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PubMed1975

DNA replication in the amphibia.

Autoradiographic techniques were used to measure rate of replication and length of the replication unit in cultured cells of Scaphiopus couchi, Bufo cognatus, Rana clamitans, and Triturus viridescens, having nuclear DNA amounts in the ratio 1:4:7:39 respectively. The autoradiographic experiments were designed to show whether the larger amounts of nuclear DNA are correlated with more rapid rates of synthesis and/or with longer replication units. -- The DNA replication rate was 2.5 mu/minute (corrected for two growing points) with 10 minutes 3H-thymidine label at 22 degrees C, but decreased with longer labelling durations. The length of the replication unit (estimated by the distance from the center of one autoradiograph to the center of the next in sequence) was most commonly in the 10-25 mu range with a 30 minute label, in all four species. The average center-to-center distance was 8 mu at 10 minutes and increased with label duration, to over 45 mu with 24 hours label. Replication was predominantly but not exclusively bidirectional. Neither rate of replication nor length of the replication unit was proportional to the amount of DNA in these species.

Amphibians↗

PubMed1976

The relationship of a specific chromosomal region to the development of the acrosome.

In early spermatids of Urodeles the chromosome segments bearing constitutive heterochromatin are localized in one half of the round nucleus; this region becomes the basal part of the long nucleus of the spermatozoon. The euchromatic chromosome segments extend toward the anterior nuclear pole in a bouquet configuration (Macgregor and Walker, 1973). In the course of spermiohistogenesis, one of the heterochromatic regions (the acrosomal chromocenter) migrates from the basal part to the anterior half of the spermatid nucleus. This heterochromatic block is identical with a species-specific, definite C-band in the karyotype. This relationship between the acrosomal chromocenter and a specific chromosomal C-band was established in Triturus cristatus, T. marmoratus, T. alpestric and Cynops pyrrhogaster. In closely related species this particular C-band lies on similar chromosomes. - While the spermatid nucleus still retains its round shape the acrosomal chromocenter despiralizes into a long heterochromatic thread (acrosomal thread). Precisely at the position of this thread the nucleus evaginates and acquires a pear-like shape. During the elongation of the nuclear protrusion the acrosomal thread remains associated with the anterior end. At termination of spermiogenesis it lies closely below the acrosome in the tip of the spermatozoon. Spontaneous aberrations which affect the acrosomal chromocenter or the thread lead to the development of spermatozoa with defective tips. - Several euchromatic segments, interspersed between the heterchromatic segments, can be recognized in the completely despiralized acrosomal thread. Genes responsible for the morphogenetic activities of both, the acrosomal chromocenter and the acrosomal thread, in the development of the spermtip, might be localized in these interspersed euchromatic segments. The existence in higher vertebrates of an acrosomal chromocenter or an equivalent chromosomal region is discussed.

Acrosome↗

PubMed1976

Ultrastructure of segmentation mitoses in cleaving newt eggs after blocking of centrospheres by quinoline.

In quinoline treated blastomeres of Triturus helveticus Raz. and Pleurodeles waltlii Michah., microtubules disappear and around the centrioles markedly enlarged dense bodies accompanied by striated bodies are accumulated, from prophase beyond metaphase. It is concluded that these bodies in untreated cells play a role in the formation of the spindle microtubules. - During metaphase the chromosome are smooth-surfaced and show a pronounced tendency to stickiness. During ana-telophase they become surrounded by nuclear membranes and form caryomeres. - Qinoline does not interfere with centriole replication, but prevents the separation if diplosomes that have been already formed, if it acts before that separation. - Some centrioles exhibit different degrees of ultrastructural disarrangement.

Animals↗

PubMed1980

Neural crest replaced by gastrula ectoderm in amphibia. Effect on neurulation, CNS, gills and limbs.

Early Axolotl gastrula ectoderm was grafted into early Triturus neural stages in place of excised neural folds at the gill and anterior trunk level. Macroscopically the young graft behaves like normal neural fold material: it follows the closing host neural plate to the dorsal midline, folds into the host's interior and, especially in the gill region, moves ventrad beneath the host's epidermis. These movements cannot be interpreted as active migration. They are the result of passive displacements by morphogenetic forces inside the embryo. Histologically the graft differentiates into neural and neural crest tissue, the quantitative relation depending on the host's region. At the gill level the graft forms mesenchyme and other neural crest elements and hardly any neural structures. In the trunk about one half of the graft forms a secondary, surplus CNS. Problems of induction, differences between gill and trunk region and between graft and normal fold behaviour are discussed. Limbs develop normally. The dorsal layer of the blastema is furnished by graft cells. Host and graft tissue can stay separate or form a combined blastema.

