Stefania Bucci

Molecular characterization of a centromeric satellite DNA in the hemiclonal hybrid frogRana esculenta and its parental species

Hybrid water frogsRana esculenta reproduce by hybridogenesis: one parental genome (ofRana lessonae) is excluded in the germ line, the other (ofRana ridibunda) is clonally transmitted to haploid gametes. The two parental species differ in that the amount of centromeric heterochromatin revealed by differential staining is much higher inRana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed inRana ridibunda genomes by the restriction endonucleaseStul, which generates a major repetitive sequence fragment of 300 and a minor one of 200 bp. This AT-rich (68%) satellite family is located at the centromeres of the five largest chromosomes (1–5) and of a medium to small heterobrachial one (8 or 9); it thus constitutes only part of the centromeric heterochromatin that characterizes allRana ridibunda chromosomes. RrS1 represents about 2.5% of the genome ofRana ridibunda; it may represent as little as 0.2% of the genome ofRana lessonae, and cannot be detected inXenopus laevis frogs orSalamandra salamandra andTriturus carnifex salamanders. Segments of the satellite sequence are similar to sequences of yeast centromeric DNA element CDEIII and of the mammalian CENP-B box. A role for RrS1 and other centromeric satellite DNAs in the germ line genome exclusion of the hybridogenetic frog hybrids, although suggested, has not yet been demonstrated.

Tests of toxicity and teratogenicity in biphasic vertebrates treated with heavy metals (Cr3+, A13+, Cd2+)

Developmental toxicity of chromium(III), aluminium(III) and cadmium(II) were evaluated by examining abnormalities and mortality in embryos belonging to different species of amphibians. Cr(III) and Al(III) are lethal at 1.5 mM concentration, and seriously affect the differentiation of central nervous system, skeleton and eye, and cause cephalic and trunk oedemas at lower concentrations, being aluminium significantly more harmful than chromium. Cd(II), tested only in P. waltl, is highly toxic: embryos exposed to concentrations ranging from 0.18 to 50 microM display malformations, delay and arrest of development in a dose dependent manner.

Gametogenesis of intergroup hybrids of hemiclonal frogs

European water frog hybrids Rana esculenta (R. ridibundaxR. lessonae) reproduce hemiclonally, by hybridogenesis: in the germ line they exclude the genome of one parental species and produce haploid gametes with an unrecombined genome of the other parental species. In the widespread L-E population system, both sexes of hybrids (E) coexist with R. lessonae (L). They exclude the lessonae genome and produce ridibunda gametes. In the R-E system, hybrid males coexist with R. ridibunda (R); they exclude either their ridibunda or their lessonae genome and produce sperm with a lessonae or with a ridibunda genome or a mixture of both kinds of sperm. We examined 13 male offspring, 12 of which were from crosses between L-E system and R-E system frogs. All were somatically hybrid. With one exception, they excluded the lessonae genome in the germ line and subsequently endoreduplicated the ridibunda genome. Spermatogonial metaphases contained a haploid or a diploid number of ridibunda chromosomes, identified through in situ hybridization to a satellite DNA marker, and by spermatocyte I metaphases containing a haploid number of ridibunda bivalents. The exception, an F1 hybrid between L-E system R. lessonae and R-E system R. ridibunda, was not hybridogenetic, showed no genome exclusion, and evidenced a disturbed gametogenesis resulting from the combination of two heterospecific genomes. None of the hybridogenetic hybrids showed any cell lines excluding the ridibunda genome, the pattern most frequent in hybrids of the R-E system, unique to that system, and essential for its persistence. A particular combination of R-E system lessonae and R-E system ridibunda genomes seems necessary to induce the R-E system type of hemiclonal gametogenesis.

A novel approach to cytotaxonomic and cytogenetic studies in the genus Triturus using monoclonal antibodies to lampbrush chromosomes antigens

Monoclonal antibodies against germinal vesicle antigens from Pleurodeles oocytes crossreact with lampbrush chromosomes of various Triturus species: C36/6, A33/22 and B71/22 bind to most lateral loops, B24/3 labels the spheres, while A1/5 and B81 give a distribution of fluorescent loops which is highly reproducible and species specific. — The antigens involved were characterized by immunoblotting of electrophoretic gels of germinal vesicle proteins and the molecular weights of those that bound to monoclonal antibodies C36/6, A33/22, B24/3 and C3/1 were determined. — The possible relationship between sites immunostained by some monoclonal antibodies and given DNA sequences distributed along the chromosomes is discussed. A new approach to cytotaxonomic and cytogenetic studies through the use of monoclonal antibodies on lampbrush chromosomes is offered, which can give new insight into the molecular mechanisms of speciation and karyological evolution in European newt species.

Differential expression of two vasa/PL10-related genes during gametogenesis in the special model system Rana

Germline cell fate decisions are primarily controlled at the post-transcriptional level with DEAD-box RNA helicases playing a crucial role in germline development. In this study, we report the identification of two DEAD-box vasa/PL10 orthologues (RlVlg and RlPL10) in a species complex of the genus Rana, characterized by hybridogenetic reproduction, an enigmatic process that involves the exclusion of an individual genome, and endoreduplication events. Both genes were expressed during the early stages of gametogenesis of R. ridibunda, R. lessonae, and their natural hybrid R. esculenta. RlVlg expression was germline specific. On the other hand, RlPL10 was also expressed in somatic tissues, although only at low levels. The two genes were expressed in different phases of mitotic and meiotic spermatogenetic divisions as demonstrated by immunostaining with an anti-H3 phosphohistone antibody. The data indicate that RlVlg and RlPL10 may represent useful markers for dissecting the molecular aspects of genome exclusion and endoreduplication of the hybridogenetic gametogenesis.

