Cis-acting loci involved in the induction and reversal of chromosome condensation in plant prophase,determined by their different replication times

Summary Anoxic UVA irradiation (300–400 nm) of cells in prophase induced their chromatin to return to the interphase decondensed form when their DNA was unifiliarly bromosubstituted. This immediate effect may be related to the incompetence of chromatin with Br-DNA when irradiated to bind proteins which induce its condensation. Hence, inhibition of protein synthesis also causes chromatin decondensation in cells with native DNA.Bromosubstitution of DNA sequences replicated in the last two thirds of the S period was as efficient as bromosubstitution of the whole genome for such an effect to take place in a nucleus. On the other hand, the irradiation accelerated the entrance into prophase of those cells in which only sequences replicated in the first third of S were bromosubstituted. Thus, early replicating loci may act as attachment sites for binding proteins preventing the induction of chromatin condensation. DNA bromosubstitution during portions of S was carried out in synchronous cell populations labelled as binucleate by a previous short caffeine treatment, inAllium cepa L. root meristems.Abbreviations Br-DNA bromosubstituted DNA - BrUdR 5-bromodeoxyuridine - UVA 300–400 nm wavelengths light - p 34 34 kDa protein codified by genecdc 2 inS. pombe or its analogues in other species     

Neurophysiological changes and dynamics of bound calcium during the development of associative learning in Helix lucorum

Neurophysiological effects and the dynamics of the content of bound calcium (Ca-b) in command neurons (LP1 and RP1) of defensive behavior during food aversion conditioning are studied inHelix lucorum. In the case of associative learning, there arises in these cells both a response to the conditioned stimulus and a nonspecific facilitation of the reactions to sensory stimuli that is characteristic for the state of sensitization. A response to the presentation of a conditioned stimulus is detected approximately 30 min after the development of long-term sensitization. The use of three or more paired stimuli reveals the characteristic dynamics of the level of Ca-b, which correlates with the neurophysiological effects and differs from the changes in Ca-b content during the development of "pure" sensitization. It is thought that the command neuron of defense behavior exhibits inHelix differences of the molecular-cellular mechanisms lying at the basis of the development of associative learning and sensitization.P. K. Anokhin Institute of Normal Physiology, Russian Academy of Sciences, Moscow. I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 691–701, November–December, 1992.     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Double fertilization inEphedra trifurca,a non-flowering seed plant: The relationship between fertilization events and the cell cycle

Summary Fertilization inEphedra trifurca was examined with a combination of light and fluorescence microscopy. Developmental analysis clearly indicates that double fertilization events, similar to those described inE. nevadensis, regularly occur during the process of sexual reproduction inE. trifurca. In addition to the typical fusion of a sperm nucleus and egg nucleus, a second fertilization event occurs between the second sperm nucleus from an individual pollen tube and the ventral canal nucleus. Both of the fertilization events take place within the confines of an individual egg cell of the female gametophyte. Microspectrofluorometric data demonstrate that each nucleus involved in a sexual fusion event proceeds through the synthesis phase of the cell cycle and increases its DNA content from 1C to 2C before the process of nuclear fusion is completed. Photometric data also confirm that the product of the second fertilization event is equal in DNA content (4C) to the zygotic nucleus derived from the first fertilization event, and is prepared to enter into mitosis as a fully functional diploid nucleus.Abbreviations DAPI 4;,6-diamidino-2-phenylindole - RFU relative fluorescence units     

Functional organization of the nucleus in the oogenesis of Chrysopa perla L. (Insecta,Neuroptera)

