Ribosomal Gene Probes Research Articles

Brazilian studies on the genetics of Schistosoma mansoni

The parasite Schistosoma is known to exhibit variations among species, strains and genera, such as, the levels of infectivity, pathogenicity and immunogenicity. These factors may differ among parasite populations according to the local epidemiological conditions. Diversity observed in Schistosoma mansoni from different geographical regions or within individuals of the same region can be determined by differences in the genotype of each parasite strain. However, until recently, finding adequate genetic markers to investigate infectivity or other epidemiological characteristics of a transmission area proved difficult. Several studies have been conducted to evaluate the genetic variability of S. mansoni, using different techniques. Intraspecific variability was observed in morphological characters, isoenzyme studies, mtDNA, ribosomal gene probes, random amplification of polymorphic DNA (RAPD) and microsatellites. The sequencing of the S. mansoni genome was the most important achievement concerning genetic approaches to the study of this parasite and may improve the development of drugs, vaccines and diagnostics of schistosomiasis. The knowledge of the genetic structure of schistosome populations in relation to epidemiological data and host variability is essential for the understanding of the epidemiology of the disease and the design of control strategies.

Purification of Entamoeba histolytica DNA containing organelles (EkhOs): a further characterization.

Entamoebu histolytic~cr is the protozoan parasite responbibk for human amoebiasis [lo]. Its life cycle is comprised of two dominant phases: the cysts that are the infective forms, and the trophozoites, the invasive forms. After ingestion, the cysts in the intestine can differentiate into trophozoites. Trophozoites can invade the epithelium and go through the portal vein to the liver, where they produce abscesses. Thus, the trophozoites are able to live in poor and rich oxygen milieu, and therefore, E. histolyticu is considered as a facultative microaerophilic protozoan. Genes and proteins participating in these changes are unknown. E. histolyticu trophozoites posses cytoplasmic DNA-containing organelles, named EhkOs [ 1,151. Crypton structures also contain DNA [4]. The pyruvate:ferredoxin oxidoreductase enzyme (EhPFO) is found in the EhkOs 1171. PFO is an enzyme in anaerobic metabolism that in Trichomo~zas vagirzulis is located in hydrogenosomes [19]. Additionally, ribosomal gene probes hybridize with EhkO's DNA [IS]. In this paper we report a method to purify EhkOs. Purified EhkOs appeared composed of small structures that associate themselves together to form EhkOs of different sizes. The presence of DNA in these organelles is demonstrated here by different techniques including propidium iodide stain, DNase-sensitivity, ['H]thymidine labeling and autoradiography, and carbon-platinum shadowed pure EhkOs.

Nature of B chromosomes in the harvest mouse Reithrodontomys megalotis by fluorescence in situ hybridization (FISH).

Using fluorescence in situ hybridization, we examined the characteristics of two types of B chromosomes in harvest mice of the genus Reithrodontomys. B chromosomes were interrogated with rDNA, telomeric repeat, LINE element and centromeric heterochromatin probes. The two types of B chromosomes share the following features: (a) telomeres present on the ends of both arms; (b) hybridization to LINE probes; (c) absence of hybridization to the ribosomal gene probes; (d) C-band-positive centromeric regions; and (e) euchromatic arms. They differ as follows: (a) the larger B element hybridizes to the centromeric heterochromatin (pMeg-1) probe whereas the smaller B element does not; (b) the amount of C-band-positive material is reduced in the smaller B chromosome relative to that present on the larger B chromosome; and (c) the smaller element is reduced in size by about a third. It is concluded that the larger B chromosome arose as a leftover centromere from centric fusion, whereas the smaller element has a different origin perhaps as an intact fragment or as an amplified region from the A chromosomes. The presence of euchromatic regions on B chromosomes may account for their survival in the karyotype.

Nucleolar fragmentation in polytene trichogen cells of Lucilla cuprina and Chrysomya bezziana (Diptera: Calliphoridae).

