INDUCTION OF VACUOLATED SPHEROPLASTS AND ISOLATION OF VACUOLES IN CYANOBACTERIA1

There is still a great deal of debate about whether cyanobacteria contain vacuoles. This might in part reflect our limited ability to isolate vacuoles. We found and isolated vacuoles from different cyanobacteria during spheroplast preparation. Lysozyme treatment induced two kinds of spheroplasts: vacuolated spheroplasts and nonvacuolated spheroplasts. Upon breakage in distilled water, vacuolated spheroplasts released transparent, spherical, and colorless vacuoles with diameters ranging from 2.3 to 16 μm. Large vacuoles could be generated by fusion of two or three small vacuoles. Additionally, large vacuoles also could engulf small ones or other cellular bodies. The isolated vacuoles could tolerate hypotonic condition, and some could be drawn into a thread. Nonvacuolated spheroplasts released few vacuoles after breaking apart. This successful confirmation and isolation of vacuoles will allow studies of the origin and function of cyanobacterial vacuoles.     

Evidence from inhibitor studies that the endophyte synthesises nitrogenase in the root nodules of Alnus glutinosa L. Gaertn.

Summary Viable spheroplasts of a marine dinoflagellate, Gonyaulax polyedra, have been prepared for the first time. This simple and rapid procedure results in a yield of over 95% intact spheroplasts. Utilizing this technique, many studies of the cell-wall-free form of this dinoflagellate are now possible.     

Analysis of Nuclear DNA content in plant cells by Flow cytometry

Flow cytometry was used to analyse the DNA content of nuclei isolated from intact plant tissues and from callus and cell suspension cultures invitro. Cell nuclei were isolated either mechanically (chopping, syringing) or by a hypotonic lysis of isolated protoplasts. Although both methods gave similar results, a slight shift to lower ploidy levels was observed after protoplast isolation from intact tissues and calli. No differences were observed if the two methods were compared using cell suspension cultures. The results showed that flow cytometry is a rapid method of nuclear DNA content analysis in intact plant tissues and variousin vitro cultures.     

渗透稳定剂的互补效应及某些渗透稳定剂对蓝藻细胞壁的降解作用

由多种盐组成的复合渗透稳定剂用于分离蓝藻原生质球的效力与单一盐溶液相比较,其作用多数显示加强,但对原生质球稳定性的影响随盐类组合而异。若干种盐类,如酒石酸铵、硫酸铵和硫酸镁,对蓝藻细胞壁表现一定的降解作用,以酒石酸铵作用最强,可用于分离原生质球。此种原生质球透明度较差,但对低渗敏感     

大豆细胞核的分离及其超显微结构的研究

本文通过对影响大豆细胞核分离诸因素的研究,介绍一种快速、简便的核分离方法。采用这种方法分离出的细胞核产量高,光镜,电镜观察证明核制品形态好,纯度较高,核膜较完整。     

Preparation and Properties of Spheroplasts from Aspergillus parasiticus with Special Reference to the de novo Synthesis of Aflatoxins

Spheroplasts were prepared from Aspergillus parasiticus NRRL 3240 using β-glucuronidase from Helix pomatia. They were osmotically fragile spherical structures which lysed when suspended in hypotonic buffers. Purity of the preparation was confirmed by phase-contrast microscopy. Maximal conversion of mycelia to spheroplasts was achieved with 48 and 72 h old cultures. Spheroplasts were metabolically active as indicated by the incorporation of labelled thymidine, uridine and leucine into DNA, RNA and proteins, respectively. A significant incorporation of [methyl-³H] thymidine into trichloroacetic acid-insoluble material suggested the presence of thymidine kinase in this organism. Spheroplasts and lysates demonstrated the ability to incorporate labelled acetate into aflatoxins. Maximum incorporation was observed in those prepared from 96 h old cultures. Lysates were more efficient in de novo aflatoxin synthesis as compared to intact mycelia and spheroplasts.     

THECAL ULTRASTRUCTURE OF THE TOXIC MARINE DINOFLAGELLATE GONYAULAX CATENELLA1

The toxic marine dinoflagellate Gonyaulax catenella Whedon & Kofoid was studied with scanning and transmission electron microscopy to describe the thecal morphology and to accurately define the taxonomic characters of the species. The closing platelet which lies in a U-shaped apical pore was revealed to be disassociable from a partly obscured apical platelet. Two previously unreported sulcal plates were charaterized and described. The entire complement of thecal plates numbered 33.     