On the basis of light, autoradiographic (uridine-³H incorporation) and electron microscopic investigation changes of nuclear structures were examined during the oogenesis in Chrysopa perla L. — In early meiotic prophase the oocyte nuclei were found to contain a large body of extrachromosomal DNA. In certain cases the latter splits up into several DNA clumps giving rise to a few (4–7) primary nucleoli, 3–5 in diameter. The primary nucleoli consist of densely packed fibrils 50–100 Å thick. They contain no granular component and are inactive in RNA synthesis. — At the beginning of large growth the extrachromosomal DNA bodies disappear and numerous electron-dense clumps, 0,5–1 in diameter, appear in the nucleus. Instead of the primary nucleoli, the nucleus now contains a great number of ring nucleoli about 0,5–1 in diameter with a granular component (granules are 150 Å). The space between them is filled up with nucleolar strands running from the surface of the ring nucleoli. — At the stage ring nucleoli of uridine^–3 H incorporation into the oocyte nucleus begins. — During later previtellogenesis and at the beginning of vitellogenesis the ring nucleoli disappear and the nucleus is filled with the network of nucleolar strands. Among them there are specific complexes. These consist of electron dense masses, of granular clusters (granules 500 Å in diameter) and large fibrillar electron light bodies. At this stage the nucleus takes the most active part in RNA synthesis. — The process of karyosphere capsule formation was studied by electron microscopy. The capsule was found to be of fibrillar nature; its structure is very peculiar and unlike any known membrane components of the cell. On the basis of cytochemical evidences the characteristics of the capsule are given. — The development of a powerful nucleolar apparatus based on the extrachromosomal DNA and a possible role of the synaptonemal complex and extrachromosomal DNA in formation of the karyosphere capsule is discussed.     

Ability of chloramphenicol to suppress phenotypically some bacterial mutations

Summary Chloramphenicol (CLP) at slightly inhibitory concentrations (3–5 g/ml) suppresses the inhibition of DNA synthesis and cell division caused by the temperature sensitive mutation dnaE486 at the nonpermissive temperature. Some other mutations can also be phenotypically suppressed by CLP. A similar effect is shown by chlortetracycline. Phenotypic suppression is caused by both these drugs in different cases than by streptomycin.     

Wall appositions induced by ionophore A 23187, CaCl2, LaCl3, and nifedipine in characean cells

Summary The formation of wall appositions (plugs) by ionophore A 23187, CaCl₂, LaCl₃, and nifedipine was studied in mature internodal cells of characeaen algae. CaCl₂ at concentrations above 10^–2M induces thick fibrillar plugs without callose inNitella flexilis. InChara corallina andNitella flexilis ionophore A 23187 (1.25×10^–5 to 5×10^–5M) and LaCl₃ (7.5×10^–5 to 2.5×10^–4M) cause flat appositions which contain callose and have a more granular structure. Plug formation by ionophore A 23187, CaCl₂, and LaCl₃ is pH-dependent and occurs beneath the alkaline regions of the cell. Nifedipine (10^–4 to 10^–5M) induces plugs inNitella flexilis after previous injury. These callose-containing wall appositions consist of a heterogeneous granular core which is covered by a fibrillar layer. The results of this work are compared with previous studies on wound wall formation and chlortetracycline (CTC)-induced plug formation which reveal that abundant coated vesicles occur only when a thick fibrillar wall layer is formed. Neither LaCl₃ nor nifedipine inhibit the formation of CaCl₂- or CTC-plugs. The unusual effects of these substances, which normally act as Ca²⁺ antagonists and therefore should prevent and not induce plug formation, are discussed. It is suggested that La³⁺ mimicks the effects of calcium and that nifedipine binding to the Ca²⁺ channels is altered in the alkaline regions of characean internodes and allows an influx of Ca²⁺.Abbreviations AFW artificial fresh water - CTC chlortetracycline - DCMU dichlorphenyldimethylurea - DMSO dimethylsulfoxide - EGTA ethyleneglycoltetraacetic acid - MES 2-(N-morpholino) ethanesulfonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - TAPS N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid     

Microscopical evidences for the presence of a nucleus inCandida albicans chlamydoconidia

By using the fluorescent, DNA specific stain DAPI (4,6-diamidino-2-phenylindole) some microscopic observations ofCandida albicans pseudomycelium and chlamydoconidia were performed. In this manner blue fluorescent dots were noted both in yeasts, psudomycelium and chlamydoconidia, so evidencing the presence of a nucleus inC. albicans chlamydoconidia.     

