Chromosomal localization and distribution of simple sequence repeats and the Arabidopsis-type telomere sequence in the genome of Cicer arietinum L.

We used fluorescence in situ hybridization to probe the physical organization of five simple sequence repeat motifs and the Arabidopsis-type telomeric repeat in metaphase chromosomes and interphase nuclei of chickpea (Cicer arietinum L.). Hybridization signals were observed with the whole set of probes and on all chromosomes, but the distribution and intensity of signals varied depending on the motif. On root-tip metaphase chromosomes, CA and GATA repeats were mainly restricted to centromeric areas, with additional GATA signals along some chromosomes. TA, A and AAC repeats were organized in a more dispersed manner, with centromeric regions being largely excluded. In interphase nuclei of the inner integument, CA and GATA signals predominantly occurred in the heterochromatic endochromocentres, whereas the other motifs were found both in eu- and heterochromatin. The distribution of the Arabidopsis-type telomeric repeat (TTTAGGG)n on metaphase chromosomes was found to be quite exceptional. One major cluster of repeats was spread along the short arm of chromosome B, whereas a second, weaker signal occurred interstitially on chromosome A. Only faint and inconsistent hybridization signals were visualized with the same probe at the chromosomal termini.

Increased specificity of colloidal silver staining by means of chemical attenuation.

A modification of the silver staining procedure of Howell and Black (1980) is reported which makes use of teleostean gelatin as protective colloid and renders a high signal-to-noise ratio. We demonstrate that this ratio can be further increased by subsequent attenuation with a chemical reducer consisting of a mixture of potassium ferricyanide (III) and sodium thiosulphate. It is shown that slight changes of the concentration of the reactive compounds of the chemical reducer make the protocol applicable to human, plant (Aliium cepa, Rhinanthus minor) as well as meiotic insect (Acheta domesticus) chromosome preparations. Due to its broad applicability, the method could find utilization in studies on chromatin and chromosome functions in many species.

Microsatellite fingerprinting in the genus Phaseolus.

The genetic variability of the genus Phaseolus was investigated by nonradioactive DNA fingerprinting. The simple repetitive sequences (GATA)4, (GACA)4, (CAC)5, and (CA)8 were used as probes to differentiate 18 species comprised of 90 genotypes. (GATA)4, (CAC)5, and (CA)8 could be detected in the genome of nearly all species, while the (GACA)4 motif occurred only in 13 species. Almost all fragments that hybridized with (GACA)4 also hybridized with (GATA)4. All but two cultivars of Phaseolus vulgaris, P. lunatus, P. acutifolius, and P. polyanthus showed specific banding patterns with (GATA)4. The other repetitive motifs revealed only limited or no intraspecific variation. In P. vulgaris, two group-specific patterns were found with (GATA)4, giving further evidence for a Middle American and an Andean origin of the P. vulgaris genotypes. The high intraspecific pattern variation that was revealed with (GATA)4 in the predominantly self-pollinating species P. vulgaris and P. lunatus can probably be explained by there being at least two primary centres of domestication and, hence, genetic diversification. In cross-pollinating species (e.g., P. coccineus), the observed intraspecific variation was, surprisingly, rather low. The present study shows that DNA fingerprinting with microsatellites successfully distinguishes among gene pools, cultivars, and, in some cases, among individuals.

Different growth rates of pigmented and white hair in the beard: differentiation vs. proliferation?

A considerable difference in growth rates of pigmented and white anagen beard hair (0.47 mm/day vs. 1.12 mm/day, on average) was measured in three individuals over a 3-year period. Nuclear differentiation in the suprabulbar region of hair shafts revealed an earlier, and more intensive, condensation of chromatin in pigmented hair than in white hair. As other differences (diameter, presence or absence of medulla) between the two hair types could be excluded, the results suggest earlier terminal differentiation of pigmented hair compared with white hair.

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment.

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.

Induction of high polyploidy in Phaseolus cell cultures by the protein kinase inhibitor, K-252a.