The location of genes coding for 18S and 28S ribosomal RNA in mitotic and polytene cells of Lucilia cuprina and Chrysomya bezziana was investigated using in situ hybridization of an 18 + 28S ribosomal gene probe and silver staining. In both species ribosomal genes were localized to secondary constriction regions in sex chromosome heterochromatin. In L. cuprina mitotic cells the probe hybridizes to a distal secondary constriction region in the short arms of the X and Y chromosomes. In C. bezziana mitotic chromosomes ribosomal genes were located in distal secondary constriction regions in the long arms of the X and Y chromosomes. In polytene trichogen cells of both species, hybridization results varied with the level of polyteny. Cells of low polyteny have a single hybridization site, but with greater polytenization, increasing numbers of extrachromosomal fragments strongly hybridize to the ribosomal gene probe. No hybridization occurs in structures representing the sex chromosomes or in the autosomes. These results indicate that fragmentation and dispersal of the nucleolus occurs during polytenization. Silver staining of both unsquashed and squashed polytene nuclei show identical behaviour of multiple, varied-sized nucleolar bodies, thus confirming the in situ hybridization results. Uridine incorporation studies in L. cuprina indicated that transcription occurs in extrachromosomal bodies similar to nucleolar fragments. Nucleolar fragmentation is more pronounced in L. cuprina males, particularly in those with the translocation T(Y;2)540. Chromosomally normal C. bezziana show nucleolar fragmentation levels similar to that in L. cuprina males. Ribosomal genes are disproportionately replicated in trichogen cells to a much greater extent than surrounding heterochromatin. Nucleolar fragmentation may be a gene amplification system, but it is not known to what degree, relative to diploid amounts, ribosomal genes replicate in trichogen cells.

Schistosoma mansoni: Use of a cloned ribosomal RNA gene probe to detect restriction fragment length polymorphisms in the intermediate host Biomphalaria glabrata

Adult susceptibility of Biomphalaria glabrata to Schistosoma mansoni infection is controlled by simple Mendelian genetics. In this study a molecular approach was used to determine the degree of genetic variation between well-defined lines of B. glabrata which are either resistant (10-R2) or susceptible (M-line) to S. mansoni infection. A cloned probe pSM389, which contains part of the S. mansoni small ribosomal RNA gene and a portion of the nontranscribed spacer was found to cross-hybridize with B. glabrata DNA and was used in Southern hybridizations to detect restriction fragment length polymorphisms (RFLPs) between the above snail stocks. Polymorphisms were noted with a variety of restriction enzymes, namely BglII, BamHI, AccI, AvaII, ClaI, EcoRI, EcoRV, KpnI, PvuII, and NcoI. Although most RFLPs were relatively minor, a significant difference was observed with EcoRV. Further analysis of the EcoRV RFLPs among other isolates of the resistant stock demonstrated that a high frequency of genetic variation exists even among isolates of the same origin, but maintained in separate laboratories. Interestingly, RFLPs in the EcoRV site were detected in DNA isolated from a single generation of selfed progeny of a single 10-R2 parent. RFLPs associated with this site were found to occur between B. glabrata and B. tenagophila, B. straminea, and B. schrammi, indicating that Southern blot analysis using ribosomal gene probes may be useful for the molecular differentiation of B. glabrata from other intermediate hosts and from morphologically similar species that are refractory to infection.

Cytoplasmic nucleoloids of Lilium male reproductive cells contain rDNA transcripts and share features of development with nucleoli

ABSTRACT Nucleolus-like inclusions in the meiocytes of Lilium sp. have been investigated at the light and electronmicroscope levels, with respect both to their composition and their formation during the meiotic divisions. Those present in the cytoplasm (cytoplasmic nucleoloids) first appear as small structures late in anaphase I. Coincidentally, small nuclear nucleoluslike inclusions (NLBs) develop on the surface of the chromosomes, and sometimes in the region between chromosomes as they are carried into the daughter nuclei. The cytoplasmic nucleoloids increase in size from late anaphase I to the dyad stage, but disappear before metaphase II. They redevelop late in anaphase II and persist, increasing in size, until the tetrad stage. The NLBs also increase in size over this period. Ultrastructural investigation indicates that both the nascent nucleoloids and NLBs are fibrous in texture as they arise in late anaphase. Elsewhere in the cytoplasm, however, large accumulations of amorphous electron-opaque material also aggregate. This material becomes associated with the nucleoloids and the NLBs during the period of their enlargement. Nucleoloids late in development, and post-telophase nucleoli are not invested by these amorphous masses. Interestingly, cytoplasmic nucleoloids and NLBs react in an identical fashion to a range of cytochemical treatments and, using in situ hybridisation at the electron-microscope level, they have been demonstrated to contain RNA sequences homologous with a wheat ribosomal gene probe. Cytoplasmic nucleoloids are thus interpreted as following an identical developmental pathway to nucleoli except in that, perhaps owing to high levels of rDNA transcription during meiosis, they condense in the cytoplasm rather than the nucleoplasm.

