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ObjectivesThis single-centre prospective randomised controlled study aimed to investigate the effectiveness of dignity therapy on spiritual well-being, demoralisation and dignity-related distress compared with standard palliative care.MethodsA total of 111 terminally ill hospice patients were randomly allocated to one of two groups: dignity therapy plus standard palliative care (intervention group) or standard palliative care alone (control group). The main outcomes were meaning, peace, faith, loss of meaning and purpose, distress and coping ability, existential distress, psychological distress and physical distress. Assessments were conducted at baseline, 7–10 and 15–20 days.ResultsFollowing randomisation, 11 dropped out before baseline assessment and 33 after post-treatment assessment. A total of 67 patients completed the study, 35 in the experimental group and 32 in the control group. Repeated measures general linear model showed significant differences between groups on peace and psychological distress over time, but not on existential distress, physical distress, meaning and purpose, distress and coping ability, meaning and faith. Specifically, patients in the dignity therapy intervention maintained similar levels of peace from baseline to follow-up, whereas patients in the control group significantly declined in peace during the same time period. Moreover, psychological distress significantly decreased from pretreatment to post-treatment in the intervention group and increased in the control group.ConclusionsDignity therapy may be an effective intervention in maintaining sense of peace for terminally ill patients. The findings of our study are of relevance in palliative care and suggest the potential clinical utility of this psychological intervention.

Introduction: Dignity Therapy (DT) is a brief, individualized, narrative psychotherapy developed to reduce psychosocial and existential distress, and promote dignity, meaning, and hope in end of life patients. Previous studies have shown that DT was effective in reducing anxiety and depression, and improving dignity-related distress. However, less is known about its efficacy on spiritual well-being. The aim of this study is to contribute to the existing literature by investigating the effects of DT on specific dimensions of spiritual well-being, demoralization and dignity-related distress in a sample of terminally ill patients. Methods: A randomized, controlled trial was conducted with 64 terminally ill patients who were randomly assigned to the intervention group (DT + standard palliative care) or the control group (standard palliative care alone). The primary outcome measures were Meaning, Peace, and Faith whereas the secondary outcome measures were (loss of) Meaning and purpose, Distress and coping ability, Existential distress, Psychological distress, and Physical distress. All measures were assessed at baseline (before the intervention), 7-10 and 15-20 days after the baseline assessment. The trial was registered with ClinicalTrials.gov (Protocol Record NCT04256239). Results: The MANOVA yielded a significant effect for the Group X Time interaction. ANOVA with repeated measures showed a significant effect of time on peace and a significant Group X Time interaction effect on peace. Post hoc comparisons revealed that, while there was a decrease in peace from pre-treatment to follow-up and from post-treatment to follow-up in the control group, there was no such trend in the intervention group. Discussion: This study provides initial evidence that patients in the DT intervention maintained similar levels of peace from pre-test to follow-up, whereas patients in the control group showed a decrease in peace during the same time period. We did not find significant longitudinal changes in measures of meaning, faith, loss of meaning and purpose, distress and coping ability, existential, psychological and physical distress. The findings of our study are of relevance in palliative care and suggest the potential clinical utility of DT, since they offer evidence for the importance of this intervention in maintaining peace of mind for terminally ill patients.

RrS1-like sequences of water frogs (genus Pelophylax) display varied genomic organization, whereas the centromeric hybridization pattern reveals species-specific differences. Using fluorescent in situ hybridization, Pelophylax cf. bedriagae, Pelophylax kurtmuelleri, and Pelophylax ridibundus showed a hybridization signal at centromeres of chromosomes 1-5, but in P. kurtmuelleri the medium-small chromosome labeled was 10 rather than 8. Pelophylax cretensis had almost 16 of 26 centromeres labeled, as did Pelophylax lessonae from Poland when its chromosomes are hybridized with a homologous probe. When StuI-digested genomic DNA was hybridized with RrS1 probe, hybridization ladders for P. ridibundus from Poland have evenly spaced steps (about 100 bp) of uniform intensity from about 200 bp upward. Steps in hybridization ladders from circum-Aegean taxa vary in intensity: larger, odd-numbered steps are often fainter. A strong double band (800/900 bp) in Anatolian P. cf. bedriagae, emphasized by a weak 700 bp band, distinguishes them from P. kurtmuelleri from the Peloponnisos, in which the 900 bp band is almost absent. The ladder in P. cretensis lacks odd-numbered steps. A and B repeats, observed originally within the RrS1 satellite of P. ridibundus, occur also in the circum-Aegean frogs and in P. lessonae, Pelophylax epeiroticus, Pelophylax saharicus, and Pelophylax shqipericus. It is plausible that AB dimers or ABB trimers rather than A or B monomers correspond to functional/evolutionary units. The presence of regions similar to yeast CDEs and mammalian CENP-B boxes suggests a role for RrS1 sequences in centromere organization.

