Biogene Amine im Gehirn vom Frosch (Rana esculenta)

Zusammenfassung Mit Hilfe der Methode zur fluoreszenzmikroskopischen Lokalisation von Catechol- und Tryptaminen wurde die Verteilung dieser Stoffe im ZNS von Rana esculenta untersucht. Catecholamin- und serotoninhaltige Neurone liegen im Nucleus reticularis mesencephali. Außerdem finden sich catecholaminhaltige Nervenzellen im Organon vasculosum hypothalami und in der Area praeoptica. Diese aminproduzierenden Zellen entsenden Zellfortsätze durch die Ependymschicht in den Ventrikel. Über diese Ausläufer erfolgt möglicherweise eine Sekretion biogener Amine in den Liquor cerebrospinalis. Catecholamin- und serotoninhaltige Axone erreichen voneinander verschiedene Kerngebiete und Areale. Neben dem periventrikulären Zellager im Tuber cinereum und in der Area praeoptica werden vor allem der ventrolaterale Teil des lateralen Septumkerns, Striatum ventrale und Epistriatum von Endstrecken catecholaminhaltiger Axone durchdrungen. Serotoninhaltige Varicositäten finden sich dagegen vor allem in Kerngebieten, die in sensorische Bahnen eingeschaltet sind (Nucleus isthmi, corpus geniculatum laterale, Area praetectalis, Tectum opticum, Thalamus dorsalis, Neostriatum). Weitere Ausbreitungsgebiete 5-Hydroxytryptamin-haltiger Fasern sind die Habenula und der Nucleus interpeduncularis, Kerngebiete, über die Erregungen aus dem limbischen System auf vegetative Zentren der Medulla oblongata geleitet werden.

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Biogenic amines in the brain of the frog (Rana esculenta)

Summary The distribution of biogenic amines in the central nervous system of Rana esculenta was investigated by means of the fluorescence-microscopical detection of catecholand tryptamines. The nucleus reticularis mesencephali was found to contain numerous neurones rich in catechol- and tryptamines. Apart from this nucleus nerve cells in the organon vasculosum hypothalami and in the area praeoptica were found to contain catecholamines. The clublike processes of these neurones penetrate the ependymal layer and extend into the ventricle. These structures are presumably responsible for a secretion of biogenic amines into the cerebrospinal fluid. Catecholamine- and serotonin-containing axons terminate on different nuclei and areas. Besides the periventricular cellular layer of the tuber cinereum and the area praeoptica, the pars ventrolateralis of the nucleus septalis lateralis, striatum ventrale and epistriatum are pervaded by terminals of catecholamine-containing neurons. Serotonincontaining varicosities are mainly to be found in nuclei, which are intercalated in sensory pathways (nucleus isthmi, corpus geniculatum laterale, area praetectalis, tectum opticum, thalamus dorsalis, neostriatum). Further areas of distribution of 5-hydroxytryptamine-fibers are the habenula and the nucleus interpeduncularis, nuclei which coordinate impulses from the limbic system projecting them on visceral centers of the medulla oblongata.

Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 M aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).     

Asynchronous meiotic rhythm as the cause of selective chromosome elimination in an interspecific <Emphasis Type="Italic">Brachiaria</Emphasis> hybrid

This paper reports the occurrence of chromosome elimination during microsporogenesis in an interspecific hybrid between a sexual diploid accession (SEX) of Brachiaria ruziziensis (2n=2x=18) and an apomictic tetraploid accession (APO) of B. brizantha (2n=4x=36). Meiosis was very abnormal in the triploid hybrid (2n=3x=27); we observed a distinct asynchrony from metaphase I to the end of meiosis. The APO and the SEX genomes did not show the same meiotic rhythm. When the former, with nine bivalents, was in metaphase I, the nine SEX univalents were not yet aligned; when the latter reached the plate, the APO genome was already in anaphase. In subsequent stages, the APO genome had reached the poles while the SEX was undergoing sister-chromatid segregation. As the SEX genome always remained temporally behind, it gave rise to one extra-nucleus in each pole. In the second division, the behavior was the same but anaphase II did not occur for the SEX genome, and only one extra-nucleus was observed in each cell in telophase II. Chromosome elimination for the SEX genome ranged from partial to total. The importance of these findings with respect to Brachiaria breeding programmes is discussed.     

A new synthetic species of Cucumis (Cucurbitaceae) from interspecific hybridization and chromosome doubling

A new synthetic species,Cucumis hytivus J.-F. Chen & J. H. Kirkbr. (2n=38), obtained by doubling the number of chromosomes in the sterile interspecific F₁ hybrid (2n=19) betweenC. sativus L. (2n=14) andC. hystrix Chakr. (2n=24), is described.     