Isolation of nuclei for chromatin analysis in fission yeast.

The methods available for analysis of the chromatin of Schizosaccharomyces pombe are time consuming (>8 h) and/or result in some degradation of the chromatin. Here we report an optimised method for the preparation of spheroplasts and the isolation of nuclei which takes <25 min and is suitable for analysis of chromatin structure by micrococcal nuclease, restriction endonuclease or by immunoprecipitation.     

POTENTIAL IMPORTANCE OF BENTHIC CYSTS OF GONYAULAX TAMARENSIS AND G. EXCAVATA IN INITIATING TOXIC DINOFLAGELLATE BLOOMS1,2,3

Thick-walled, nonmotile cysts (termed hypnocysts) of two dinoflagellates were isolated from estuarine sediments in Cape Cod, Massachusetts, and germinated to produce their respective motile, thecate stages. Hypnocysts from Orleans district were identified as Gonyaulax excuvata (Braarud) Balech sensu Loeblich & Loeblich. Visually identical hypnocysts from Falmouth district were provisionally identified as Gonyaulax tamarensis Lebour. Both species were toxic. A geographic survey in September detected hypnocysts in only the sediments of locations where toxic blooms developed the preceding and following Spring. Laboratory incubation (16 C) of hypnocysts from sediment samples stored in the dark (5 C) for 6 mo initiated excystment by the temperature increase, with no appreciable effect from light regime, nutrient, or chelator concentrations. Motility of excysted germlings was optimum in highly chelated medium and in the presence of light. We conclude that hypnocysts of both tasa are important in seeding recurrent annual blooms, synchronizing early bloom development with vernal warning of seawater and increasing the geographic range of the species. We suggest that many red tides in New England and eastern Canadian waters are initiated through the displacement of motile estuarine populations into nearshore area by tidal advection and surface runoff, although the potential existence and importance of offshore cyst reservoirs cannot be discounted. Evidence is presented that hypnocysts are probable sexual zygotes whereas the thin-walled cysts readily formed in laboratory cultures (pellicle cyst) are asexual. Pellicle cysts are of limited durability, do not overwinter in nature, and therefore do not play a significant role in initiating toxic blooms.     

Purifying nuclei.

Nuclear isolation methods exist since over 50 years and even today new procedures and amendments of standard methods are published. They can be classified into nonaqueous and aqueous methods. The latter can be subdivided into isotonic, hypertonic and hypotonic systems. In most cases the aqueous isolation renders nuclei closer to their physiological status in the cell. A standard method for the hypotonic isolation of nuclei is presented and the methodology of nuclear isolation is discussed.     

Complemental Effect of Osmotic Stabilizers and Their Roles in Degradation Cell Walls of Blue-Green Algae

The compound osmotic stabilizers consisted of several salt solutions exerted a greater effect on the isolation of blue-green algae spheroplasts than a single salt solution. However, the effect of compound osmotic stabilizers on the spheroplast stability could be multiphasic. Some osmotic stabilizers, such as the solution of (NH4) 2C4H406, (NH4) 2SO4 and MgSO4, exerted degradation on cell walls of the blue-green alga; among which the (NH4) 2C4H406 solution (0.15 mol/L) had the greatest degradation resulting in formation of spheroplasts. The spheroplasts were sensitive to hypotonic condition but were less transparent.     

A METHOD FOR ISOLATING INTACT MITOCHONDRIA AND NUCLEI FROM THE SAME HOMOGENATE,AND THE INFLUENCE OF MITOCHONDRIAL DESTRUCTION ON THE PROPERTIES OF CELL NUCLEI

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0–6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl₂ is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The α-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0–6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase — contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.     