The study of hybridogenesis is expected to shed light on the role played by the so‐called ‘asexual reproduction’ in vertebrate evolution, since hybridogenesis, gynogenesis and parthenogenesis are restricted to lower vertebrates. Hence, it seemed interesting to define the stage and time of germline differentiation in reproductively unstable organisms which, to accomplish functional gamete maturation, can change the normal approach to meiosis by means of unusual cytological steps, such as genome elimination and endoreduplication. The cytogenetic aspects of ‘asexual’ meiosis have already been thoroughly studied. At present, molecular investigations are in progress to characterise the single steps of germline differentiation in a hybridogenetic green frog system, and the results obtained so far can be considered promising, as shown in this work. Vasa, PL10 and Y‐box homolog genes, first isolated in Rana (Pelophylax), are expressed during early phases of gametogenesis in R. (P.) ridibunda and R. (P.) lessonae ...

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, wa...

A family of interspersed repetitive elements, RlBamHI, with sequence similarity to the transposase of hAT DNA transposons, occurs in genomes of eight western Palaearctic water frog taxa and the brown frog Rana dalmatina, but was not detected in Xenopus laevis or Salamandra salamandra. RlBamHI elements are not tandemly arrayed, are dispersed across all chromosomes although not uniformly distributed, and based on dot-blot hybridizations may constitute as much as approximately 10% of the genomes of Rana lessonae, Rana ridibunda, and Rana perezi, but only approximately 1% of that of Rana saharica. Eleven nucleotide sequences of a 572 bp fragment from the nine taxa are very similar (pairwise differences 0.4–8.1% for nucleotides, 0.6–18.2% for amino acids), and all share a single open reading frame across the whole RlBamHI fragment. The reading frame is maintained despite several indels, most of which are multiples of 3 bp, but a pair of which in one species alters and, after 13 codons, restores the reading frame. It is possible that the reading frame is selectively maintained, suggesting recent or even present transposition capacity. The amino acid sequences encoded by RlBamHI elements, but not the nucleotide sequences themselves, reveal the similarity of RlBamHI to members of the widespread hAT superfamily of DNA transposons. Amino acid sequence comparisons permitted no convincing phylogenetic placement of RlBamHI among 32 representative hAT transposons across organisms, probably reflecting both the genetic divergence of RlBamHI elements and the paucity of aligned residues available for analysis. Among DNA transposons, a genomic fraction of 10% is extraordinarily high. Phylogenetic analyses of Rana RlBamHI sequences match poorly with independent genetic and molecular phylogenies; the elements compared probably are not orthologous, which renders their sequences inadequate for reconstructing organismal phylogenetic histories.

We investigated the induction of apoptosis by cadmium in NIH 3T3 murine fibroblasts. Apoptosis was triggered effectively by 10 microM CdCl2 within 24 h, under which conditions cell viability was reduced by 50%. Cadmium-induced apoptosis was demonstrated by both morphological and biochemical analysis. We have shown that cadmium concentrations of 5-20 microM caused nuclear fragmentation. Moreover, internucleosomal DNA fragmentation was evoked by 10-25 microM CdCl2 within 24 h, as detected by the formation of ladder patterns in DNA electrophoresis. Since the induction of programmed cell death occurs together with modifications in the cell cycle, we examined the ability of cadmium to block cell divisions by using a 5-bromo2-deoxy-uridine incorporation assay. Our results indicate that about 40% of treated cells are blocked in G0-G1 phase when exposed to 10 microM cadmium for 27 h. Finally, we addressed the question of whether the effect of cadmium could be prevented by suppressing apoptosis. Over-expression of the anti-apoptotic protein Bcl-2 in NIH 3T3 cells protects against cadmium toxicity, thus suggesting a role for Bcl-2 in the regulation of cadmium-induced apoptosis.

TAFs are thought to play an essential role in eukaryotic RNA polymerase II transcription by mediating the expression of distinct subsets of genes. TAFII60/70 was studied in yeast, Drosophila and humans: in the present work, we analyzed the homologue PwTAFII70 in Pleurodeles. The gene is expressed in ovarian oocytes and throughout development, and the level of expression decreases in late embryos. The transcripts are localized in the animal hemisphere of the fertilized eggs and in the animal blastomeres of embryos at cleavage; later PwTAFII70 mRNA is expressed in the neural plate and folds. TAFII70 protein, which is present in fertilized eggs and throughout development, progressively shows a lower level of expression starting from the neurula stage.