Genetic and morphological variability in a population of Diphyllobothrium dendriticum (Nitzsch, 1824)

Plerocercoids of Diphyllobothrium dendriticum were isolated from whitefish (Coregonus clupeaformis) from McGregor Lake, Alberta. Adult D. dendriticum were obtained by infection of golden hamsters (Mesocricetus auratus). Analysis of morphological variables indicated high variability and some overlap with D. latum. Correlations in morphology were due to the covariance of shape variables. Isozyme analysis also showed high variability, with two thirds of the enzymes studied being polymorphic. Correlations with morphology indicate a relationship between rare patterns for malate dehydrogenase and esterase and increased size of adults. Southern hybridisation with ribosomal gene probes produced two common patterns and a rare intermediate form, indicating that the species is variable even within a local population. Correlations between rDNA pattern type and morphological variables supported the hypothesis of a genetic base for the morphological variability.

A molecular karyotype of Eimeria tenella as revealed by contour-clamped homogeneous electric field gel electrophoresis

DNA from sporozoites of Eimeria tenella was resolved by pulsed field gel electrophoresis into nine chromosomal bands. Some bands of this molecular karyotype contained more than one chromosome as determined by the relative intensity of both staining with ethidium bromide and hybridisation to an E. tenella telomeric probe. Haploid forms of the parasite must be presumed to contain at least 12 chromosomes. The two smallest chromosomes were about 1.1 and 1.4 megabases. Most chromosomes were in excess of 3 Mb with the largest over 5 Mb as determined by comparison with the co-migration of chromosomes from Schizosaccharomyces pombe. A 5S ribosomal gene probe hybridised to a single chromosomal band.

A genomic change associated with the development of resistance to hycanthone in Schistosoma mansoni

Ribosomal gene probes were used to investigate the genetic basis of drug resistance in schistosomes in a model where resistance to the anthelmintic hycanthone (HC) is generated by exposing immature worms to the drug. Two strains of Schistosoma mansoni, JHU and NMRI, were used. Drug resistance could be produced in the JHU strain by treatment with HC, but was also found to occur spontaneously. In contrast, it was not possible to detect or produce resistance to HC in the NMRI strain. A genomic alteration accompanied the development of resistance. The change was evidenced by the occurrence of restriction fragment length polymorphisms (RFLPs) when Southern blots of genomic DNA from HC-resistant worms were hybridized with the ribosomal probe pSM389, which contains part of the small rRNA gene plus non-transcribed spacer (NTS) sequence. The most reliable marker of HC-resistance was a 3.6-kb BamHI fragment which was present and heritable in 7 drug-resistant lines derived from the JHU strain but absent from the parent JHU population and from NMRI parasites. The universal absence of the 3.6-kb RFLP in HC-sensitive individuals and its presence in the drug-resistant progeny suggest that resistance results from an induced change in the population rather than from selection of HC-resistant parasites. The rRNA gene sequence responsible for detecting the 3.6-kb RFLP appears to be localized either to the NTS or to the 5′ end of the small rRNA gene, since hybridization to a probe containing sequence from the rRNA gene contiguous and downstream from the insert of pSM389 failed to reveal the RFLP. These results show that the development of resistance to HC is accompanied by a genomic rearrangement.

Differentiation between Diphyllobothrium dendriticum and D. latum using isozymes, restriction profiles and ribosomal gene probes

Plerocercoids of Diphyllobothrium dendriticum from cisco (Coregonus artedii) and whitefish (C. clupeaformis) and of Diphyllobothrium latum from pike (Esox lucius) and walleye (Stizostedion vitreum) were collected at Quigly Lake, Manitoba. Adult tapeworms were obtained by experimental infection of hamsters (Mesocricetus auratus) and were used to verify species identity. Isozyme analysis showed no differences between adult and plerocercoid stages. The enzymes esterase and malate dehydrogenase differed between species. Comparison of DNA restriction fragment profiles showed species specific differences, as did Southern hybridization analysis using nematode ribosomal gene probes.

Ribosomal Gene Number Variability in Douglas-Fir

We studied genetic variability for the number of copies of nuclear ribosomal RNA genes in 54 trees derived from seed collected throughout most of the natural range of Douglas-fir (Pseudotsuga menziesll [Mirb.] Franco). Slot-blots were hybridized with a radiolabeled heterologous ribosomal gene probe, and the relative gene number was estimated from densitometer scans of the resulting autoradiographs. Genetic variability for ribosomal gene number was substantial and resided almost entirely within populations; variance among trees accounted for half the total variance. Gene number increased with latitude, elevation, and longitude. The tendency for gene number to increase northward concurs with cllnes reported for genome size in Douglas-flr, in other conifers, and among wild and cultivated herbaceous plant species.

