jeanflop/post_ocr_correction2 huggingface dataset:
mm in diameter) are very numerous and contain two forms of spores: a round peripheral monokaryotic form (3.5 µm) and a reniform central diplokaryotic form (4 µm). Cysts extracted from host tissues were first washed three times by centrifugation at 90 g in phosphate buffer saline (PBS). Then, they were crushed mechanically in a Polytron homogenizer to release the spores. Following several washings in PBS to eliminate the fragments of fish tissues, the spores were purified by centrifugation at 540 g for 30 min through a discontinuous gradient consisting of layers of 25, 50, 75 and 100 % Percoll [15]. They were finally washed in PBS by three centrifugations at 540 g and stored in the same buffer at 4 °C. Spore germination The germination of spores of microsporidia can be induced by several environmental parameters that differ from one species to another. A suspension containing 4.108 spores in PBS was centrifuged at 540 g for 5 min. The pellets were suspended in 1 ml of mixture consisting in equal parts of PBS and 2 % low melting point agarose, maintained at 37 °C. After cooling, the small pieces of agarose containing spores were removed from the mold and put in germination solution for 20 min at room temperature. Two media were defined, according to the species: 3M urea for G. atherinae, 0.1 M KCl/3% (v/v) H2O2 for S. lophii. With this treatment, the microsporidian spores eject their intact DNA through the polar filament. The pieces of agarose were then rinsed twice 10 min in 10 ml of PBS to eliminate the germination solution. Preparation of DNA and pulsed field gel electrophoresis The small pieces of agarose were immersed in lysis buffer, consisting of 500 mM EDTA, 1 % N-lauryl sarcosine and 2 mg/ml proteinase K, for 48 h at 42 °C. Then, they were stored at 4 °C. Before electrophoresis, the pieces of agarose containing DNAs of microsporidia and Saccharomyces cerevisiae strain 288C (size standards) were equilibrated 5 times in 10 mM Tris, 1 mM EDTA, pH 8 (10 ml) over 30 min. The agarose gels (1.5 %) were
in germination solution for 20 min at room temperature. Two media were defined, according to the species: 3M urea for G. atherinae, 0.1 M KCl/3% (v/v) H2O2 for S. lophii. With this treatment, the microsporidian spores eject their intact DNA through the polar filament. The pieces of agarose were then rinsed twice 10 min in 10 ml of PBS to eliminate the germination solution. Preparation of DNA and pulsed field gel electrophoresis The small pieces of agarose were immersed in lysis buffer, consisting of 500 mM EDTA, 1 % N-lauryl sarcosine and 2 mg/ml proteinase K, for 48 h at 42 °C. Then, they were stored at 4 °C. Before electrophoresis, the pieces of agarose containing DNAs of microsporidia and Saccharomyces cerevisiae strain 288C (size standards) were equilibrated 5 times in 10 mM Tris, 1 mM EDTA, pH 8 (10 ml) over 30 min. The agarose gels (1.5 %) were cast in TBE buffer (0.05 M Tris, 0.025 M sodium borate, 1 mM EDTA, pH 8.5) and electrophoresed at 15 °C in 5 liters recirculating TBE. They were run on a CHEF electrophoresis apparatus at 150 V using 90 sec pulse for 72 h. Gels were stained in 0.4 µg/ml ethidium bromide overnight and then photographed under UV transillumination using Polaroid film. On graph paper, the sizes of yeast chromosomes in kilobase pairs were reported according to their run measured on Polaroid film. The resulting curves were then used to determine the size of S. lophii and G. atherinae chromosomal DNA molecules. The genome size was estimated by calculating the sum of individual chromosomal sizes. Results and discussion Having successfully induced in vitro germination of two microsporidia species, G. atherinae and S. lophii, we have resolved their molecular karyotypes using the PFGE technique (Fig. 1). The banding patterns for the two species show great differences in number, size and staining intensity. For G. atherinae, 16 chromosomal DNA bands, ranging between 420 and 2,700 kb, were observed against 12 from 230 to 980 kb for S. lophii (Table I). The number and distribution of chromosomes in S. lophii thus appear lower
ethidium bromide overnight and then photographed under UV transillumination using Polaroid film. On graph paper, the sizes of yeast chromosomes in kilobase pairs were reported according to their run measured on Polaroid film. The resulting curves were then used to determine the size of S. lophii and G. atherinae chromosomal DNA molecules. The genome size was estimated by calculating the sum of individual chromosomal sizes. Results and discussion Having successfully induced in vitro germination of two microsporidia species, G. atherinae and S. lophii, we have resolved their molecular karyotypes using the PFGE technique (Fig. 1). The banding patterns for the two species show great differences in number, size and staining intensity. For G. atherinae, 16 