Somatic polyploidy of species-specific and tissue-specific degrees occurs in almost all plant species studied so far, but nearly nothing is known about the control mechanisms switching the mitotic cycle to an endoreduplication cycle. In order to search for a possible role of the cdc2 kinase, cell suspension cultures of the Runner bean, Phaseolus coccineus (Leguminosae) were treated with K-252a, an inhibitor of protein kinase activity. The treatment resulted in continuous cell cycles without mitosis, and hence induced polyploidy levels up to 2048C. It is, therefore, suggested that phosphorylation of a protein kinase, probably of the cell cycle-important p34(cdc2) type, is involved in the control of endoreduplication.

Phytohemagglutinin gene expression during seed development of the runner bean, Phaseolus coccineus.

The expression of phytohemagglutinin (PHA) genes was studied in various tissues during the development of the seeds of Phaseolus coccineus cv. Hammond's Dwarf Scarlet by means of northern hybridization and reverse transcription polymerase chain reaction (RT-PCR). The expression is highly development-dependent, starting in the late stage (cotyledons begin to fill the endosperm cavity, i.e. 17 to 24 days after pollination), and tissue-specific. The highest levels of PHA-mRNA are found in the cotyledons (at a very late stage), much lower levels in the embryo axis. Very low levels could be detected in earlier stages of the endosperm, the integument, the funiculus, and probably also in the embryo suspensor. Some PHA mRNA was found in the cotyledons of dry seeds, indicating the presence of undegraded transcripts in mature seeds.

Immunocytochemical localization of the storage protein phaseolin in the embryo-suspensor of Phaseolus coccineus.

Antibodies against the bean storage protein, phaseolin, were induced in rabbits. Sections of bean embryos of various age were incubated with the antibodies, and exposed to fluorescein isocyanate (FITC) conjugated goat anti-rabbit immunoglobulin. Distinct positive signals were found in vacuoles of the suspensor giant cells. Immunofluorescent staining of protein bodies in cotyledon cells, histochemical staining reactions of the sections, as well as gel electrophoretic analysis of the proteins, isoelectric focusing and Western blotting confirmed the specificity and reliability of the immunochemical staining of the suspensor cells. The results give evidence for synthesis of storage proteins in the embryo-suspensor and indicate a function so far not detected of this embryonal nutritive organ.

Polyploidy in differentiation and evolution.

Somatic and generative (germ-line) polyploidy are more widely spread phenomena among living organisms than generally thought. The occurrence of polyploidization and related events in normal and pathological differentiation, their recognized main functions, as well as the structural specificities of polyploid nuclei are reviewed, and the relationship between ontogenetic and phylogenetic events is discussed. The mechanisms leading to the polyploid state, as well as other processes resulting in a genomic condition different from the diploid one (such as DNA under-replication, gene amplification, and chromatin elimination), are briefly sketched. The various changes in chromosomal DNA described are, in conclusion, seen as evidence supporting the paradigm of a "fluid" or dynamic organization of the eukaryotic genome, as being part of a cybernetic feedback regulation system of gene expression. A model is proposed that unifies the aspects of DNA variation, chromatin structure, and diversification in ontogenesis and phylogenesis.

Light-stimulated ultraweak photon reemission of human amnion cells and Wish cells.

Photon reemission in the ultraweak intensity range that is observed after irradiation of cell suspensions with light, reveals characteristic differences between normal human amnion cells and transformed Wish cells from the same parental tissue. The reemission kinetics, approximated best by a hyperbolical process, were studied as a function of cell density, showing that: malignant Wish cells have a photon storage capacity that is not improved by increasing the cell density; and that normal amnion cells exhibit a photon storage capacity that strongly increases with increasing cell density. The interpretation of this effect and the nature of the emitter are discussed.

[The structure of eukaryotic chromosomes]. / Die Struktur der Eukaryonten-Chromosomen.

Recent models for the organization of the chromosomes of eukaryotes in mitosis and interphase are discussed with respect to their condensation hierarchy. Possible functional interpretations are indicated. At present, an alternating coiling and loop formation, induced by histones and nonhistone proteins, respectively, appears to be the most probable structure. The relationship of DNA properties to chromatin structure and function is also briefly discussed.

Transcription of repetitive DNA in condensed plant chromatin.

Polyadenylated transcripts homologous to highly repetitive DNA were found in root tips of Vicia faba by Northern blot hybridization. Electron microscope autoradiography using [3H]uridine as a probe revealed transcription of condensed chromatin in various higher plants. This is consistent with the general rule that highly repetitive DNA is located within condensed chromatin, but it is new that this chromatin fraction is active in RNA synthesis to a considerable amount. Semi-quantitative comparison of the intensity of transcription in species with widely differing 2C DNA contents by means of light microscope autoradiography revealed an inverse relationship between the amount of 2C DNA (and condensed chromatin), and the rate of RNA synthesis.

