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Length and interspersion of repetitive and non repetitive DNA sequences in four amphibian species with different genome sizes.

The interspersion period of repetitive and unique sequences was analyzed by two different methods, electron microscopy and agarose gel electrophoresis, for four Amphibian species with different nuclear DNA content, namely the Anura Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) and the Urodela Triturus cristatus (23 pg) and Necturus maculosus (52 pg). Within each of the two subclasses it has been found that interspecific differences, in DNA content, due to variations in the amount of repetitive sequences, do not involve variations in length of the interspersed repetitive sequences. They remain about 380 base pairs. Furthermore, the unique sequences length has been found to be shorter in Bufo (760 base pairs) than in Xenopus (1600) and in Necturus (880) than in Triturus (1340). A study of the interspersion period has shown that the great difference in DNA content between Anura and Urodela, which had been previously shown not to have involved changes in the relative amounts of the various sequence classes, does not involve changes in the interspersion period.

Animals↗

Immunological identity of proteins that bind stored 5S RNA in Xenopus oocytes.

In small oocytes of Xenopus laevis, the three most abundant proteins are isolated as basic polypeptides with molecular weights of 48 kD (P48), 43 kD (P43) and 40 kD (P40, also known as transcription factor IIIA). All three proteins share common properties in being able to bind specifically ribosomal 5S RNA molecules and influence, in different ways, their rates of production and utilization. It has been shown by biochemical analysis and immunological characterization that the three proteins are structurally distinct and are most probably the products of different genes. Immunostaining and radio-immunoassays indicate that both P48 and P43 have diverged considerably in structure between the amphibian genera Xenopus and Triturus. Antibodies raised against the transcription factor for Xenopus laevis 5S RNA genes (P40/TFIIIA) do not cross-react with the transcription factor isolated from oocytes of the closely related species Xenopus borealis. A protein equivalent of TFIIIA is not found in 5S RNA-containing RNP storage particles of Triturus oocytes. The functions of the three Xenopus oocyte proteins in transporting 5S RNA between different cellular compartments are considered in the light of these variations.

Animals↗

Influence of longitudinal whole animal clinorotation on lens, tail, and limb regeneration in urodeles.

Two species of newts (Urodela) and two types of clinostats for fast clinorotation (60 rpm) were used to investigate the influence of simulated weightlessness on regeneration and to compare results obtained with data from spaceflight experiments. Seven or fourteen days of weightlessness in Russian biosatellites caused acceleration of lens and limb regeneration by an increase in cell proliferation, differentiation, and rate of morphogenesis in comparison with ground controls. After a comparable time of clinorotation the results obtained with Triturus vulgaris using a horizontal clinostat were similar to those found in spaceflight. In contrast, in Pleurodeles waltl using both horizontal and radial clinostats the results were contradictory compared to Triturus. We speculate that different levels of gravity or/and species specific thresholds for gravitational sensitivity could be responsible for these contradictory results.

Animals↗

Activation of masked neural determinants in amphibian eggs and embryos and their release from the inducing tissue.

The neural-archencephalic (forehead) inducing activity of the microsomes and the high speed supernatant from Xenopus laevis ovaries and eggs was tested on gastrula ectoderm of Triturus alpestris. Both fractions have a very small inducing activity which increases considerably after autolysis or treatment with dissociating agents. A comparison with the inducing activity of the high speed supernatant from gastrulae suggests that neural inducing factors are synthesized in the ovary, stored in a masked form and activated, in part, during gastrulation. When the supernatant proteins from eggs and gastrulae were subjected to size-exclusion HPLC the neural inducing activity was eluted in different size classes, suggesting a limited proteolysis of a precursor during early embryogenesis. Further experiments have shown that treatment of the blastoporal lip from early Triturus gastrulae with actinomycin D or cycloheximide which inhibit the synthesis of RNA and protein respectively, diminishes the neural-archencephalic (forehead) inducing activity of the blastoporal lip. Treatment with actinomycin D or cycloheximide followed by ethanol, which, besides other structures, impairs the plasma membrane, does, however, not reduce the inducing activity. This may suggest that components which are needed for the transport of the neuralizing factor are diminished, when the synthesis of mRNA and protein is inhibited. Such components are probably needed for the release of the neuralizing factor from the blastoporal lip, the inducer tissue.

Animals↗

Increase in liver pigmentation during natural hibernation in some amphibians.