chromosomal DNA bands, ranging between 420 and 2,700 kb, were observed against 12 from 230 to 980 kb for S. lophii (Table I). The number and distribution of chromosomes in S. lophii thus appear lower than in G. atherinae. Our estimate of genome size is 19.5 Mb for G. atherinae. In fact, the 620 and 730-kb bands show an above average staining intensity. It cannot be excluded that non-homologous chromosomes of the same size co-migrate, which leads to an underestimate of genome size. Inversely, a few bands show a low staining intensity, e.g., that migrating at 540 kb. Depending on the chromosome composition in GC bases, variations in the rate of fixation of ethidium bromide could also explain such staining differences. For S. lophii, the genome size would be close to 6.2 Mb, all bands of its karyotype having the same staining intensity. The important differences observed between the electrophoretic patterns suggest that these two species are very distant from each other. This is not surprising since they belong to two different genera, and G. atherinae is monomorphic while S. lophii is dimorphic. Munderloh et al. [11] and Malone and McIvor [12] realized molecular karyotypes on species of Microsporidia from insects, belonging to three different genera (Nosema, Vavraia and Vairimorpha) (Table II). The estimates of genome size for three Nosema species were 7.4 to 10.5 Mb, with 8 to 13 bands ranging from 290 to
few bands show a low staining intensity, e.g., that migrating at 540 kb. Depending on the chromosome composition in GC bases, variations in the rate of fixation of ethidium bromide could also explain such staining differences. For S. lophii, the genome size would be close to 6.2 Mb, all bands of its karyotype having the same staining intensity. The important differences observed between the electrophoretic patterns suggest that these two species are very distant from each other. This is not surprising since they belong to two different genera, and G. atherinae is monomorphic while S. lophii is dimorphic. Munderloh et al. [11] and Malone and McIvor [12] realized molecular karyotypes on species of Microsporidia from insects, belonging to three different genera (Nosema, Vavraia and Vairimorpha) (Table II). The estimates of genome size for three Nosema species were 7.4 to 10.5 Mb, with 8 to 13 bands ranging from 290 to 1,810 kb in the former case [11] and, from 410 to 1,390 kb in the latter case [12]. The molecular karyotype of Vairimorpha sp. comprised 8 bands from 720 to 1,790 kb and the genome size was 9.2 Mb. The size of the Vavraia oncoperna genome would be either 8 or 10.2 Mb with respectively 14 or 16 bands. Such an intraspecific variation is related to the infestation of two different hosts. It should be stressed that all these genomes are smaller than that found for G. atherinae, one species we studied. In fact, we found the two extreme cases within Microsporidia. e.g., G. atherinae with the largest genome and S. lophii with the smallest one. In numerous eukaryotic cells, the development of PFGE [15] allowed investigations of whole genome and chromosomal DNA polymorphism. This technique has allowed the resolution of molecular karyotypes of yeast species [16-18], and initial typing of some species of Saccharomyces [19]. The genome sizes for various yeasts fluctuate from 12.3 to 13.9 Mb [20]. However, important variations in Candida genomes should be noted, ranging from 12 to 31 Mb with 8 to 18 bands [21]. In the amoeboflagellate Naegleria gruberi [22], the genome has been
or 10.2 Mb with respectively 14 or 16 bands. Such an intraspecific variation is related to the infestation of two different hosts. It should be stressed that all these genomes are smaller than that found for G. atherinae, one species we studied. In fact, we found the two extreme cases within Microsporidia. e.g., G. atherinae with the largest genome and S. lophii with the smallest one. In numerous eukaryotic cells, the development of PFGE [15] allowed investigations of whole genome and chromosomal DNA polymorphism. This technique has allowed the resolution of molecular karyotypes of yeast species [16-18], and initial typing of some species of Saccharomyces [19]. The genome sizes for various yeasts fluctuate from 12.3 to 13.9 Mb [20]. However, important variations in Candida genomes should be noted, ranging from 12 to 31 Mb with 8 to 18 bands [21]. In the amoeboflagellate Naegleria gruberi [22], the genome has been sized at 19.6 Mb with 23 bands. The genome size of Giardia lamblia [23], a flagellate considered as one of the most ancient eukaryotes, ranges between 10.6 and 11.9 Mb and varies geographically. Small nuclear genomes are also found in Pneumocystis species, AIDS opportunist parasites [24-26]. The molecular karyotypes of this genus are very different with 5 to 20 bands from 295 to 1,500 kb, and the genome size varies between 8 and 16 Mb. However, there are strong