Binding of dantrolene-Na to the ciliated membrane ofParamecium aurelia

Summary Dantrolene-Na is a muscular relaxant which binds to sarcoplasmic reticulum (SR) with high affinity and decreases the availability of Ca²⁺ channels. The binding of fluorescent compounds, dantrolene-Na, nifedipine and chlortetracycline to the ciliary membrane ofParamecium aurelia has been studied. Dantrolene at the concentrations of 1.9 · 10^–5, 3.8 · 10^–5 and 7.9 · 10^–5 M manifested a punctuated binding pattern to the cell membrane. Isolated cilia also bound dantrolene at their basal portion, whereas deciliated cell bodies lost their dotted binding pattern. Chlortetracycline showed a similar but weaker fluorescent staining. Nifedipine treated cells revealed no sign of fluorescent binding to the membrane and was only taken up in food vacuoles.Based on these observations we propose that dantrolene binding regular arrays ofParamecium cell membrane could be identical to granular plaques observed by electron microscope. The possible functioning of these structures as Ca²⁺ reservoirs is also discussed.     

Genetic processes in intergeneric cell hybrids Atropa + Nicotiana

Summary The genetic constitution of the cell hybrids Atropa belladonna + Nicotiana chinensis, obtained by cloning of individual heteroplasmic protoplast fusion products (Gleba et al. 1982) and cultured in vitro for 12 months, has been studied. The study comprised 11 hybrid cell clones of independent origin and included analysis of a) chromosome number, size, morphology, and relative position in metaphase plates, b) multiple molecular forms of the enzymes esterase and amylase, and c) relative nuclear DNA content. The data obtained permit us to conclude that, after one year of unorganized growth in vitro, the cells of most (8) clones had retained chromosomes of both parents, while species-specific elimination of nearly all Atropa chromosomes had occurred in three clones. About half of the non-segregating clones possess 120–150 chromosomes including 50–70 of Atropa and 50–90 of Nicotiana. Other clones are polyploid and possess 200–250 chromosomes with a predominance of either Atropa or Nicotiana chromosome types. Only a few chromosomal changes (reconstituted chromosomes, ring chromosomes) have been detected. In some metaphase plates, chromosomes of the two parents tend to group separately, indicating non-random arrangement of chromosomes of the two parents within the hybrid nucleus. Cytophotometric studies of the relative nuclear DNA content showed that distribution histograms for cell clones were similar to those of non-hybrid cultured cells. Cell populations were relatively homogenous and do not indicate any genetic instability as a result of hybridization between remote plant species. Biochemical analysis of isoenzyme patterns confirmed that in most cell clones, species-specific multiple molecular forms of esterase and amylase from both parents were present, i.e. genetic material of both parental species was expressed in the cell hybrids.Dedicated to Professor G. Melchers with gratitude     

Chromatin condensation dynamics and implications of induced premature chromosome condensation

Somatic cell cycle is a dynamic process with sequential events that culminate in cell division. Several physiological activities occur in the cytoplasm and nucleus during each of the cell cycle phases which help in doubling of genetic content, organized arrangement of the duplicated genetic material and perfect mechanism for its equal distribution to the two daughter cells formed. Also, the cell cycle checkpoints ensure that the genetic material is devoid of damages thus ensuring unaltered transmission of genetic information. Two important phenomena occurring during the cell cycle are the DNA condensation and decondensation cycles in the nucleus along with the cyclic expression and functioning of certain specific proteins that help in the same. Several protein families including Cyclins, cyclin dependent kinases, condensins, cohesins and surivins ensure error free, stage specific DNA condensation and decondensation by their highly specific, controlled orchestrated presence and action. Understanding the molecular mechanisms of chromatin compaction towards formation of the structural units, the chromosomes, give us valuable insights into the cellular physiology and also direct us to techniques such as premature chromosome condensation. The techniques of inducing ‘prophasing’ of interphase cells are undergoing rapid advances which have multidimensional applications for basic research and direct applications.     

Composition of the cell wall polysaccharides in some geophilic dermatophytes

The main polysaccharide fractions from cell wall material of several geophilic dermatophyte species were characterized as a glucomannan (F1S) which amounted to 4.0–6.5% and a glucan-chitin complex representing 44.2–71.0%. The neutral sugar content of fraction F1S in these species was mannose (38.7–78.2%), galactose (0.3–7.3%) and glucose (3.2–8.2%) except inM. fulvum (21.9%) andE. stockdaleae (12.5%). Small proportions of xylose, about 1%, were found in this fraction except inM. fulvum which reached 7.8% and inM. nanum which lacked xylose. The main products detected after Smith degradation were glycerol and glucose. From fraction F1S ofM. fulvum a glucan (18.3%) and a mannan (41.5%) were obtained. These two polysaccharides could be used as chemotaxonomic characters for the definition of this group of fungi.     