Two repetitive DNA families were detected in the genomes of Palaearctic green water frogs. The first family, named RrS1, is a centromeric satellite DNA, which allows discrimination between the genomes of Rana ridibunda and Rana lessonae, thence representing a useful molecular tool to determine timing and modes of genome exclusion from the germ line cells of the hybrid R. es‐culenta. The second repetitive family, named Rana/Pol III, consists of short, tandemly arrayed sequences, scattered throughout the genomes, and resembling SINE retroposons in their structure. The Rana/Pol III family is present in the genomes of R. lessonae, R. ridibunda, and their hybrid R. esculenta, as well as in R. shqiperica, R. epeirotica, R. cretensis, and the Italian taxon. These sequences are also present in the Iberian species R. perezi, although less abundant, but appear to be lacking in the North African species R. saharica. The distribution of Rana/Pol III elements in the genomes of Palaearctic green water frogs is...

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. wall, is highly toxic: embryos exposed to concentrations ranging from 0.18 to 50 μM display malformations, delay and arrest of development in a dose dependent manner.

A cDNA encoding a protein (B24) belonging to the Mcm/P1 family was isolated from the newt Triturus carnifex. In eukaryotes, the members of the Mcm/P1 family are essential factors in the DNA replication process. B24 protein (TcMcm3) is present in salamandrid ovarian oocytes and early embryos; its role was tested by injecting specific anti-B24 monoclonal antibodies into the cytoplasm of one blastomere of two-cell stage embryos. The injected blastomere encountered cleavage arrest either soon after the injection or following one or two divisions; later, it degenerated. Instead, the uninjected blastomere went on developing and organizing a hemi-embryo, which does not grow beyond the tailbud stage. These results are consistent with the hypothesis that the B24 protein is involved in DNA replication at cleavage. The B24 protein studied here appears to play a specific role in early development; other variants of the Mcm3 group seem to be employed by different adult tissues.

In many organisms, the specification of cell fate and the formation of embryonic axes depend on a proper distribution of maternal mRNAs during oogenesis. Asymmetrically localized determinants are required both for embryonic axes and germline determination in anuran amphibians. As a model system of these processes, we have used a species complex of the genus Pelophylax (Rana), characterized by a hybridogenetic reproduction that involves events of genome exclusion and endoreduplication during meiosis in both sexes. With the aim of characterizing the still largely unknown molecular events regulating Pelophylax gametogenesis, we have isolated in this animal model homologues of the deleted in AZoospermia-like (DAZl) and pumilio gene families (named RlDazl and RlPum1, respectively), which encode posttranscriptional regulators. Expression pattern analysis of these genes showed that RlDazl is exclusively expressed in gonadal tissues, whereas RlPum1 is expressed in both somatic tissues and gonads. In situ hybridization carried out on gonads revealed that the two transcripts were asymmetrically localized along the animal–vegetal (A–V) axis of oocytes. In particular, the RlDazl transcript progressively collected to the vegetal pole during oogenesis, whereas the RlPum1 mRNA was preferentially enriched at the animal hemisphere. In adult testes, RlDazl and RlPum1 were expressed in specific phases of spermatogenetic divisions as shown by immunostaining with anti-H3 phosphohistone antibody. Our results indicate that RlDazl and RlPum1 represent two early indicators of oocyte polarity in this hybridogenetic vertebrate model. Additionally, RlDazl share with vertebrate DAZ- like genes a germ cell-specific expression pattern. J. Exp. Zool. (Mol. Dev. Evol.) 316:330–338, 2011. © 2011 Wiley-Liss, Inc.

Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca(2+) homeostasis seems to have an important role during Cd(2+) injury, but the precise assessment of Ca(2+) signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca(2+) changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca(2+) signals and caused a 40% inhibition of sarcoplasmic-ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.

