from mammalian tissues. It exhibited a broad pH optimum and was unaffected by 2 mM ethylenediaminetetra-acetate. In contrast, it was inhibited reversibly by 1 mM cyanide and irreversibly by prior incubation in 40 mM 3-amino-1:2:4-triazole for 1 hr or heating at 80 degrees C for 15 min. 2. Catalase activity was highest in the unembryonated "egg" and decreased dramatically as development proceeded. 3. Catalase activity in adult body wall muscle was similar to that in rat skeletal muscle, but dramatically lower than that in rat liver. Catalase activity was barely detectable in A. suum testis. 4. Cytochrome-c peroxidase activity did not appear to be present in adult A. suum muscle mitochondria.     

DNA synthesis in the early embryo of the nematode Ascaris suum.

We have used microspectrofluorimetry to measure the rate of DNA synthesis in the first two embryonic cell cycles of the parasitic nematode Ascaris suum. The S-phase of the early Ascaris cell cycles occupies at most 1 hr; G2 phase is prominent and occupies approximately 11 hr; no G1 phase could be detected. These results contrast with our previous measurements made with embryos of the free-living nematode Caenorhabditis elegans, in which the earliest cell cycles consist of simple alternations between S and M phases.     

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.     

Ecdysteroids during embryonation of eggs of Ascaris suum

1. The optimal temperature for in vitro development of fertilized eggs of Ascaris suum was 24 degrees C. 2. Samples (2 X 10(7) eggs) were obtained from in vitro embryonating cultures every 3 days for 4 weeks; lipids were extracted, partially purified, fractionated with HPLC and analyzed for ecdysteroids by radioimmunoassay. 3. Free ecdysone and 20-hydroxyecdysone (20-HE) were at low levels (less than 20 pg) in freshly excised eggs and rose to maximal values on day 6 of embryonation. 4. Conjugated ecdysone and conjugated 20-HE rose to maximal values on day 9. 5. Both free and conjugated ecdysteroids were undetectable from days 15 to 27 of cultivation. 6. These profiles indicate that ecdysteroids might have a selective role in nematode embryonation and/or tanning of the egg shell.     

Morphology and behaviour of sex chromosomes during meiosis in Ascaris suum.

The morphology and behaviour of sex chromosomes was studied in A. suum during meiosis. It was found that the five sex chromosomes have their proper characteristic. The largest is submetacentric, of 2 microns mean length. The second largest is acrocentric, mean length of 1.4 mu. The third largest is metacentric, 1.2 mu mean length. The fourth and the fifth are metacentric, of mean length of 1 mu. In primary and secondary spermatocyte cells the sex chromosomes are close to each other, most often in the peripheral part of the cell. During anaphase I the pentad sex chromosomes lie freely between the two sister cells. It is assumed that in anaphase II the five sex chromosomes divide equally and are regularly distributed in the daughter cells. It was found that the chromosomes set of female Ascaris in metaphase I contains 24 bivalent chromosomes n = 24 and of male Ascaris 19 bivalents and 5 univalents. It is assumed that the univalent chromosomes, found in spermatocyte cells, determine sex.     

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC₅₀ value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity.     

Ascaris suum: development of intestinal immunity to infective second-stage larvae in swine

The development of protective immunity to Ascaris suum was examined in pigs naturally exposed to eggs on a contaminated dirt lot. Pigs became almost totally immune to second-stage larvae migrating from the intestines because few larvae from a challenge inoculum could be found in the lungs, and liver white-spot lesions (an immunopathologic response to migrating larvae) were absent. Blood from these pigs contained lymphocytes that responded blastogenically to larval antigens in vitro, while the serum contained antibody to larval antigens. Immunity was related to parasite exposure and not to the age of the host, and was not affected by the removal of adult A. suum from the intestines. Naturally exposed pigs responded to a variety of A. suum antigens with an immediate-type skin reactivity, and their intestinal mucosa contained relatively large numbers of mast cells and eosinophils. Other pigs were maintained on a dirt lot not contaminated with A. suum eggs and the effects of common environmental conditions on development of resistance to A. suum were studied. Resistance also developed in these pigs because 72% fewer larvae were detected in their lungs following a challenge exposure than in control pigs confined indoors on concrete floors and challenged similarly. This response was not expressed at the intestinal level, however, because their livers had numerous, intense white-spot lesions. To verify that the intestinal immunity that developed in pigs after natural exposure to A. suum was a direct result of homologous infection and not related to other stimuli encountered on a dirt lot, pigs maintained indoors on concrete floors, free from inadvertent helminthic infection, were inoculated orally with A. suum eggs daily for 16 weeks. Intestinal immunity was induced because larvae from a challenge inoculum were not detected in the lungs, and few white-spot lesions appeared on the livers of these pigs. Apparently, continual exposure of the intestinal mucosa to larvae eventually elicits the appropriate effector components necessary to prevent larval migration from the intestines.     