Human U3 small nucleolar RNA genes are localized to the nucleoplasm

U3 RNA, an abundant, conserved, capped, small RNA localized to the nucleolar compartment of eukaryotic cells, is implicated in the processing of pre-ribosomal RNA. The genes for U1 and U2 snRNAs ( s mall n uclear RNAs) are clustered and present in the nucleoplasmic DNA; however, the localization of U3 snRNA genes is not known. DNAs, isolated from HeLa cell nuclei and nucleoli, were hybridized with labeled probes corresponding to 1) the 5′-flanking and 2) the coding regions of the human U3 snRNA gene. The intensity of signals obtained with both the probes were 5–6 fold greater in nuclear DNA, compared to nucleolar DNA. With ribosomal gene probe, the nucleolar DNA had sixfold more intense signal than nuclear DNA. These results indicate that genes for U3 snRNA are in the nucleoplasm. Therefore, U3 snRNA, like 5S ribosomal RNA, appears to be synthesized in the nucleoplasm and transported to the nucleolus.

Anti-schistosomal drugs: observations on the mechanism of drug resistance to hycanthone, and on the involvement of host antibodies in the mode of action of praziquantel.

This paper reports recent observations from our laboratory dealing with the anti-schistosome drugs hycanthone (HC) and praziquantel (PZQ). In particular, we discuss a laboratory model of drug resistance to HC in Schistosoma mansoni and show that drug sensitive and resistant lines of the parasite can be differentiated on the basis of restriction fragment length polymorphisms using homologous ribosomal gene probes. In addition, we summarize data demonstrating that effective chemotherapy of S. mansoni infection with PZQ in mice requires the presence of host anti-parasite antibodies. These antibodies bind to PZQ treated worms and may be involved in an antibody-dependent cellular cytotoxicity reactions which result in the clearance of worms from the vasculature.

Outcome after prenatal detection of a sporadic, unstable translocation t(5;21).

Amniotic fluid cultures from a 37 year old woman showed a sporadic 46,XX,t(5;21)(5qter----5p13 or p14::5pter----5p13 or p14::21p12----21qter) complement. In the majority of metaphases the 5p fragment was attached to the stalks of chromosome 21; however, in 9% of metaphases, the fragment was loosely attached by a 'thread' and in 6% it was completely detached. Silver staining and in situ hybridisation with a homologous ribosomal gene probe, which localises to stalk regions (nucleolar organisers, NOR) of human acrocentric chromosomes, failed to show a reciprocal exchange. Prognosis was uncertain because the possibility that the 5p fragment might have been lost in some cell lines could not be excluded. Nonetheless, the parents elected to continue the pregnancy. The translocation was confirmed in blood specimens obtained both at birth and at 1 year of age and showed similar instability. However, the proband shows no anomalies and is developing normally at 1 year.

Multiple ribosomal gene sites revealed by in situ hybridization of Xenopus rDNA to Triturus lampbrush chromosomes.

A variety of 3H-labelled ribosomal gene probes were hybridized in situ to the nascent transcripts of lampbrush chromosomes from the crested newt, Triturus cristatus carnifex. The probes were from Xenopus laevis and included rDNA isolated by CsCl gradient centrifugation, recombinant plasmids and purified restriction fragments of rDNA. All the probes gave essentially the same result. About 10-15 loop pairs were distinctly labelled in each preparation, almost all of them located on the heteromorphic arms (HTAs) of chromosome 1. Ribosomal gene probes were also hybridized in situ to the DNA of denatured mitotic chromosomes from some of the individuals used to provide lampbrush preparations. Minor, scattered sites of hybridization were found in the HTAs, but the main clusters of ribosomal genes were found on chromosomes 6 and/or 9, in agreement with previous determinations of nucleolus organizer position in this species. However, the nucleolus organizers were not sites of labelled loops in lampbrush transcript hybridizations.--We have incubated isolated lampbrush-stage nuclei in media containing alpha-amanitin and labelled RNA precursors. Although extrachromosomal nucleolar genes incorporated label, supposedly due to transcription by RNA polymerase I, no lampbrush loops were labelled.--It appears that in T. c. carnifex there are ribosomal gene sequences at the main nucleolus organizers and at a number of sites scattered along the HTAs. The ribosomal genes at the nucleolus organizers are not extended in the form of actively transcribing loops unlike the ribosomal sequences on the HTAs, which are heavily labelled in transcript hybridization. The ribosomal sequences on the HTAs appear not to be transcribed by the same RNA polymerase that transcribes the ribosomal genes of extrachromosomal nucleoli.