Mitotic chromosomes of the European water frogs Rana ridibunda and Rana lessonae, the parental species of Rana esculenta, differ significantly in their centromeric regions: when C-banded or when made fluorescent, the centromeres of R. ridibunda (and of the ridibunda chromosomes in R. esculenta) are visible as a conspicuous dark granule or as a conspicuous fluorescent spot; the centromeres of R. lessonae (and of the lessonae chromosomes in R. esculenta) are inconspicuous or not fluorescent. Lampbrush chromosomes of these three taxa are described in detail for the first time; those of R. ridibunda and R. lessonae differ significantly in morphostructural characters such as conspicuousness of centromeres and number, form, and location of giant loops as well as in chiasma frequency. Chromosomes of the two parental species can thus be distinguished when present in lampbrush complements of hybrids. Reproduction in both sexes of natural R. esculenta lineages is hemiclonal: only the unrecombined genome of one parental species, usually R. ridibunda, is transmitted to haploid gametes (hybridogenesis). In 18 hybrids from natural populations of Poland, somatic tissues had allodiploid complements with chromosomes from each parental species. In contrast, spermatocytes I of five males and oocytes I of seven of eight females (221 of 222 oocytes) were autodiploid and contained only R. ridibunda chromosomes that formed n bivalents. These 12 hybrids thus were hybridogenetic. A single female hybrid had oocytes I (33 of 34) with genomes of both parental species; they showed various disturbances including tetraploidy, reduced number of chiasmata, and incomplete synapsis resulting in univalents. This individual thus was not hybridogenetic. The irregular lampbrush patterns indicate that such hybrids will have severely reduced fertility and most of their successful gametes will result in allotriploid progeny.

European water frog hybrids Rana esculenta (R. ridibunda×R. 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.

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.

The ability of cadmium to disrupt calcium homeostasis has been known since a long time, but the precise cellular targets of its toxic action are still debated. A great problem in the interpretation of data has been associated with the ability of cadmium to strongly bind traditional calcium probes. Aequorin, the well-characterized calcium-sensitive photoprotein, was used as intracellular calcium indicator during cadmium injury in NIH 3T3 murine fibroblasts. NIH 3T3 cells were transfected with a cDNA construct containing aequorin fused to a truncated glutamate receptor, which directs the probe to the outer surface of intracellular membranes. At first, we tested if different cadmium concentrations were able to modify the rate of light emission by aequorin showing that cadmium concentrations15 microM were ineffective on aequorin luminescence. Hence, aequorin chimeras revealed as a useful tool in the analyses of Cd2+/Ca2+ interference. To directly investigate the role of Cd2+ in Ca2+ homeostasis, we have started to selectively measure the free Ca2+ concentration in different cell compartments. Here, we report that cadmium reduces the transient free calcium signal after stimulation of cells with bradykinin. Further studies are in progress to clarify the role of mitochondria and endoplasmic reticulum in cadmium-induced alterations of Ca2+ homeostasis in order to link signal transduction modifications with the onset of apoptosis induced by cadmium exposure.

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 th...

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.

The localization of the TATA-binding protein (TBP) associated factor II70 (TAFII70) in the germinal vesicle (GV) of newt oocytes was investigated. In spreads of GV content, anti-hTAFII70 monoclonal antibody (mAb) stained Cajal bodies (CBs) that were either attached to specific sites on the lampbrush chromosomes or free in the nucleoplasm. To confirm this localization the PwTAFII70 cDNA was cloned and myc-tagged transcripts injected into the oocyte cytoplasm. Newly translated PwTAFII70 protein was detected a few hours later in the Cajal bodies. These data support the hypothesis that Cajal bodies are the assembly sites of the transcription machinery of the oocyte nucleus. TAFII70 protein can play a role in lampbrush transcription; alternatively TAFII70 can be considered a component in the subset of TFIID complexes that do not function during oogenesis, but are accumulated in the oocyte for later use during early development.Key words: TAFII70, Cajal body, lampbrush chromosomes, RNA transcription and processing, newts, Pleurodeles.

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) and Cr (0.75 and 1.5 mmol 1-1) were toxic to P. waltl development and both the ions affected planarian regeneration at concentrations ranging from 0.25 to 1.5 mmol 1-1. Cadmium was highly toxic to amphibian embryos at concentrations ranging from 0.18 to 50 μmol 1-1. This metal ion was also highly toxic to planarians, but it did not significantly affect the regeneration process. The results indicate that bioassays with these two organisms are useful to assess the toxic potential of aquatic pollutants.