Ambystoma↗

PubMed1975

Neural crest and early fore limb development in amphibia.

Anterior trunk neural folds were exchanged between neurula stages 15 to 16 (Harrison) of several Triturus species and Ambystoma mexicanum. Donor neural crest cells migrated ventrad before and during initial bud formation. They lined the early lumb epidermis and became incorporated into the young blastema. Donor melanophores and non-melanophores contributed substantially to host limb buds.

Ambystoma↗

PubMed1995

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study.

Neuroanatomic comparison of the binding capability of 2-[125I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[125I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.

Animals↗

PubMed1975

In situ hybridization of ribosomal DNA labelled with 125iodine to metaphase and lampbrush chromosomes from newts.

Methods are described for in situ hybridization of ribosomal DNA from Xenopus laevis, labelled in vitro with 125iodine, to mitotic and lampbrush chromosomes from Triturus cristatus carnifex. The hybridization reaction was carried out in a mixture containing 50% formamide, 4 X SSC, 0.1 M KI, at 37 degrees C, or in 2 X SSC, 0.1 M KI at 65 degrees C. Autoradiographs of mitotic metaphases from 2 males showed labelling over the middle of the short arm of one chromosome IX in each metaphase. In some cases, a region near the end of a longer chromosome was also labelled. In a lampbrush preparations, labelling was confined to a region identified as about 53 units, near the middle of the short arm of both halves of bivalent IX. The usefulness of the technique and the significance of the labelling of only 1 of the 2 chromosomes IX in mitotic preparations are discussed.

Animals↗

PubMed1976

DNA reassociation kinetics in relation to genome size in four amphibian species.

DNA reassociation kinetics were studied, by means of the hydroxyapatite chromatography method, for four species of Amphibians with different nuclear DNA content: Xenopus laevis (3 pg DNA per haploid genome) and Bufo bufo (7 pg) of the Anura subclass and Triturus cristatus (23 pg) and Necturus maculosus (52 pg) of the Urodela subclass. Within each subclass the two species studied were found to have about the same absolute amount of unique DNA. The differences of total nuclear DNA can be accounted for by quantitative variations of the repetitive sequence classes, at least in part due to changes in the number of copies of the various sequences. On the contrary the great difference in nuclear DNA between the two subclasses, Anura and Urodela, involves all sequence classes in parallel; the slowly reassociating fraction appears to be unique in spite of a tenfold difference in absolute amount. The dependence of reassociation kinetics on DNA fragment length for the four species indicates for all of them an interspersed organization of the various sequence classes.

Amphibians↗

PubMed1995

Expression of alpha SM actin in terrestrial ectothermic vertebrates.

alpha-Smooth muscle (alpha SM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-alpha SM-1. Immunoreactivity to this antibody has been shown to be localized in the NH2-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian (Triturus vulgaris, Rana esculenta) and three reptilian species (Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti-alpha SM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-alpha SM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine alpha SM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac-EEED, but not Ac-DEED, as occurs in mammals. alpha SM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-alpha SM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle (P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution alpha SM actin arose earlier than previously proposed.

Actins↗

PubMed1986

The influence of furosemide and Co2+ on electrolyte and water transport in newt distal tubule and frog skin.

The technique of stationary microperfusion of the early distal tubule used in experiments on male newts (Triturus vulgaris) revealed the intratubular effect of CoCl2 and furosemide on the ion transport. CoCl2 in concentration 5 X 10(-4) M increased Na and Cl reabsorption, reduced Ca, Mg and K reabsorption, and increased (TFexp/TFo)in from 1.14 +/- 0.04 to 1.68 +/- 0.27 (p less than 0.001). CoCl2 in concentration 5 X 10(-5) M enhanced Na and Cl reabsorption, without affecting transport of other ions. 6 X 10(-4) M furosemide decreased reabsorption of water, Na, Cl, Ca and Mg. The combined effect of Co2+ and furosemide inhibited reabsorption of Mg and Ca to a greater extent, but stimulated Na reabsorption. Experiments on frog skin showed that being added to the apical membrane of cells, Co2+ stimulates short circuit current and potential differences, whereas furosemide reducing Cl absorption induces only hyperpolarization. Experimental findings allow to assume that Co2+ on the outer surface of the apical membrane of the distal tubule cells blocks the selective Ca and Mg channels and activates Na channels.

Animals↗

PubMed1976

Analysis of chromatin-associated fiber arrays.