Neither a germ line-specific nor several somatically expressed genes are lost or rearranged during embryonic chromatin diminution in the nematode Ascaris lumbricoides var. suum

Ascaris lumbricoides var. suum is a parasitic nematode of pigs. Its embryos undergo chromatin diminution between the third and fifth cleavages, resulting in the loss of about 30% of the DNA from all somatic precursor cells while the germ line DNA stays intact. Most of the eliminated DNA has been shown to be satellite sequences. Theodor Boveri [(1910) In "Festschrift fur R. Hertwig, III," Vol. 3, pp. 131-214, Fischer] proposed that functions essential only to the germ line might be lost from the soma. We have examined this proposal by cloning a gene encoding the major sperm protein (MSP) using a cloned MSP gene from Caenorhabditis elegans as a probe. The MSP appears to be expressed only in the testis of Ascaris, as it is in Caenorhabditis. Actin and alpha tubulin were also cloned to serve as somatically expressed gene controls. By probing Southern blots of somatic and germ line DNA with these cloned genes, it was found that none of them was lost or rearranged during chromatin diminution. Thus at least one germ line-specific gene is neither lost nor rearranged during chromatin diminution. We also found that the two nematode species differ widely in their numbers of both MSP and actin genes. Caenorhabditis has greater than 30 MSP genes, but Ascaris has no more than three; whereas Ascaris has many more actin genes than Caenorhabditis.     

DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of oxytricha

We have measured the reassociation kinetics of DNA from the micronucleus and from the macronucleus of the hypotrichous cillate Oxytricha. The micronuclear DNA reassociates with at least a two-component reaction, indicating the presence of both repeated and non-repeated sequences. The kinetic complexity of micronuclear non-repeated DNA is in the range of 2 to 15 × 10¹¹ daltons; the haploid DNA content of the micronucleus is 4 × 10¹¹ daltons (0.66 pg), measured microspectrophotometrically. The DNA of the macronucleus reassociates as a single second-order reaction, with a kinetic complexity of 3.6 × 10¹⁰ daltons. A comparison of the kinetic complexities of micronuclear and macronuclear DNAs suggest a 5 to 30 fold reduction in DNA sequence complexity during the formation of a macronucleus from a micronucleus. Macronuclear DNA is in pleces with an average molecular weight of 2.1 × 10⁶ daltons. Since the kinetic complexity of macronuclear DNA is 3.6 × 10¹⁰ daltons, the macronucleus must contain about 17,000 different kinds of DNA pieces.Each macronucleus contains 3.5 × 10¹³ daltons (58 pg) of DNA, indicating that each sequence must be present about 1000 times per macronucleus or 2000 times per cell.     

Reassembly of nucleosomal histone octamers during replication of chromatin

We have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were density-labeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of single-stranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-substituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.     

Factors contributing to the in vitro development of Ascaris suum from second-stage larvae to mature adults

When a three-step roller culture system was used, second-stage larvae of Ascaris suum, artificially hatched from eggs, developed in high numbers to the fourth stage, and a few to young and mature adults. The culture system consisted of (1) Medium KW-2 supplemented with 10 mM L-cysteine for the first 4 days, and with 5 mM L-cysteine for the following 7 days; (2) followed by Medium API-18 for 7 days; and (3) thereafter, by Medium API-1 supplemented with hemin (bovine) at a concentration of 24 micrograms/m1. Cultures were gassed with 95% nitrogen-5% carbon dioxide for the first 4 days and 85% nitrogen-5% oxygen-10% carbon dioxide thereafter, and incubated at 39 C. Two mature females that produced unfertilized eggs and a mature male with spermatozoa were the most advanced stages attained. The mature females were obtained in 67 and 73 days; and the largest female measured 110 mm. The latter produced 1,356,000 unfertilized eggs, from days 67 to 125. The mature male was obtained in 80 days; it measured 77 mm long and had paired spicules that were 1.5 mm long. Development of A. suum in three other culture systems showed that deletion of Medium API-18 or its substitution by Medium KW-2 limited development to late fourth stage and early, young adults, respectively; and the use of Medium API-1 without hemin limited development to early fourth stage.