Nuclei of amphibian oocytes contain spheres, either attached at specific loci of lampbrush chromosomes or free in the nucleo‐plasm, the structure and function of which have been investigated for long time. These spheres were initially shown to contain components involved in pre‐mRNA splicing, pre‐rRNA processing and histone pre‐mRNA 3’ end formation. Later, the spheres were labelled by B24/3 monoclonal antibody, which recognizes a maternal protein of 104 kDa. Recently, a cDNA clone encoding protein B24 has been isolated in Triturus carnifex and analysis of the nu‐cleotide sequence has shown that B24 protein belongs to the Mcm/P1 family, which is thought to play an important and conserved role in initiation of DNA replication. To test the function of B24 protein microinjections of B24/3 mAb were made into one blastomere of two and four‐cell stage embryos. The injected cell degenerated and only hemi‐embryos or underdeveloped embryos were obtained, because B24 protein plays an important role in cell...

Abundant natural interspecies hybrids between the European water frog Rana ridibunda and at least three other taxa reproduce hemiclonally, by hybridogenesis: the non-ridi- bunda genome is excluded in the germ line before meiosis, and the unrecombined ridibunda ge- nome is transmitted to haploid gametes. In contrast, natural hybrids between Rana ridibunda and either of two Balkan species (Rana shqiperica and Rana epeirotica) do not show such genome exclusion. This plausibly results from failure of Balkan Rana ridibunda genomes to "induce" such exclusion in the germ line of hybrids, from "resistance" of Rana shqiperica and Rana epeirotica genomes to such exclusion in hybrids with an "inducing" Rana ridibunda genome, or both. We tested the second hypothesis by examining lampbrush chromosome patterns in oocytes of hybrids that in the soma contain one "inducing" ridibunda genome and one genome of either of the two Balkan species. Several lampbrush chromosome markers (e.g., presence and location of certain giant loops and conspicuousness and width of centromeres) discriminate sets of Rana ridibunda chromosomes from those of Rana shqiperica and Rana epeirotica. Based on such markers, nine diploid female hybrids between Rana ridibunda or Rana esculenta from natural hybridogenetic lineages (Rana ridibunda ◊ Rana lessonae, making ridibunda gametes) from central Poland and either Rana shqiperica or Rana epeirotica each contained both parental genomes in primary oo- cytes; the bivalents showed reduced numbers of chiasmata compared with parental species. It follows that none of these hybrids was hybridogenetic. This conclusion is confirmed, for two hy- brids between Rana epeirotica and either Rana ridibunda or Rana esculenta, by protein electro- phoretic comparison of somatic tissues with primary oocytes, all of which evidenced allelic markers of both parental species. Because Rana ridibunda genomes that are known to induce germ line genome exclusion when combined in hybrids with Rana lessonae genomes were used, these data provide the first compelling evidence for resistance of Rana shqiperica as well as Rana epeirotica genomes to such exclusion. J. Exp. Zool. 279:163-176, 1997. © 1997 Wiley-Liss, Inc.

Hybrid water frogs Rana esculenta reproduce by hybridogenesis: one parental genome (of Rana lessonae) is excluded in the germ line, the other (of Rana 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 in Rana ridibunda. An abundant, tandemly arrayed, centromeric satellite DNA, designated RrS1, is revealed in Rana ridibunda genomes by the restriction endonuclease Stul, 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 all Rana ridibunda chromosomes. RrS1 represents about 2.5% of the genome of Rana ridibunda; it may represent as little as 0.2% of the genome of Rana lessonae, and cannot be detected in Xenopus laevis frogs or Salamandra salamandra and Triturus 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.

The spermatogenesis of 9 F1 hybrids of Triturus cristatus carnifex female x T. vulgaris meridionalis male was studied in squash preparations of testicular fragments, treated by the C-staining method. The chromosome number of these hybrids was examined in spermatogonial metaphases and found to be diploid. The two parental sets were always recognized, which means that a regular, although heterospecific, amphimixis occurred (2n = nfemale + nmale). Meiotic prophase I is greatly altered owing to a failure of typical chromosome pairing and chiasma formation. At metaphase I and/or meta-anaphase I, the effects of the hybrid combination of the 2 specific parental sets are clearly visable. Most primary spermatocytes contain only univalents. A few show chromosome associations (bivalents, trivalents and, more rarely, quadrivalent chains) besides univalents. Such associations are of 2 types: (a) intragenomal associations = associations of 2 chromosomes by a terminal (a1) or subterminal chiasma (a2); (b) intergenomal associations = associations of 2 chromosomes by a terminal (b1) or subterminal chiasma (b2). Univalents segregate at random while the associations often lag on the equatorial plane or migrate entire to a spindle pole. Primary spermatocytes with chromosome multivalents can encounter greater difficulties in accomplishing the first cytokinesis. Secondary spermatocytes are numerically and qualitatively unbalanced; however, some of them undergo spermiogenesis and can give rise to a small number of sperms, generally abnormal and never united in bundles. --Problems related to the occurrence of "anomalous" chiasmata and of intra- and inter-genomal homologies are discussed.