Arterial supply of the choriocapillaris of anuran amphibians (Rana temporaria,Rana esculenta)

Summary The pattern of the vascular supply to the choroid of the frog eye was studied in toto with the use of the injection-replication-SEM technique. The choroid of anuran amphibians is composed mainly of the choriocapillaris. In both species studied (Rana temporaria, Rana esculenta), an independent arterial supply to the choriocapillaris supplemented that from the ciliary arteries. This additional vascular route arises from the optic artery, a separate branch of the arteria infundibularis superficialis. The optic artery, accompanied by its vein within the vascular sheath of the optic nerve, joins the rich arterial capillary network of the choriocapillaris and supplies the posterior pole of the ocular bulb. The superficial capillary network displays a dense collar around the entrance of the optic nerve into the eye and is composed of a circular meshwork of small capillaries, several layers deep. More peripherally, however, it becomes single layered. This capillary network, as a whole, establishes numerous connections with the adjacent choriocapillaris at the posterior pole of the ocular bulb. In anuran amphibians the complex arrangement of both arterial systems supporting the choriocapillaris may be regarded as a more complete equivalent of the short posterior ciliary arteries of mammals.     

Inferred chromosome morphology of the ancestral genome ofTriticum

The lengths of the A, B, and D genomes of common wheat,Triticum aestivum, were measured from the karyotype. Relative to the B genome, standardized as length 1.000, the lengths of the A and D genomes were 0.835 and 0.722, respectively. The lengths of the chromosome arms in the A and D genomes were then multiplied by the appropriate constants so that the total lengths of each genome also equalled 1.000. These calculations revealed that homoeologous chromosomes in wheat, with a few exceptions, have similar sizes and arm ratios. The arm lengths of the three homoeologues in each homoeologous group were then averaged. These average chromosomes turned out to be remarkably similar, in size and arm ratio, to their homoeologues in the E genome ofElytrigia elongata. This evidence and data on cross-compatibility and morphological characteristics suggested that the genusTriticum is a result of adaptive radiation from the perennial genusElytrigia, specifically from the complex of species possessing the E genome or one closely related to it.     

Analysis of metaphase I chromosome association in species of the genus Aegilops

Summary Metaphase-I chromosome associations in every diploid and polyploid species of the genus Aegilops were studied using C-banding in order to analyse the cytogenetic behaviour of the whole complement as well as of specific genomes in different polyploid species. Differences were observed in the frequency of associations per cell among different species of the same ploidic level and even between species sharing the same genomic constitution. Differences were also found between different genomes within the same polyploid species and between the same genome when present in several diploid and polyploid species. Several factors proposed as having an influence on the frequency of metaphase-I associations, such as chromosome morphology, C-heterochromatin content, genetic control and genome interactions, are discussed. Most of the polyploid Aegilops species showed a diploid-like behaviour at metaphase I although multivalents involving homoeologous associations were occasionally observed in Ae. biuncialis, Ae. juvenalis and Ae. crassa(6x); therefore, the Aegilops diploidising genetic system is not equally effective in all polyploid species.     

Complete elimination of maternal mitochondrial DNA during meiosis resulting in the paternal inheritance of the mitochondrial genome in Chlamydomonas species

Summary. The non-Mendelian inheritance of organellar DNA is common in most plants and animals. In the isogamous green alga Chlamydomonas species, progeny inherit chloroplast genes from the maternal parent, as paternal chloroplast genes are selectively eliminated in young zygotes. Mitochondrial genes are inherited from the paternal parent. Analogically, maternal mitochondrial DNA (mtDNA) is thought to be selectively eliminated. Nevertheless, it is unclear when this selective elimination occurs. Here, we examined the behaviors of maternal and paternal mtDNAs by various methods during the period between the beginning of zygote formation and zoospore formation. First, we observed the behavior of the organelle nucleoids of living cells by specifically staining DNA with the fluorochrome SYBR Green I and staining mitochondria with 3,3′-dihexyloxacarbocyanine iodide. We also examined the fate of mtDNA of male and female parental origin by real-time PCR, nested PCR with single zygotes, and fluorescence in situ hybridization analysis. The mtDNA of maternal origin was completely eliminated before the first cell nuclear division, probably just before mtDNA synthesis, during meiosis. Therefore, the progeny inherit the remaining paternal mtDNA. We suggest that the complete elimination of maternal mtDNA during meiosis is the primary cause of paternal mitochondrial inheritance. Correspondence and reprints: Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 901-0213, Japan.     