The amount/distribution of liver melanin in 3 amphibian species (Rana esculenta, Triturus a. apuanus, Triturus carnifex) was studied during 2 periods of the annual cycle (summer activity-winter hibernation) by light and electron microscopy, image analysis and microspectrofluorometry. The increase in liver pigmentation (melanin content) during winter appeared to be correlated with morphological and functional modifications in the hepatocytes, which at this period were characterised by a decrease in metabolic activity. These findings were interpreted according to the functional role (e.g. phagocytosis, cytotoxic substance inactivation) played by the pigment cell component in the general physiology of the heterothermic vertebrate liver and, in particular, in relation to a compensatory engagement of these cells against hepatocellular hypoactivity during the winter period.

Amphibians↗

The newt ribozyme is part of a riboprotein complex.

Strand-specific transcripts of a satellite DNA of the newts, Notophthalmus and Triturus, are present in cells in monomeric and multimeric sizes. These transcripts undergo self-catalyzed, site-specific cleavage in vitro: the reaction requires Mg2+ and is mediated by a "hammerhead" domain. Transcription of the newt ribozyme appears to be performed by RNA polymerase II under the control of a proximal sequence element and a distal sequence element. In vitro, the newt ribozyme can cleave in trans an RNA substrate, suggesting that in vivo it might be involved in RNA processing events, perhaps as a riboprotein complex. Here we show that the newt ribozyme is in fact present as a riboprotein particle of about 12 S in the oocytes of Triturus. In addition, reconstitution experiments and gel-shift analyses show that a complex is assembled in vitro on the monomeric ribozyme molecules. UV cross-linking studies identify a few polypeptide species, ranging from 31 to 65 kDa, associated to the newt ribozyme with different affinities. Finally, we find that an appropriate oligoribonucleotide substrate is specifically cleaved by the riboproteic activity in S-100 ovary extracts.

Animals↗

Histone gene number in relation to C-value in amphibians.

We have compared the number of copies of sequences complementary to a cloned Xenopus histone H4 coding sequence in the genomes of Xenopus, Triturus and Ambystoma, amphibian species with widely different C-values (3, 23 and 38pg DNA/haploid genome respectively). Quantitative autoradiography indicates that H4 sequence constitute a greater proportion of the genome the larger that genome is. Measurement of the absolute copy-number by reassociation kinetic analysis indicated 47 +/- 10, 636 +/- 21 2685 +/- 349 copies per haploid genome each in Xenopus, Triturus and Ambystoma respectively. Whilst this confirms a trend of increasing copy-number with increasing C-value, the two are not directly proportional and some other factors must contribute to determining the number of copies of these genes.

Ambystoma↗

Projections of color coding retinal neurons in urodele amphibians.

Optic fiber projection in the brain of Salamandra salamandra was investigated by degeneration techniques. Terminal fields are described in the thalamus and in the optic tectum. Microelectrode recordings were performed from ganglion cells in the retina and from their terminals in the thalamus and tectum in Salamandra and Triturus alpestris. 'On' cells showed maximal sensitivity either in the blue or in the yellow spectral region; they project to the thalamus. Color coding 'on-off' cells project to the tectum opticum. In Triturus a seasonal change in these neurons occurs. Probably due to transition of vitamin A2 into vitamin A1 the spectral sensitivity is different. In springtime blue-red opponent-color neurons were recorded, in fall however, blue-yellow neurons were found.

Animals↗

[Analysis of the structural features of the sperm chromatin of amphibia].

Deoxyribonucleoproteid (DNP) organisation of the sperm from amphibian species (Rana temporaria, Triturus vulgaris, Bufo bufo) with different sperm chromosomal basic proteins has been analysed by nuclease fragmentation. Electrophoretic distribution of chromatin fragments reveals regular organisation of DNP in the sperm of the species investigated. In spermal DNP of the frog Rana temporaria, 3 sub-fractions of histone H1 were found. In the sperm of the newt Triturus vulgaris and the toad Bufo bufo, abundant arginin-rich histones H3 and H4 as well as the presence of low molecular weight protamine-like proteins were noted. Spermal chromatin of all the species studied exhibits high content of chromosomal RNA which is resistant to the effect of exogenous RNAse.

Animals↗

[Furosemide-sensitive transport of chloride through the apical membrane of cells of the proximal tubule of the triton kidney].