SEXUAL REPRODUCTION IN THE TOXIC DINOFLAGELLATE GONYAULAX MONILATA1

The sexual cycle of Gonyaulax monilata Howell was observed in stationary cultures and in nitrogen-deficient medium. The armored, isogamous gametes fuse in a characteristic manner with cingula at oblique angles. Nuclear fusion lags slightly behind cytoplasmic fusion. The zygote enlarges for several days. The dark, double-flagellated planozygote encysts within 1–3 wk. Early hypnozygotes are round to ovoid and contain lipid and one or two large golden-yellow globules. As the hypnozygote matures, the globules become smaller and the cytoplasm darkens and pulls from the wall. All cysts examined contained only one nucleus. A very dark, uninucleate post-hypnozygotic cell escapes through an archeopyle and within 24 h divides into daughter cells which divide in 24–48 h forming a small chain. The production of thick walled zygates in culture implies that such resting stages in marine sediments could serve as a source stock for blooms. This species causes toxic red tides and the existence of benthic “seed beds” consisting of hypnozygotes is now plausible.     

CHARACTERIZATION AND MOLECULAR PHYLOGENYOF A PROTEIN KINASE cDNA FROM THE DINOFLAGELLATE GONYAULAX (DINOPHYCEAE)1

Degenerate primers corresponding to conserved protein kinase motifs were used to amplify potential kinase DNA fragments from a Gonyaulax polyedra Stein cDNA library using PCR. One PCR fragment, potentially encoding a CAMP-dependent protein kinase, was used as a probe to isolate a near full-length cDNA from the library. The nucleic acid sequence of the entire cDNA clone had a high homology to the catalytic subunit of cAMP-dependent protein kinase (cAPK subfamily and affiliated members. Northern blot analysis showed that the corresponding mRNA had a size (about 1.4 kb) and a relative high abundance consistent with a cAPK homologue. Southern blot analysis showed that while there are roughly 30 copies of the kinase gene per genome, the pattern of restriction fragments is inconsistent with the hypothesis of a large gene family. Phylogenetic analyses comparing the deduced amino acid sequence from the Gonyaulax cDNA with other cAPK sequences place Gonyaulax close to the slime mold Dictyostelium discoideum. This is the first phylogenetic analysis of dinoflagellates based on protein sequence, and the results are in agreement with similar analyses based on rRNA sequences.     

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

ADAPTATION OF CERATIUM FURCA AND GONYAULAX POLYEDRA (DINOPHYCEACE) TO DIFFERENT TEMPERATURES AND IRRADIANCES: GROWTH RATES AND CELL VOLUMES1

Growth rates and cell volumes of Ceratium furca Ehrenberg and Gonyaulax polyedra Stein were determined during the log phase of growth in cultures which had been extensively adapted to one of three temperatures and five irradiances. At each temperature, curves for the growth rate vs. irradiance for both species had light-limited and light-saturated regions. Three properties of these curves characterized the response of each species to temperature: the light-saturated growth rate, the irradiance at which growth became light-saturated and the compensation irradiance for growth. For both species, the first two properties generally decreased with declining growth temperature, while the compensation irradiance declined for Ceratium but had a V-shaped response pattern for Gonyaulax. The light-saturated growth rates were generally higher for Ceratium than for Gonyaulax, while the irradiance at which growth became saturated and the compensation irradiance were lower for Ceratium. The changes in cell volume associated with the irradiance and temperature of growth were very different for Ceratium and Gonyaulax. The cell size of Gonyaulax increased as irradiance and temperature decreased, while cell volumes of Ceratium did not change with temperature but were smallest at the highest and lowest growth irradiances. In general, the growth rate patterns were similar for Ceratium and Gonyaulax, while those for cell size were different. The maximum growth rate, the irradiance at which growth became saturated, the compensation irradiance, and the cell volume all showed that Ceratium grew at the same rate or faster than Gonyaulax over the entire range of irradiances and temperatures examined.     

Isolation of Polyribosomes from Yeast During Sporulation and Vegetative Growth

Exponentially growing and sporulating cells of Saccharomyces cerevisiae have been subjected to a variety of conditions which mechanically disrupt the cell in an effort to establish conditions which permit the recovery of intact polyribosomes. Grinding cells for 10 s with glass beads in a Bronwill cell homogenizer was sufficiently gentle to yield a polyribosome content in exponentially growing cells which was similar to values obtained from yeast spheroplasts. Polyribosome patterns in sporulating yeast were similar to those from exponentially growing cells. This technique is fast, reproducible over a wide range of cell concentrations, and eliminates the need to make spheroplasts to recover intact polyribosomes.