Cytogenetic studies on an intergeneric hybrid betweenAgropyron tsukushiense andElymus mollis

Elymus mollis is distributed widely from Korea to Japan, Kamchatka and Alaska, the northern part of U.S.S.R., and Northern and Eastern Canada, Greenland and Iceland. This species is tetraploid (2n=28). A strain of this species collected in Hokkaido was crossed withAgropyron tsukushiense var.transiens collected in Mishima. From this cross, 22 F₁ plants were produced. Crossability calculated from the number of hybrid plants produced and the number of floret pollinated was 30.6%. The shape of the F₁ spikes was of theAgropyron type but the glumes were hairy as were those of theElymus parent. One of the characteristics distinguishingElymus fromAgropyron is the production of two spikelets at almost all nodes of the rachis. This character was not expressed in the F₁ plants. All pollen grains of the F₁ plants were completely abortive. The average chromosome pairing at the MI of the PMCs of the F₁ amounted to 2.03 bivalents and 30.95 univalents. Almost all bivalents ranging from one to seven were rod-shaped connected with interstitial or terminal chiasma. These results indicate a lack of genomic homology between the three genomes ofA. tsukushiense and the two genomes ofE. mollis. Contribution No. 37 from the Plant Germ-plasm Institute, Faculty of Agriculture, Kyoto University, Kyoto, Japan.     

Homoploid hybrid speciation and genome evolution via chromosome sorting

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.     

Mitochondrial genome size variation and restriction fragment length polymorphisms in threePhaseolus species

Summary Restriction patterns of mitochondrial DNA (mtDNA) from threePhaseolus species were examined to estimate their relative genome sizes and to determine the level of interspecific variability and relatedness. Three restriction endonucleases that produced relatively simple profiles were identified and used to determine the genome size of the three species. Taking into account fragment stoichiometries, the average estimates across enzymes were 456, 324, and 400 kb, respectively, forP. vulgaris, P. coccineus, andP. acutifolius. Restriction fragment length polymorphisms (RFLPs) differentiated the species when the mtDNAs were digested with seven endonucleases and hybridized with five cosmid clones covering ca. 200 kb of mtDNA sequences. Proportions of shared restriction fragments between every two species were computed as F-values and demonstrated thatP. vulgaris andP. coccineus are more related to each other than either is toP. acutifolius, and that the latter has a similar degree of relationship to the other two species.     

Cohabitation of KP and full-length P elements in the genome of MR strains inducing P-M-like hybrid dysgenesis in Drosophila melanogaster

Summary P strains of Drosophila melanogaster are characterized by the presence of both full-length and deletion derivatives of the transposable element P in their genome, and by their ability to induce the syndrome of hybrid dysgenesis among the progeny of certain intra-strain crosses, when introduced through the male parents. In contrast, strains belonging to the M' class, and which were also found to bear P element-homologous sequences, lack this ability and this has been attributed to the presence in the genome of most of these strains of a distinct class of deletion derivatives termed KP, which can suppress the action of functional P factors. Here we demonstrate that KP elements are present, next to full-length ones, in the genome of at least three strains which induce P-M-like dysgenic symptoms, including GD sterility. KP elements form the majority of the P-homologous sequences in the strains MR-h12, 23.5/CyL ⁴ and the latter's derivative 23.5 ^*/Cy. While the first one is a genuine P strain and the second one depicts a strong P cytotype, the third is a genuine M' strain. The hybrid dysgenesis induced by the two 23.5 MRF strains seems to be due, not primarily to the P elements, but to the action of hobo elements.     

Intermitochondrial junctions in the heart of the frog,Rana esculenta

Summary In muscle fibers of the frog heart, junctions between outer membranes of adjacent mitochondrial profiles are occasionally found. In thin sections of embedded tissue and of mitochondrial pellets, the intermitochondrial junctional space is 5.4±0.15 nm; the external leaflets of the membranes are joined by periodic structures separated from each other by 16.3±0.29 nm. There are 65.3±2 periodic structures per m of membrane measured on a section perpendicular to the junction. After cryofracture, the outer membrane is cleaved into two parts. Closely packed, parallel rows of large particles and furrows are found either on the P-, or on the E-faces. The rows of particles are 11±0.3 nm thick and are separated from each other by 16.5±0.46 nm, their density being 65±2.28 per m of the membrane. In junctional areas, rows of particles on one membrane correspond with the furrows on the other membrane. Intermitochondrial junctions appear to be real structures and not artifacts due to preparation procedures. The conditions of their occurrence are discussed.     

Ultrastructure and development of the gills in<Emphasis Type="Italic"> Rana dalmatina</Emphasis> (Amphibia,Anura)

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Ortons larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.     