Electron microscopic examination of chromatin from embryonic nuclei of Oncopeltus fasciatus and Drosophila melanogaster reveals arrays of chromatin associated fibers. The lengths and spacings of these fibers were analyzed to provide a basis for defining and interpreting regions of transcriptionally active chromatin. The results of the analysis are consistent with the interpretation of some fibers as nascent RNA with associated protein (RNP). The chromatin segments underlying these fiber arrays were classified as ribosomal or non-ribosomal transcription units according to definitions and criteria described by Foe et al. (1976). Nascent fibers on active ribosomal transcription units were analyzed and compared for Drosophila melanogaster, Triturus viridescens, and Oncopeltus fasciatus. A common feature of the fiber patterns on ribosomal TUs is that origin-distal fibers exhibit greater length variability and a lower slope relative to proximal fibers. The region of increased variability in fiber lengths is correlated with the expected location of 28S ribosomal RNA sequences in the distal half of each ribosomal transcription unit. Because 28S ribosomal RNA appears to contain more extensive regions of base sequence complementarity, we suggest that the length of ribosomal RNP fibers is influenced under our spreading conditions by the secondary structure of the nascent RNA. In order to calculate the RNA content of RNP fibers, chromatin morphology was used to estimate lengths of transcribed DNA. The packing ratio of DNA in chromatin, which we express as the length of B-structure DNA divided by length of chromatin, is 1.1-1.2 and 1.6 for the DNA in active ribosomal and non-ribosomal chromatins, respectively. These DNA packing ratios are used to determine the extent to which nascent RNP fibers are shorter than the transcribed DNA (expressed as DNA/RNP length ratio). For non-ribosomal transcription units and for proximal fibers of ribosomal transcription units. DNA/RNP length ratios are relatively constant within each array. However, considerable variability in this ratio (4-23) is observed for different arrays of fibers. Possible sources of this variability are considered by comparing ratios derived from the presumably identical ribosomal transcription units. Further analysis of the morphology of nascent fibers may elucidate the contributions of proteins and successive RNA sequences to RNP structure.

Animals↗

PubMed1995

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

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.

Animals↗

PubMed1983

A study of chromosomal organization of repetitive DNA sequences by in situ hybridization.

Chromosomal DNA restriction fragments from Triturus cristatus carnifex, were cloned in pBR322. Five clones containing repetitive DNA sequences were analysed in terms of size, repetition frequencies, GC contents and interspersion patterns. All the data suggest that the cloned sequences are typical for the major repetitive classes found in carnifex and represent members of individual repetitive families. All five cloned sequences hybridize in situ to nascent RNA transcripts on lampbrush loops present in the heteromorphic region of chromosome 1. One of the cloned sequences is interesting in that it shows individual variation. The least repeated sequences are transcribed at many more loci than the more highly repeated sequences and are better represented in the total ovarian RNA.

Animals↗

PubMed1996

Effect of a dipeptide inhibiting ubiquitin-mediated protein degradation nerve-dependent limb regeneration in the newt.

The dipeptide Leu-Ala, which inhibits ubiquitin-mediated protein degradation, has been shown to act in vitro as an inhibitor of neurite outgrowth of PC12 cells (Hondermarck et al. [1992] Biochem. Biophys. Res. Commun. 189:280). Using agarose beads as vehicles, we tested, in vivo, the effect of this dipeptide (and the inactive inverse, Ala-Leu, as a control) on limb regeneration in the newt (Triturus cristatus), a nerve-dependent developmental process. Leu-Ala inhibited the growth of mid-bud blastemas without altering blastema differentiation, while Ala-Leu had no effect. Cytological observations of dipeptide-treated blastemas using Bodian staining or neurofilament antibodies showed that all the blastema tissues were unmodified except with regard to innervation. Leu-Ala-treated blastemas were devoid of nerve fibers in the epidermal cap, while the mesenchyme distal to the dipeptide impregnated bead exhibited fewer nerve fibers than did Ala-Leu-treated blastemas, which were similar to the control nontreated blastemas. Thus, Leu-Ala, in reducing blastema innervation, inhibits its growth in the same manner as surgical denervation.

Animals↗

PubMed1997

Lampbrush chromosomes and chiasmata of sex-reversed crested newts.