The effect of furosemide on transtubular electrical potential (Vt) as well as on the potentials of apical (Va) and basolateral (Vb) membranes of the Triturus vulgaris proximal nephron cells was studied in alteration of the electrochemical potential gradient Na+ and Cl- through the apical membrane. Furosemide did not change the Va and Vb provided the concentration of NaCl in the lumen of proximal canal was the same as in the blood plasma. When the NaCl concentration diminished in the lumen furosemide increased Va and Vb, the Vt remaining unchanged. The effect of furosemide is considerably lesser when the Cl- concentration alone diminishes in the lumen. The data obtained suggest presence of furosemide-sensitive transport of Cl- in the apical membrane of proximal canal cells in the Triturus kidney. This membrane seems to have systems of electrogenic transport of Cl- insensitive to furosemide but depending on the Na+ electrochemical potential gradient.

Animals↗

Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). IV. Salamandridae.

Cloacae were examined from male and female salamanders representing 12 genera and 22 species in the Salamandridae. All female salamandrids possess numerous sperm storage glands, spermathecae, in the roof of the cloaca, but intergeneric variation exists in the occurrence of additional cloacal glands. Pleurodeles and Tylototriton possess both vent and anterior ventral glands, and secondary loss has occurred of vent glands in all other genera and anterior ventral glands in Chioglossa, Cynops, Paramesotriton, and Triturus The most highly derived cloaca occurs in Euproctus asper, in which the cloacal tube extends through a conical projection, and ventral glands secrete onto the dorsolateral surface of the projection rather than into the cloaca. Marked intergeneric variation occurs in males in conformation of the cloacal cavities and in extent of the dorsal gland. In Cynops, Euproctus, Pachytriton, Paramesotriton, Taricha, and Triturus, the pseudopenis (a broad, posteriorly projecting evagination of the dorsal roof) fills much of the cavity of the anterior cloacal chamber. In most salamandrids, distal ends of the dorsal glands occur lateral to pelvic glands in the anterior end of the cloaca, and dorsal gland tubules descend to secretory sites at the posterior end of the vent. Salamandra and Mertensiella possess a unique, bifurcated dorsal gland in which distal ends of tubules lie dorsal to the other cloacal glands, and proximal ends curve ventrally in the anterior end of the cloaca to secretory sites along the cloacal orifice. Cladistic analyses indicate that the variation in presence of anterior ventral glands is due to homoplasy. The occurrence of female vent glands, bifurcated dorsal glands, and the pseudopenis supports a phylogeny based upon non-cloacal characters.

Animals↗

Induction and the Turing-Child field in development.

The central problem in biological development is the understanding of epigenesis. The dominant theory of development in the last 80 years that also purports to explain epigenesis is induction theory. It suggests that development is driven by sequential inductions where each "induction" (in one sense of the word induction) is effected by the action of an inducing part of the embryo on a responding part of the embryo. The theory stems from Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) who transplanted a tissue from the dorsal blastopore lip of Triturus into the ventral ectoderm of another gastrula and thus initiated and "induced" (in another sense of the word induction) gastrulation and embryogenesis in the ventral side of the host that became a double embryo (siamese twins). We explain this induction, i.e. the formation of the double embryo, according to the Child theory and the Turing-Gierer-Meinhardt theory when it is also assumed that cAMP and ATP are the Turing activator and inhibitor, respectively. Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) also suggested that the ingressing mesoderm induces the overlying ectoderm to form the neural plate and neural tube. This 'neural induction', the 'primary embryonic induction', became the cornerstone of induction theory, i.e. of the sequential induction concept referred to above. But we argue that the metabolic gradients that precede and accompany neurulation, as obtained by Child, also for Triturus, arise through a Turing self-organization if it is assumed that cAMP and ATP are the Turing morphogens, and these gradients are the cause and primary event of neurulation. Thus there is no need to invoke the 'neural induction'. It is argued that fundamental events such as gastrulation and also organ formation are caused by the Turing-Child field and not by sequential induction. Similar principles, such as bud formation caused by a radial metabolic pattern that transforms to a longitudinal pattern, govern the formation, for example, of the mouth and the gut. The formation and localization of bottle cells is explained according to the Child-Turing field and modern biochemistry. The chemical metabolic pre-pattern precedes, and causes, morphogenesis and differentiation as envisaged by Turing. The Spemann and Mangold (W.Roux' Arch.f.Entw.d.Organis.u.mikrosk.Anat.100 (1924) 599) transplantation experiment when performed on a sea urchin duplicates not only the phenotype but also the metabolic (reduction) pattern. These experimental results, by Horstadius, predicted by Child, follow from the Turing-Gierer-Meinhardt theory if it is assumed that cAMP and ATP are the Turing morphogens. If the transplantation is performed not onto the whole sea urchin but onto only a part of it, that manifests only a part of the metabolic pattern, then from the part a phenotypic whole underlain by a normal and a whole metabolic pattern can be rescued. These experimental results of Horstadius follow from Turing theory if cAMP and ATP are the Turing morphogens. Understanding how to transform a part into a whole can be valuable in regenerative medicine. Unspecific induction of a secondary amphibian embryo is similar to the induction of posterior structures at the anterior pole of an insect, and the "double abdomen" (and Kalthoff's experimental results) of the midge Smittia resulting from UV irradiation of the anterior pole, can be explained by Meinhardt theory of unspecific induction if ATP is the Turing morphogen. When not working on regeneration, Child investigated intact living organisms and his observation method was not disruptive to normal development, whereas workers in induction theory work with pieces and in general disrupt normal development. We conclude that the Turing-Child field causes all development and explains epigenesis. Sequential induction does not explain epigenesis and does not exist in normal development. But induction in the sense of a transplantation leading to double embryo or rescuing a whole phenotype from a part is of interest.