Distribution of gonadotropin-releasing hormone-like peptides in the brain during development of juvenile male Rana esculenta

Summary The distribution and density of cell bodies and fibers immunoreactive to GnRH-like peptides were investigated in the brain of male juvenile frogs (Rana esculenta) during postmetamorphic development. An immunohistochemical technique was used, involving antisera raised against 4 variants of GnRH: mammalian GnRH, chicken GnRH-I, chicken GnRH-II and salmon GnRH. A comparison of the immunohistochemical distribution at 8 different developmental stages shows that the maximum density of immunoreactive-GnRH elements, and the full distributional complexity of this system, is attained at the completion of spermatogenesis. Immunoreactive-GnRH cell bodies first appear in the anterior preoptic area during the metamorphic climax, and then appear sequentially in the medial septal area, tegmentum and, lastly, in the retrochiasmatic area and olfactory bulb when immunoreactive-fibers also reach the cerebellum. The GnRH system reacts positively to antisera for all 4 GnRH variants, but immunoreactivity for chicken GnRH-I is the weakest.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Toward a bacterial genome technology: integration of theEscherichia coli prophage lambda genome into theBacillus subtilis 168 chromosome

A novel approach to the cloning large DNAs in theBacillus subtilis chromosome was examined. AnEscherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome ofB. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In theB. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kbB. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in theB. subtilis chromosome and possible applications of this technique are discussed.     

Analysis of the Brassica oleracea genome by the generation of B. campestris-oleracea chromosome addition lines: characterization by isozymes and rDNA genes

Summary This study aimed at generating chromosome addition lines and disclosing genome specific markers in Brassica. These stocks will be used to study genome evolution in Brassica oleracea L., B. campestris L. and the derived amphidiploid species B. napus L. B. campestris-oleracea monosomic and disomic chromosome addition plants were generated by crossing and backcrossing the natural amphidiploid B. napus to the diploid parental species B. campestris. The pollen viability of the derived sesquidiploid and hyperploid ranged from 63% to 88%, while the monosomic and disomic addition plants had an average pollen fertility of 94% and 91%, respectively. The addition lines were genetically characterized by genome specific markers. The isozymes for 6PGD, LAP, PGI and PGM, and rDNA Eco RI restriction fragments were found to possess the desired genome specificity. Duplicated loci for several of these markers were observed in B. campestris and B. oleracea, supporting the hypothesis that these diploid species are actually secondary polyploids. A total of eight monosomic and eight disomic addition plants were identified and characterized on the basis of these markers. Another 51 plants remained uncharacterized due to the lack of additional markers. rDNA genes were found to be distributed in more than one chromosome, differing in its restriction sites. Intergenomic recombination for some of the markers was detected at frequencies between 6% and 20%, revealing the feasibility of intergenomic gene transfer.     

Tomato genome is comprised largely of fast-evolving, low copy-number sequences

Summary Fifty random clones (350–2300 bp), derived from sheared, nuclear DNA, were studied via Southern analysis in order to make deductions about the organization and evolution of the tomato genome. Thirty-four of the clones were mapped genetically and determined to represent points on 11 of the 12 tomato chromosomes. Under moderate stringency conditions (80% homology required) 44% of the clones were classified as single copy. Under higher stringency, the majority of the clones (78%) behaved as single copy. Most of the remaining clones belonged to multicopy families containing 2–20 copies, while a few contained moderately or highly repeated sequences (10% at moderate stringency, 4% at high stringency). Divergence rates of sequences homologous to the 50 random genomic clones were compared with those corresponding to 20 previously described cDNA (coding sequence) clones. Rates were measured by probing each clone (random genomics and cDNAs) onto filters containing DNA from various species from the family Solanaceae (including potato, Datura, petunia and tobacco) as well as one species (watermelon) from another plant family, Cucurbitaceae. Under moderate stringency conditions, the majority of the random clones (single copy and repetitive) failed to detect homologous sequences in the more distantly related species, whereas approximately 90% of the 20 coding sequences analyzed could still be detected in all solanaceous species. The most highly repeated sequences appear to be the fastest evolving and homologous copies could be detected only in species most closely related to tomato. Dispersion of repetitive sequences, as opposed to tandem clustering, appears to be the rule for the tomato genome. None of the repetitive sequences discovered by this random sampling of the genome were tandemly arranged — a finding consistent with the notion that the tomato genome contains only a small fraction of satellite DNA. This study, along with a companion paper (Ganal et al. 1988), provides the first general sketch of the tomato genome at the molecular level and indicates that it is comprised largely of single copy sequences and these sequences, together with repetitive sequences are evolving at a rate faster than the coding portion of the genome. The small genome and paucity of highly repetitive DNA are favourable attributes with respect to the possibilities of conducting chromosome walking experiments in tomato and the fact that coding regions are well conserved among solanaceous species may be useful for distinguishing clones that contain coding regions from those that do not.