variations in staining intensity of some bands and it is difficult to provide a good estimate of the genome size in certain cases. Spraguea lophii appears to have the smallest genome among all the protozoans so far examined. In Cryptosporidium, a unique interspecific polymorphism has been shown to occur between C. baileyi and C. parvum, yet all strains of C. parvum exhibit the same DNA banding pattern [27]. In other studied parasitic Protozoa, such as Leishmania and Trypanosoma, the polymorphism is more important, with a high degree of inter- and intraspecific variability [28-31]. To study the polymorphism of G. atherinae, we have resolved the molecular karyotypes of five cysts treated separately. The cysts were extracted from five different fishes caught in
this genus are very different with 5 to 20 bands from 295 to 1,500 kb, and the genome size varies between 8 and 16 Mb. However, there are strong variations in staining intensity of some bands and it is difficult to provide a good estimate of the genome size in certain cases. Spraguea lophii appears to have the smallest genome among all the protozoans so far examined. In Cryptosporidium, a unique interspecific polymorphism has been shown to occur between C. baileyi and C. parvum, yet all strains of C. parvum exhibit the same DNA banding pattern [27]. In other studied parasitic Protozoa, such as Leishmania and Trypanosoma, the polymorphism is more important, with a high degree of inter- and intraspecific variability [28-31]. To study the polymorphism of G. atherinae, we have resolved the molecular karyotypes of five cysts treated separately. The cysts were extracted from five different fishes caught in different Languedoc ponds. Considering that every cyst arose from the infestation of one host cell by one spore, we have assimilated everyone as a strain of G. atherinae. Four strains display identical banding patterns. One strain is characterized by the absence of the 2,380 kb-band (Fig. 7, lane 2). This may be due either to a partial deletion leading to a comigration or to a loss of this chromosome. In this case, PFGE alone is insufficient to interpret the absence. We hope that PFGE coupled with DNA restriction analysis and hybridization with specific probes will provide additional information. Acknowledgments: the authors are greatly indebted to G. Bouix, F. Coste and B. Romestand, Laboratoire de Parasitologie et Immunologie, Université Montpellier 2, for the facility given to collect the G. atherinae and S. lophii spores. VERSION ABRÉGÉE Les Microsporidies, phylum des Microspora [1], sont des protozoaires parasites intracellulaires obligatoires dont les spectres d'hôtes sont extrêmement larges, recouvrant tout le règne animal, l'Homme compris [2]. La transmission de ces parasites se fait par l'intermédiaire d'une spore très résistante, dotée d'une paroi chitineuse. En réponse à des stimuli particuliers au site de germination [9], elle expulse son sporoplasme infectieux dans le cytoplasme d'une cellule-hôte,
may be due either to a partial deletion leading to a comigration or to a loss of this chromosome. In this case, PFGE alone is insufficient to interpret the absence. We hope that PFGE coupled with DNA restriction analysis and hybridization with specific probes will provide additional information. Acknowledgments: the authors are greatly indebted to G. Bouix, F. Coste and B. Romestand, Laboratoire de Parasitologie et Immunologie, Université Montpellier 2, for the facility given to collect the G. atherinae and S. lophii spores. VERSION ABRÉGÉE Les Microsporidies, phylum des Microspora [1], sont des protozoaires parasites intracellulaires obligatoires dont les spectres d'hôtes sont extrêmement larges, recouvrant tout le règne animal, l'Homme compris [2]. La transmission de ces parasites se fait par l'intermédiaire d'une spore très résistante, dotée d'une paroi chitineuse. En réponse à des stimuli particuliers au site de germination [9], elle expulse son sporoplasme infectieux dans le cytoplasme d'une cellule-hôte, par l'intermédiaire d'une longue structure tubulaire appelée « filament polaire ». La majeure partie du cycle de l'agent infectieux se déroule alors dans la cellule-hôte. De très petite taille, ces Eucaryotes unicellulaires sont dépourvus de mitochondries [3] et possèdent des ribosomes de type procaryotique, 70S [4, 5]. L'analyse des séquences d'ARN ribosomiques (de taille 16S et 23S) a fait suggérer une divergence très précoce des Microsporidies par rapport aux autres Eucaryotes, au cours de l'évolution [6, 7, 8]. On small genomes in eukaryotic organisms Peu de travaux ont été consacrés à l'étude de l'organisation génomique des Microsporidies. Le développement de techniques de germination in vitro a facilité l'extraction de l'ADN chromosomique intact, autorisant l'établissement de caryotypes moléculaires au moyen de l'électrophorèse en champ pulé (PFGE) [10]. Les caryotypes électrophorétiques de cinq espèces différentes, parasitant des Insectes, sont actuellement connus [11, 12]. Ils présentent entre 8 et 16 bandes d'ADN chromosomique, et la somme des tailles moléculaires correspondantes procure des évaluations de taille du génome haploïde allant de 7,4 à 10,2 Mb. Nous présentons des résultats de séparation par PFGE des molécules d'ADN chromosomique chez deux espèces de Microsporidies parasites de poissons : Glugea atherinae Berrebi, 1978 (parasite de Atherina boyeri