Bioassays for testing effects of Al, Cr and Cd using development in the amphibian Pleurodeles waltl and regeneration in the planarian Dugesia etrusca

Abstract Historically, water quality studies have been directed toward obtaining physical and chemical measurements on toxicants occurring in the aquatic environment. At present, bioassays are increasingly used as sensitive indicators of pollutant toxicity, since they are rapid, inexpensive, applicable to a variety of toxicants and allow several acute and chronic endpoints to be assessed simultaneously. The analysis of the potential toxicity of heavy metals was conducted on two aquatic model systems, an amphibian, Pleurodeles waltl, and a freshwater planarian, Dugesia etrusca. Aluminum, chromium and cadmium were chosen since human activity has led to a sharp increase of these metals in the environment. Environmentally realistic and supra-environmental concentrations were assayed in short-term toxicity tests. The effects of these metal ions were evaluated by the analysis of both malformations and lethality in amphibian embryos and alterations to planarian regeneration. Aluminum (0.15, 0.75 and 1.5 mmol 1-1...

Negative environmental factors and biodiversity: The case of the hybridogenetic green frog system from Lake Trasimeno

Abstract South of the Po Valley in Italy, green frogs constitute a hybridogenetic system, represented by one parental species (Rana lessonae) and a hybrid form (R. esculenta). Lacking one parent (R. ridibunda), the hybrid is a reproductive parasite on the coexisting parent: progeny from matings between hybrids are inviable; the hybrid reproduces hybridogenetically by first eliminating the R. lessonae genome from the germ‐line cells prior to meiosis, reduplicating the R. ridibunda set, and then transmitting the R. ridibunda genome hemiclonally to gametes. Hybridity is restored in each generation, since R. esculenta gametes are fertilised by gametes from R. lessonae. In order to ascertain whether there are any differences in susceptibility to environmental disturbances between the parental species and the hybrid form, the green frogs living in Lake Trasimeno (central Italy) were analysed. In the absence of reliable morphological systematic characteristics, hybrids and nonhybrids were distinguished by means of a species‐specific centromeric satellite DNA, and the percentages of R. lessonae and R. esculenta specimens at each sampling station were calculated. The results indicate that the parental host is more susceptible to negative environmental factors, which could threaten the whole local hybridogenetic system.

Effects of cordycepin on morphology and RNA synthesis of amphibian lampbrush chromosomes

Abstract The action of certain inhibitors of RNA and protein syntheses on the morphology of the loops of lampbrush chromosomes in amphibian oocytes was studied. Cordycepin, which affects the synthesis of nuclear heterogeneous RNA, induces a complete retraction of the loops into the chromomeres. On the other side puromycin and cycloheximide do not induce any changes in the morhology of lambrush chromosomes.

Molecular investigations in western Palearctic water frogs

Abstract In the green frog hybrids there exists an exclusive cytogenetic reproductive mechanism, referred to as hybridogenesis, whose evolutionary value is still under debate. As a contribution to the knowledge of the possible link between reproductive strategies and phylogenetic relationships, the analysis of repetitive DNA elements in the genomes of the Balkan green frogs, Rana epeirotica and R. shqiperica was carried out to compare their molecular DNA organization with those already known for R. ridibunda and R. lessonae. The Balkan species were chosen, because they give rise to spontaneous, although infrequent and not hybridogenetic hybrids with R. ridibunda. The elements of the RrSl family, a centromeric satellite DNA previously isolated in R. ridibunda, are clustered at centromeric regions also in both Balkan species, although the molecular organization of the RrSl sequences proved to be most similar in R. ridibunda and R. epeirotica. Furthermore, a repetitive DNA family (R1BamHI) is interspersed throughout the genome and is highly conserved in all species examined. Notably, the RIBamHI sequences, correlated to members of the hAT family, represent the first transposable elements found in the genus Rana. In the light of the results obtained so far, further studies are in progress with the aim of isolating and characterizing new families of repeated DNA, as additional results are required to compare the genomes of the different frog species and infer phylogenetic deductions on the genome evolution accompanying the speciation process and occurring inside the Palearctic green frog system.

Isolation and expression of RlYB2, a germ cell‐specific Y‐box gene in Rana

Y‐box proteins are a highly conserved family of gene expression regulatory factors. During gametogenesis they may play a dual role, as both transcriptional activators of germ cell‐specific genes and as translational repressors of stored maternal transcripts. We report the identification of RlYB2, a Y‐box homolog gene specifically expressed in the germ cells of green frogs belonging to Rana esculenta complex, a model system characterized by a hybridogenetic gametogenesis. In developing germ cells of the hybrid R. esculenta, arisen by natural cross of the parental species R. ridibunda and R. lessonae, one set of the parental genomes is excluded and the remaining one, first endoreduplicates and then is transmitted to gametes. In situ hybridizations performed on gonadal tissues showed that the RlYB2 transcript was widely expressed in the ooplasm at early stages of oogenesis in both the parental species and hybrids. Interestingly, a hybridization signal, presumably related to RlYB2‐like nascent transcripts, was observed in nuclei of stage II oocytes. The presence of RlYB2 mRNA during early oogenesis suggests that this gene may be involved in regulating the transcription and/or translation of maternal mRNAs in this special vertebrate model system.