The Immunoregulatory Effects of Endoglucanase Preparations on Plant Resistance to Fungal Infections

The effects of enzymatic preparations—pectomacerin, hemicellulase, andTrichoderma viride 13/10 cellulase—on plant immunological status were studied using two pathosystems, carrot root–white rot agent (Sclerotinia libertiana) and carrot root–black rot agent (Rhizopus nigricans) as examples. It was demonstrated that these preparations reduced the plant damage by infections, namely, decreased the permeability of cell membranes in the infected tissue and stimulated its defense responses, which were expressed as a stable elevation in the content of phenolic compounds and formation of tissue protective barriers.     

Protective effect of sodium selenite on ofloxacin-induced loss of chloroplast DNA inEuglena gracilis

Some xenobiotics induce permanent loss of chloroplasts inEuglena gracilis which results in the growth of white mutant colonies (bleaching effect). The effect of ofloxacin and sodium selenite on the organization of chloroplast DNA ofE. gracilis was studied by fluorescent microscopy. In the presence of ofloxacin DNA-specific staining with DAPI revealed a decrease in chloroplast DNA content and in the number of nucleoids per chloroplast. This was accompanied with a decrease of chloroplast number per cell and with the loss of stigma. Spectrophotometry of chlorophyll extract revealed changes in th ratio of chla: chlb. The presence of sodium selenite protected chloroplast DNA against the inhibitory effect of ofloxacin.     

Color recovery after photoconversion of H2B::mEosFP allows detection of increased nuclear DNA content in developing plant cells

Many higher plants are polysomatic whereby different cells possess variable amounts of nuclear DNA. The conditional triggering of endocycles results in higher nuclear DNA content (C value) that in some cases has been correlated to increased cell size. While numerous multicolored fluorescent protein (FP) probes have revealed the general behavior of the nucleus and intranuclear components, direct visualization and estimation of changes in nuclear-DNA content in live cells during their development has not been possible. Recently, monomeric Eos fluorescent protein (mEosFP) has emerged as a useful photoconvertible protein whose color changes irreversibly from a green to a red fluorescent form upon exposure to violet-blue light. The stability and irreversibility of red fluorescent mEosFP suggests that detection of green color recovery would be possible as fresh mEosFP is produced after photoconversion. Thus a ratiometric evaluation of the red and green forms of mEosFP following photoconversion could be used to estimate production of a core histone such as H2B during its concomitant synthesis with DNA in the synthesis phase of the cell cycle. Here we present proof of concept observations on transgenic tobacco (Nicotiana tabacum) Bright Yellow 2 cells and Arabidopsis (Arabidopsis thaliana) plants stably expressing H2B::mEosFP. In Arabidopsis seedlings an increase in green fluorescence is observed specifically in cells known to undergo endoreduplication. The detection of changes in nuclear DNA content by correlating color recovery of H2B::mEosFP after photoconversion is a novel approach involving a single FP. The method has potential for facilitating detailed investigations on conditions that favor increased cell size and the development of polysomaty in plants.     

EPR Spectroscopic Study of the Interaction of E. coliCells with Ascorbic Acid and Sodium Nitrite

Ascorbic acid, an effective modulator and regulator of cell metabolism, was shown to induce the production of nitric oxide inE. colicells. This process was detected by EPR spectroscopy as the generation of a spectral signal typical of nitrosyl–iron–sulfur centers (Fe–S–NO) under anaerobic conditions. Incubation of E. colicells in the presence of ascorbic acid under aerobic conditions was shown to be accompanied by sodium nitrite formation. It is suggested that ascorbic acid is capable of supporting the system of energy supply to cells in hypoxia caused by reduced oxygen content or treatment with sodium nitrite.     