Abstract The karyotype of Neurergus strauchii shows pericentric and subterminal C-bands heterochromatin and nucleolar organizers on chromosome IX. This site is revealed by both a C-band and a chromomycin A3 fluorescent band; the GC-specific fluorochrome also highlights additional sites of fluorescence. The lampbrush chromosomes have a general morpho-structural feature known for Triturus lampbrush chromosomes, but giant loops are lacking. Nucleolar organizer and sphere sites are the main cytogenetic markers in the lampbrush karyotype of Neurergus. The implication of these results to our understanding of cytotaxonomy and evolutionary biology of Palearctic Salamandrids is discussed.

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.

A combined chromosome and C-heterochromatin polymorphism in pair 12 in the complement of the newt species, T. italicus is described. The C-heterochromatin polymorphism is presumably due to a loss in the proximal C-band, whereas the chromosomal polymorphism has its origin in two different independent pericentric inversions both including the centromere and the proximal C-band of chromosome 12. The double-inversion polymorphism has a wide distribution over the range and follows a clear bipolarity between a northern area where the karyotype is homomorphic for the standard type of pair 12 (ST/ST) and an opposite area where the ST type is completely replaced by variant M1 and M2 metacentric chromosomes 12. Various karyophylogenies are possible, but the simplest and the most probable presumes an ancestral karyotype of ST/ST and a mechanism of gradual replacement of the heterobrachial chromosome ST by two independent pericentric inversions. The present data are discussed in relation to existing theories on karyological evolution of Urodeles and the functional significance of telocentric chromosomes suggested by Sessions et al. (1982).

Chromosome number, karyotype and chromosomal banding pattern of Mertensiella caucasica were studied in differentially stained mitotic preparations. The main feature of the C-banded appearance concerns the centromere regions, which are heavily stained by the Giemsa and are outlined by proximal C-bands. This morpho-structural character seems to be peculiar of the genome of M. caucasica, when compared with the C-banding pattern shown by the chromosomes of Salamandra, considered the most closely related form. Therefore, a preliminary cytotaxonomic survey seems to support the opinion that the Caucasian salamander can be referred to a well defined genus Mertensiella rather than a subgenus of Salamandra.

Spermatogenesis in the F1 hybrid (2n=24=12 female + 12 male) between the closely related newt species T. cristatus carnifex and T. marmoratus was apparently normal up to pachytene. Many unpaired chromosomes were present at diplotene and a typical diakinesis was lacking. Primary spermatocytes at meta- and meta-anaphase contained up to 12 regular intergenomal bivalents and a corresponding number of univalents when less then 12 II. Most chiasmata were terminal or subterminal, some intercalary. Chiasmata between corresponding heterospecific chromosomes can be reported as true: real crossing over has taken place, proving the presence of primary chromosomal homologies between the 2 sets of the parental species. Evidence for recombination is based on the segregation of particular markers (i.e., subterminal C-bands and NORs) observed in certain chromosomes at metaphase II. One chromatid of single chromosomes can show the T. cristatus "pheno-type" and the other the T. marmoratus phenotype". A few primary spermatocytes contain a certain number of irregular associations (intragenomal or intrahaploid bivalents, irregular intergenomal bivalents, chromosome multivalents) joined by chiasmata which can be defined as anomalous. Other abnormalities concern the occurrence of interlocked bivalents which occasionally show an anomalous exchange between heterologous chromatids. Cytogenetic criteria useful to evaluate the taxonomic relationships between different species have been discussed as well as some possible trends in chromosome evolution and speciation within the genus Triturus.

SUMMARYThe karyological knowledge of the Carpathian newt T. mpntandoni has been improved by the study of the mitotic chromosomes banded by the C-banding method and the analysis of the ovarian giant lampbrush chromosomes. The karyotype appears very similar, although not overlapping to that of the allied species T. vulgaris, as it results also from the analysis of hybrid complements. A modification of the content of the three species groups or Artenkreise including the European newts appears necessary, also on the basis of recent hybridological investigations.