Adenosine Triphosphate↗

Early functional organization of spinal neurons in developing lower vertebrates.

The spinal neurons in the embryos and young larvae of two amphibians (Xenopus and Triturus) and two fish (Oryzias and Brachydanio) are described and compared. They can be placed into a limited number of common neuron classes: Rohon-Beard sensory, dorsolateral and dorsolateral commissural sensory interneurons, inhibitory ascending interneurons, two classes of inhibitory commissural interneuron, excitatory descending interneurons, motoneurons and possible sensory Kolmer-Agdhur neurons. In Triturus and other urodeles, there are also giant dorsolateral commissural sensory interneurons. The functions of the spinal neurons in simple flexion responses and swimming are considered in relation to evidence mainly from the Xenopus tadpole.

Amphibians↗

Ultrastructure and permeability of nuclear membranes.

The fine structures of nuclear envelopes known to have different permeability properties were compared. Membranes of salivary gland cell nuclei of Drosophila (third instar) and Chironomus (prepupae), which are strong barriers to ion diffusion, and membranes of oocyte nuclei (germinal vesicle) of Xenopus and Triturus, which are much more ion-permeable, show no essential difference in size, frequency, and distribution of their membrane gaps ("pores") which could account for the marked disparities in membrane permeability. The gaps are occupied by diffuse electron-opaque material with occasional central regions of strong opacity. This material may possibly account for the high diffusion resistance of Drosophila and Chironomus nuclear envelopes, where the resistance is far too great to allow free diffusion through the gaps. But material of this kind is also present in the more permeable nuclear envelopes of Xenopus and Triturus oocytes, and there are no convincing structural differences discernible with the techniques employed.

Animals↗

Ribonucleoprotein particles in the amphibian oocyte nucleus. Possible intermediates in ribosome synthesis.

Studies of the sedimentation properties of RNP(1) material from the nucleus of the amphibian oocyte have indicated (1) that there are few, if any, 78S ribosomes in the nucleus, (2) that there are smaller particles sedimenting at 50-55S and 30S, and (3) that the larger of these is the precursor of the 60S subunit of the cytoplasmic ribosomes. Although the nature of the 30S material is not completely clear, it probably includes precursor particles to the 40S ribosomal subunit. Heavy (50-55S) particles are predominant in immature oocytes of Triturus viridescens, whereas in immature oocytes of Triturus and Amblystoma mexicanum they are reduced greatly in amount, but are still detectable. Double-labeling studies of RNA and protein reveal that both types of particle incorporate uridine-(3)H, but that the 50-55S material of immature oocytes does not incorporate (14)C-labeled amino acids. However, other evidence exists that favors the RNP nature of this material. Sedimentation analyses after SDS extraction show that 50-55S particles contain 40 and 30S RNA, whereas 30S particles contain 20S RNA. These types of RNA represent at least 80% of all the extractable nuclear RNA. The 50-55S particles are probably heterogeneous, including both particles containing mostly 40S RNA and particles containing only 30S RNA.