Effect of protein synthesis inhibitors on neuronal mechanisms of sensitization inHelix snail

A sensitizing treatment with 5–10% quinine solution causes short-term (lasting 50–70 min) and long-term (lasting several hours) changes in the activity of the command neurons for defensive behavior (LPl1 and PPl1) in the snailHelix lucorum. The short-term effects are characterized by a depolarizing shift in membrane potential, increased excitability, and an initial increase in the content of bound calcium (Ca-c) in the neurons. The long-term effects appear as facilitation of synaptic components of neuronal responses to sensory stimuli without any changes in excitability and in membrane potential, and also as a repeated increase of Ca-c content. Treatment with anisomycin or cycloheximide during sensitization acquirement prevents development of long-term sensitization.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 109–115, March–April, 1993.     

Evolution of thedec-1 eggshell locus inDrosophila. II. Intraspecific DNA sequence analysis reveals length mutations in a repetitive region inD. melanogaster

Summary Thedec-1 eggshell gene inDrosophila melanogaster encodes follicle cell proteins required for proper eggshell assembly. As shown by Southern and Northern analyses thedec-1 gene occurs in four alleles (Fcl-4) among wild-type strains. Its second exon has a distinct feature in the form of 12 repeats with 78–91 nucleotides; the first five show nearly 100% homology. DNA sequence comparison of the repeated region of the alleles revealed that the length polymorphisms are caused by changes in the numbers of the first five repeats. The results suggest that the alleles have been generated by unequal intragenic crossing-over and/or slippage during DNA replication and that the allelic length variants have arisen independently. The possiblilty that the most common allele,FC1, has a selective advantage over the other alleles is discussed.     

A histochemical approach to the knowledge about the neuron nucleus: The “pre-alarm chromatin”

Chromatin as a functional whole.Since the nineteen-fifties (1,2), studies on the histochemistry of the nucleus have been based on its concept as a whole: measurement of the DNA content, and the ratio between nucleus size and cell size appeared to be (and were in effect) an indication of the functional status of the single cell and of the cell population. Two decades later, the already well-known morphological distinction between the chromatins as euchromatin and heterochromatin was reinterpreted on the basis of the degree of spiralization of the nucleosomal fiber and its complexity (3). Subsequently, considerable information about the non-random interphasic position of the chromosomal domains in the nucleus was obtained by in situ hybridization, and the successive reconstruction of their location in the nucleus by image processing with Normarski optics and rotating stage or by confocal microscopy (4–8).Moreover, immunological studies using monoclonal antibodies raised against the splicing factors acting on nuclear pre-mRNAs in discrete nuclear regions (spliceosomes) (9,10), lent support to the notion that the chromatin machinery operates as a whole.Invited contribution to the Joint Meeting of the Universities of Milan, Pavia and Ulm, located in the European Regions of Lombardy (Italy) and Baden-Wurttemberg (Germany), on Cellular and Molecular Aspects of Neuronal Development, Ulm, April 5–8, 1992.     

Intracellular fate and nuclear targeting of plasmid DNA

One of the major steps limiting nonviral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of the transfected cells. The nuclear localization signal (NLS) of the SV40 large T antigen is known to efficiently induce nuclear targeting of proteins. We have developed two chemical strategies for covalent coupling of NLS peptides to plasmid DNA. One method involves a site-specific labeling of plasmid DNA by formation of a triple helix with an oligonucleotide–NLS peptide conjugate. After such modification with one NLS peptide per plasmid molecule, plasmid DNA remained fully active in cationic lipid-mediated transfection. In the other method, we randomly coupled 5–115 p-azidotetrafluorobenzyllissamine–NLS peptide molecules per plasmid DNA by photoactivation. Oligonucleotide–NLS and plasmid–lissamine–NLS conjugates interacted specifically with the NLS-receptor importin . Plasmid–lissamine–NLS conjugates were not detected in the nucleus, after cytoplasmic microinjection. Plasmids did not diffuse from the site of injection and plasmid–lissamine–NLS conjugates appeared to be progressively degraded in the cytoplasm. The process of plasmid DNA sequestration/degradation stressed in this study might be as important in limiting the efficiency of nonviral gene transfer as the generally recognized entry step of plasmid DNA from the cytoplasm into the nucleus