SUMMARYThe lampbrush phase of 6 F1 female hybrids of Triturus cristatus carnifex ♀ × T. vulgaris meridionalis ♂ and 1 female hybrid of the reciprocal cross was analyzed in several growing oocytes. The chromosome constitution of these hybrids was found to be diploid and derived from a regular, although heterospecific amphimixis (2n= n♀ + n♂). All the oocytes contained 24 unpaired lampbrush chromosomes, among which the single univalents of the 2 parental sets were recognized by their cytogenetic markers (loops, spheres, NORs, other objects and lateral projections). Lampbrush univalents can be joined to one another by transitional fusions of spheres and telomeres and, occasionally, by very rare chiasmata which may be defined as anomalous since they form between heterologous chromosomes and probably represent a further impediment to subsequent meiotic stages. Problems concerning chromosomal homologies and cytogenetic aspects of sexuality and sex determination in these species hybrids have been discussed in th...

1. 1. Vitellogenin has been identified by polyacrylamide gel electrophoresis as one of the major protein fraction in the serum of estrogenized female newts. 2. 2. Antibody to purified vitellogenin has been used to determine immunoelectrophoretically the kinetics of vitellogenin accumulation in the serum following administration of a single dose of 17β estradiol. 3. 3. In vivo incorporation studies with [ 35 S] methionine have shown that, as vitellogenin is synthesised and secreted in the circulating fluid, less and less radioactivity is detectable in the intracellular hepatic melieu. 4. 4. Loss of radioactivity from the liver is causally related to appearance of vitellogenin in the serum. In vitro incorporation studies are equally indicative of this relationship. 5. 5. The data are interpreted as indicating that estrogen administration to female newt causes the liver to synthesize vitellogenin with increasing efficiency.

The maps of the lampbrush chromosomes of Triturus marmoratus oocytes were constructed on the basis of their lengths and major morphological characters such as giant fusing loops, dense matrix loops, lumpy objects, axial granules, lateral globules and reflected fusions; a nucleolus organizing region occurs subterminally on the right side of chromosome X. — Bivalent I appears morphologically asymmetrical, its two partners being of different lengths and bearing heteromorphic loops and other heterozygous structures: this heteromorphism may indicate that the two partners of bivalent I represent the ZW heterochromosomes of the species. Finally, an autoradiographic study has been performed in order to ascertain the pattern of 3H-uridine incorporation shown by the most typical landmarks and nucleoli.

Sono stati studiati i «lampbrush chromosomes» di mutanti semi-albini diTriturus cristatus carnifex. Il rilevamento degli organizzatori nucleolari ha permesso il completamento delle mappe cromosomiche della specie.

An ammoniacal silver staining technique was applied to mitotic metaphase chromosomes of 2 species of Newts (Triturus). The method is useful for identifying nucleolar organizer regions. In addition, it reveals other sites of unknown significance.

The hematic yold precursor--vitellogenin--has been identified immunochemically in the serum of estrogenized females of the nest Triturus cristatus by employing an antiserum prepared against yold proteins.

L'identificazione dei centromeri, delle regioni nucleolo-organizzatrici e di quelle sfera-organizzatrici ha permesso il completamento della morfologia dei «lampbrush chromosomes» diTriturus alpestris apuanus.

A chromosomal variation, changing shape and C-banding pattern of chromosome XII of Triturus italicus was detected among the offspring of two F1 hybrid families of T. italicus female x T. vulgaris meridionalis male. In both families a number of individuals appeared to have a metacentric instead of the expected subtelocentric chromosome XII of T. italicus.--Investigations in three well separated localities in the range of the species showed the polymorphism to have a wide distribution and to be part of a complex pattern involving at least two inversions and (presumably) deficiencies of large C-bands. At meiosis, the shape of bivalent XII, and the location and frequency of chiasmata in the bivalent varied with the karyomorph involved. It is suggested that large rearrangements may still play an important role in the karyological evolution of Triturus.

The study of the mechanisms of speciation and of evolution in the animal world is a subject of wide biological interest which in recent years has become the object of an increasingly vast literature. Protein, chromosome and anatomical evolution as well as changes in the patterns of gene expression represent some of today’s most extensively studied themes which require strict in­terdisciplinary approaches. In each animal group, therefore, any conclusion regarding “phyletic relationship” can be drawn only on the basis of all the chromosomal and genetic, as well as biochemi­cal and biometrical, evidence (White 1973a).

Diploid F1 hybrids between Rana ridibunda from Adriatic SW Yugoslavia and Polish R. lessonae, and between Polish R. ridibunda and an unnamed species from SW Yugoslavia are shown by electrophoresis and examination of lampbrush chromosomes to contain both parental genomes in their diploid oocytes I. In contrast, central European R. ridibunda genomes in diploid hybrids, whether F1s or from natural hemiclonal lineages, induce exclusion of the lessonae genome in the germ line, only ridibunda translation products and endoreduplicated ridibunda chromosomes being detected in their diploid oocytes I. The data demonstrate geographic variation of R. ridibunda in the ability of its genomes to induce clonal gametogenesis in interspecies hybrids. They also suggest that genomes of the unnamed Yugoslavian species may be resistant to such exclusion.