Triturus cristatus carnifex provides a particularly clear example of sexual dimorphism for chiasma frequency and localisation. Oocytes from normal XX females routinely carry one proximal chiasma on each arm of their lampbrush bivalents. Spermatocytes from normal XY males have more numerous and relatively distal chiasmata. Lampbrush chromosomes from the oocytes of sex-reversed XY neofemales are found to resemble those from normal oocytes in having one proximal chiasma on each bivalent arm. A comparison of particular markers on the heteromorphic long arm of chromosome 1 provides evidence to equate the lampbrush 1A to somatic 1A, and confirms previous reports that lampbrush chromosome 1A is slightly longer than 1B. The XY sex bivalent of neofemales does not show any obvious heteromorphy of recognised marker loops.

Animals↗

PubMed1978

Clustered and interspersed repetitive DNA sequences in four amphibian species with different genome size.

We have compared the amount of clustered and interspersed repetitive sequences in the genome of four Amphibia with different DNA contents per haploid nucleus: two Anura (Xenopus laevis, 3 pg and Bufo bufo, 7 pg) and two Urodela (Triturus cristatus, 23 pg and Necturus maculosus, 52 pg). High molecular weight DNA of the four species was denatured and reassociated to the same Cot in order to obtain duplex sequences with a similar reiteration frequency. Single-stranded DNA was digested off with the Aspergillus S1 nuclease. DNA was then fractionated according to the molecular weight through an agarose A-50 column. We found that the amount of long repetitive sequences is roughly proportional to the genome size in the four species, while the number of short (about 300 base pairs) repetitive sequences is increased many-fold in the species with the larger DNA content, both in Anura and in Urodela.

Amphibians↗

PubMed1981

[Somatotopic relations between the olfactory mucosa and the olfactory bulb in the triton].

The axons of receptors located in the olfactory neuroepithelium are known to synapse with mitral cell dendrites in the glomerular layer of the ipsilateral olfactory bulb, but few previous studies showed a clear-cut topological organization of this projection. We therefore injected horseradish peroxidase (HRP) into restricted areas of the olfactory mucosa in adult tritons, Triturus cristatus. The animals were perfused with glutaraldehyde 17 h later; the mucosa, the olfactory nerve and bulb were removed, serially cut and treated with diaminobenzidine to reveal the localization and extent of the injection and the areas in the glomerular layer of the bulb to which the injected HRP had been transported by anterograde axonal flow. Small injections of HRP tended to label a small area in the bulb, while larger injections projected to a wider zone. The results also indicated the existence of an orderly projection, with a transposition of the dorso-ventral axis of the neuroreceptor sheet into an essentially anteroposteriorly oriented axis in the bulb. Thus, the dorsally located areas of the olfactory mucosa project to the most anterior part of the glomerular layer, whereas the vomero-nasal mucosa, which is located near the most lateral part of the ventral mucosa, projections to the posterior enlargement of the glomerular layer in the bulb. The data also showed that nerve bundles within the olfactory nerve maintain are orderly position which depends upon their origin. It is postulated that this somatopic organization reflects the development undergone by the peripheral olfactory system during ontogenesis.

Animals↗

PubMed1988

The effect of heat shock on the morphology of amphibian lampbrush chromosomes.

Isolated oocytes of Triturus cristatus carnifex cultured in Barthe's medium respond to a sudden increase in temperature of 35 or 37 degrees C by the rapid condensation of their lampbrush chromosomes. The degree of condensation depends on the severity of the heat shock and is accompanied by the retraction of the transcriptionally active lampbrush loops. The oocytes were kept at 20 degrees C for 12 h after the period of heat shock, before the chromosomes were isolated. The minimum period required to effect loop retraction in immature oocytes of 0.7 mm in diameter was 10 min at 35 degrees C; although more extensive and complete retraction occurred if the oocytes were incubated for periods of up to 40 min. Chromosomes from more mature oocytes (1.4 mm in diameter) require longer periods of heat shock before undergoing condensation. The oocytes themselves do not show any obvious morphological changes after heat shock, although the germinal vesicles are initially too fragile to isolate manually for several hours. When the oocytes are returned to ambient temperatures, they can be cultured in Barthe's medium for several days, enabling the chromosomes to be isolated and studied. Generally, the chromosomes and loops from immature oocytes remain in a condensed state for about 48 h but then the chromosomal axis begins to relax and the loops begin to re-form. Depending on the severity of the initial heat shock, complete recovery of the normal lampbrush morphology is attained after a few days. The re-formed loops are morphologically indistinguishable from untreated loops in control preparations and are of the same length on average. This response of lampbrushes to heat shock is a reliable and repeatable process and should therefore become a valuable model system for the study of chromatin and chromosome structure during changes in transcriptional activity. The time taken for the lampbrushes to recover their normal morphology, is discussed in terms of the return to normal cellular transcription.

Animals↗