Amino Acids↗

An electron microscope study of the salamander thyroid during hormonal stimulation.

Cytological changes in thyroid glands following administration of thyroid-stimulating hormone (TSH), were studied in adult salamanders, Ambystoma tigrinum, Triturus torosus, and Triturus viridescens by electron and light microscopy. Thyroids from untreated salamanders contained large follicles, faintly basophilic colloid, low follicle cells with flattened nuclei, and scant, slightly basophilic cytoplasm. After TSH administration the cell height and nuclear volume increased. Cytoplasmic basophilia was markedly increased and follicle lumina were reduced. In electron micrographs, stacks of ergastoplasmic lamellae appeared near the nucleus occasionally in contact with the nuclear membrane. In more advanced stages of stimulation, lamellar arrays were largely replaced by small disoriented vesicles and larger vacuoles containing colloid-like material. Sections of obliquely oriented ergastoplasmic membranes contained rows of extremely fine particles. Microvilli increased in size and number and Golgi structures became more extensive. Homogeneous osmiophilic droplets increased in size and abundance. Some of the smaller droplets were seen associated with the Golgi zone. Droplets similar in size and density frequently contained closely packed, whorled lamellae. Mitochondria showed no structural changes but occurred in aggregates interposed between the nucleus and highly folded portions of the basal cell membrane.

Ambystoma↗

Are there insertions in the ribosomal DNA of vertebrates?

We have analysed the Eco RI restriction pattern of rDNA of the newt Triturus vulgaris and of some other amphibian species by Southern blotting and hybridization with nick-translated Xenopus rDNA prepared from the recombinant plasmids pXlr11 and pXlr12 (21). After hybridization with r11, the 28S coding fragments become visible in two bands, a prominent one of 5.3 kb and a weak band of 5.9 kb representing about 8% of the 28S genes. The evidence obtained so far by additional digestions with Bam HI and Bgl II indicates that in this species and in Triturus helveticus the coding regions of the 5.9 kb fragments are interrupted by an insertion 0.6 kb in length located in a 1.6 kb Bgl II fragment at the 3' end of the Eco RI fragment, which we believe to be the first described in a vertebrate.

Animals↗

Mitochondrial DNA rates and biogeography in European newts (genus Euproctus).

Sequence divergence for segments of three mitochondrial DNA (mtDNA) genes encoding the 12S and 16S ribosomal RNA and cytochrome b was examined in newts belonging to the genus Euproctus (E. asper, E. montanus, E. platycephalus) and in three other species belonging to the same family (Salamandridae), Triturus carnifex, T. vulgaris, and Pleurodeles waltl. The three Euproctus species occur (one species each) in Corsica, Sardinia, and the Pyrenees. This vicariant distribution is believed to have been determined by the disjunction and rotation of the Sardinia-Corsica microplate from the Pyreneean region. Because time estimates are available for the tectonic events that led to the separation of the three landmasses, we used sequence data to estimate rates of evolution for the three gene fragments and investigated whether they conform to the rate-constancy hypothesis. By the Tajima (1993, Genetics 135:599-607) test, we could not detect rate heterogeneities for the ribosomal genes and for transversions in the cytochrome b gene. Assuming that these sites are evolving linearly over time and that cessation of gene flow occurred simultaneously with vicariant events, we compared the time of divergence estimated by molecular distances with the divergence times based on the geological estimates. Because we have two estimates of divergence time from the geological record, the split of Corsica/Sardinia from the Pyrenees and the split of Corsica from Sardinia, we could compare ratios of molecular divergence with the ratio of geological time divergence. The ratios are very similar, indicating that the molecular clock hypothesis cannot be rejected. These geological events also allowed us to calculate absolute rates of evolution for ribosomal and cytochrome b genes and compare them to rates for the same regions in other salamandrids and other vertebrates. Ribosomal mtDNA rates are comparable to those reported for other vertebrates, but cytochrome b rates are 3-7 times lower in salamanders than in other ectotherms. From a phylogenetic perspective, our data suggest that the cladogenic events leading to species formation in Euproctus and Triturus occurred very closely in time, indicating that the two genera may not be monophyletic. A duplication of the cytochrome b gene in T. carnifex was found, and the implications of this finding for mtDNA phylogenetic studies are discussed.

Animals↗