Biophoton emission. New evidence for coherence and DNA as source.

The phenomenon of ultraweak photon emission from living systems was further investigated in order to elucidate the physical properties of this radiation and its possible source. We obtained evidence that the light has a high degree of coherence because of (1) its photon count statistics, (2) its spectral distribution, (3) its decay behavior after exposure to light illumination, and (4) its transparency through optically thick materials. Moreover, DNA is apparently at least an important source, since conformational changes induced with ethidium bromide in vivo are clearly reflected by changes of the photon emission of cells. The physical properties of the radiation are described, taking DNA as an exciplex laser system, where a stable state can be reached far from thermal equilibrium at threshold.

Computer-aided diagnosis of plant species by chromatin ultrastructure and genome analysis.

Nuclear ultrastructure in non-cycling cells of plants is fairly species specific, whereby the most important parameter is the percentage of condensed chromatin. Therefore, the automated analysis of electron micrographs of ultrathin sections of plant nuclei allow recognition of the species embedded. Flow-sorted, fluorochrome stained nuclei permit the detection of small evolutionary changes in genome organization (e.g. in cell cultures). Moreover, new computer programs for the analysis and comparison of denaturation and renaturation curves allow the diagnosis of plant species at the genome level and to detect differences in certain DNA fractions between species.

Condensed chromatin in the cell nuclei of the human uterine cervix: squamous and columnar epithelium, non-pregnant and pregnant state, and adenocarcinomata.

The proportion of chromatin in the condensed state was comparatively determined by ultrastructural-stereological techniques in the normal squamous and columnar epithelium of the human uterine cervix, the columnar epithelium of a pregnant woman, and in the case of two adenocarcinomata. Significant differences were found. Together with autoradiographic data, the results allow a functional interpretation of the changes in chromatin organization during carginogenesis.

A physical (electromagnetic) model of differentiation. 2. Applications and examples.

The electromagnetic model of cell differentiation originated by Nagl and Popp (1983) was applied to two biological events, chromatin condensation and photoperiodicity (oscillations). Some experiments are suggested, which may help to refute or verify, respectively, the role of ultraweak photon emission in differentiation and the control of gene activity.

A physical (electromagnetic) model of differentiation. 1. Basic considerations.

There are a number of biological phenomena and events that cannot yet be adequately described, such as cell growth and differentiation, which may be controlled by physical factors. Fröhlich (1980) has discussed the principles of dissipative structures as applied to electromagnetic interactions in relation to basic couplings in biological systems. Recently, increasing evidence of photon storage and ultraweak photon emission from living systems, particularly from DNA, has suggested the concept of an electromagnetic model of differentiation, based on the known quantum optical properties of nucleic acids. This model has the advantage over all ideas so far published, that it is (1) simple; (2) universally applicable to events in living matter, because it is consistent with both the quantum mechanical and the thermodynamic properties on the one hand, and the known biological and biochemical data and phenomena at the other hand; (3) it not only describes the phenomena and events in terms of pure mathematical parameters, but it can also explain them; and (4) it escapes the difficulty of finding basic control mechanisms, which themselves do not need a regulator, ad infinitum.

Loss of DNA and euchromatin in senescing leaf cells of Allium.

The leaves of the chive, Allium schoenoprasum, have an average life-span of 53 days, then they fade from top to bottom, in the same sequence as cells originated. Starting during the adult phase, the amount of DNA per nucleus decreases significantly. Nuclei of senescent cells exhibit about 15% less DNA than nuclei of juvenile cells. Electron-microscopic investigations have shown that the diffuse chromatin is lost from the nuclei, followed by shrinkage of the space left. Quantitation was achieved by measurement of the percentage of condensed chromatin per nucleus. Senescence starts in different tissues, and in individual cells of a tissue, asynchronously. Chloroplasts undergo structural changes after initiation of aging in the nuclei. The short life-span, in spite of a relatively high DNA content (11.8 pg), is suggestive of a programmed senescence in Allium leaves.