The chromosomes of Euproctus montanus and E. platycephalus were studied by means of the C-banding method and the AS-SAT technique which are useful for identifying the single pairs of the complement and for recognizing nucleolar organizer regions. According to the morpho-structural characteristics shown by the specific karyotypes, it has been possible to draw some cytotaxonomic deductions concerning the karyological evolution within the insular group.

Specific banding patterns can be produced on the mitotic chromosomes of the newt species Triturus vulgaris meridionalis and T. italicus by using the Giemsa stain technique. These bands are most useful cytogenetic markers in karyotyping, since they facilitate identification of the individual elements of the complements. Evaluation of the shape of chromosomes as well as of the banding patterns produced by the Giemsa stain indicates that the karyotypes of T. vulgaris meridionalis and T. italicus are differentiated: hence the specific distinction of the two Salamandrids, still debated by taxonomists, appears supported by chromosome evidence. — Most of the bands seem to correspond to the heterochromatic tracts observable on mitotic chromosomes from embryos and larvae either untreated or submitted to cold treatment. Besides, the comparison of mitotic karyotypes and lampbrush maps shows that the bands located near the centromeric regions of mitotic chromosomes probably correspond to the so-called bars visible on either side of centromeres of lampbrush chromosomes, while some of the subterminal bands may correspond to the sphere.

The highly repetitive Rana/Pol III family consists of short, tandemly arrayed sequences, scattered throughoutthe genomes of Palearctic green water frogs. The repeat unit is about 250 bp in length and is a composite element: itcontains a SINE-like retroposon with a tRNA structure, flanked by two short direct repeats, and the occurrence of twointernal repeats gives evidence that an additional transposition event may have inserted a segment within the alreadytransposed element. Rana/Pol III family is present in the genomes of Rana lessonae, R. ridibunda, and their hybridform R. esculenta, as well as in R. shqiperica, R. epeirotica, R. cretensis, and the Italian taxon. These sequences arealso present in the Iberian R. perezi, although less abundant, but appear to be lacking in the north African speciesR. saharica. The distribution of Rana/Pol III in the genomes of Palearctic green frogs is in agreement with the phyletichistory based on genetic data. The evolutionary pattern proposed for the genus Rana enables us to suppose that thehybridogenetic mechanism is one of the factors accounting for the possible horizontal transfer of Rana/Pol III elementsfrom the central-north Europe species toR. perezi.Key words: repetitive DNA, SINE-like retroposons, hybridogenesis,Rana esculenta group.Resume : La famille de sequences tres repetitives Rana/Pol III comprend de courtes sequences en tandem qui sontdispersees dans l’ensemble du genome chez les grenouilles vertes du genre Rana. L’unite repetee mesure environ 250pb et s’avere d’une nature composite; elle comprend un retrotransposon de type SINE qui presente une structured’ARNt, lequel est borde de deux courtes sequences repetees directes. De plus, la presence de deux sequences internesrepetees suggere qu’un evenement de transposition additionnel aurait pu inserer un segment a l’interieur d’un elementtranspose. La famille de sequences Rana/Pol III est presente chez le Rana lessonae,leR. ridibunda, leur hybrideR. esculenta de meme que chez le R. shqiperica, R. epeirotica, R. cretensis et le taxon italien. Ces sequences sontegalement presentes chez l’espece iberique R. perezi, bien qu’en moins grand nombre, alors qu’elles semblent etreabsentes chez l’espece nord-africaineR. saharica. La distribution des sequences Rana/Pol III chez ces especes est enaccord avec la phylogenie etablie a l’aide des donnees genetiques. Le type d’evolution propose chez le genre Ranapermet aux auteurs de supposer que le mecanisme hybridogenetique est l’un des facteurs qui pourrait expliquer lepossible transfert horizontal d’elements Rana/Pol III des especes d’Europe du Centre-Nord vers leR. perezi.Mots cles : ADN repetitif, retrotransposon de type SINE, hybridogenese, especes du groupe Rana esculenta.[Traduit par la Redaction] Bucci et al. 511

E' stata descritta la diversa morfologia dei cromosomi mitotici diTriturus alpestris apuanus, studiati in blastomeri e in cellule somatiche di individui adulti.

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