d'ARN ribosomiques (de taille 16S et 23S) a fait suggérer une divergence très précoce des Microsporidies par rapport aux autres Eucaryotes, au cours de l'évolution [6, 7, 8]. On small genomes in eukaryotic organisms Peu de travaux ont été consacrés à l'étude de l'organisation génomique des Microsporidies. Le développement de techniques de germination in vitro a facilité l'extraction de l'ADN chromosomique intact, autorisant l'établissement de caryotypes moléculaires au moyen de l'électrophorèse en champ pulé (PFGE) [10]. Les caryotypes électrophorétiques de cinq espèces différentes, parasitant des Insectes, sont actuellement connus [11, 12]. Ils présentent entre 8 et 16 bandes d'ADN chromosomique, et la somme des tailles moléculaires correspondantes procure des évaluations de taille du génome haploïde allant de 7,4 à 10,2 Mb. Nous présentons des résultats de séparation par PFGE des molécules d'ADN chromosomique chez deux espèces de Microsporidies parasites de poissons : Glugea atherinae Berrebi, 1978 (parasite de Atherina boyeri Risso, 1810) et Spraguea lophii Doflein, 1898 (parasite de Lophius piscatorius Linné, 1798). Les caryotypes électrophorétiques de ces deux espèces sont nettement différents. Pour G. atherinae, 16 bandes sont réparties de 420 à 2 700 kb (Tableau I) et la taille du génome serait de 19,5 Mb, si l'on fait abstraction des différences d'intensité constatées pour certaines bandes. Il ne peut pas être exclu que deux chromosomes non homologues de même taille soient présents dans une bande d'intensité supérieure à la moyenne. Pour S. lophii, 12 bandes de même intensité sont observées entre 230 et 980 kb (Tableau I). Le génome de cette espèce ne serait alors que de 6,2 Mb. Parmi les Microsporidies étudiées, ces deux espèces représentent donc deux cas extrêmes, G. atherinae ayant le plus grand génome et S. lophii le plus petit. On sait que, chez les levures, la taille du génome varie de 10 à 31 Mb, selon l'espèce considérée [16, 21, 25]. Chez le flagellé Giardia lambelia, elle a été évaluée à 10,6-11,9 Mb [23]. Quant à Pneumocystis, la taille du génome est comprise entre 8 et 16 Mb [24-26]. Le génome de la Microsporidie S. lophii apparaît donc comme le plus petit génome
abstraction des différences d'intensité constatées pour certaines bandes. Il ne peut pas être exclu que deux chromosomes non homologues de même taille soient présents dans une bande d'intensité supérieure à la moyenne. Pour S. lophii, 12 bandes de même intensité sont observées entre 230 et 980 kb (Tableau I). Le génome de cette espèce ne serait alors que de 6,2 Mb. Parmi les Microsporidies étudiées, ces deux espèces représentent donc deux cas extrêmes, G. atherinae ayant le plus grand génome et S. lophii le plus petit. On sait que, chez les levures, la taille du génome varie de 10 à 31 Mb, selon l'espèce considérée [16, 21, 25]. Chez le flagellé Giardia lambelia, elle a été évaluée à 10,6-11,9 Mb [23]. Quant à Pneumocystis, la taille du génome est comprise entre 8 et 16 Mb [24-26]. Le génome de la Microsporidie S. lophii apparaît donc comme le plus petit génome nucléaire connu d'un Eucaryote. Un important polymorphisme de taille et même de nombre de molécules d'ADN chromosomique est fréquemment observé chez les souches d'une même espèce au sein de genres de protozoaires parasites, tels Leishmania et Trypanosoma [31, 32]. Notre étude sur cinq souches de Glugea atherinae a plutôt révélé une faible variabilité. Toutefois, une bande de 2380 kb est absente chez l'une de ces souches. Des analyses de restriction d'ADN et des hybridations sont à réaliser pour tenter de comprendre la cause de cette variation. Acknowledgments: the authors are greatly indebted to G. Bouix, F. Coste and B. Romestand, Laboratoire de Parasitologie et Immunologie, Université Montpellier 2, for the facility given to collect the G. atherinae and S. lophii spores. REFERENCES 1. Sprague V. 1977. Classification and phylogeny of the Microsporidia. In: Bulla L. A. Jr., Cheng T. C, Eds. Comparative Pathology, 2, New York: Plenum Press, 510 p. 2. Margiliet A. M., Strano A. J., Chandra R., Neafie R., Blum M., McCully R. M. 1973. Disseminated microsporidiosis in an immunologically compromised infant. Arch. Pathol. 95: 145-50. 3. Vavra J. 1965. Étude au microscope électronique de la morphologie et du développement de quelques microsporidies. C. R. Acad. Sci. Paris
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