Chloroplast and mitochondrial DNA in a brown alga Egregia menziesii.

Chloroplasts and mitochondria of the brown alga Egregia menziesii were studied with the electron microscope. In both organelles, 15-25-A fibrils with DNA characteristics are found within areas of electron transparency. In each chloroplast there are two DNA-containing areas, one at each tip of the chloroplast. This localization, the shape and size of each DNA-containing area, and its close association with lamellae in a nondividing chloroplast are noted. One or occasionally two DNA-containing areas are found within the mitochondrion and they are compared with a similar structure in the chloroplast.

Chitinous fibrils in the lorica of the flagellate chrysophyte Poteriochromonas stipitata (syn. Ochromonas malhamensis).

Ordered microfibrils are formed on the membrane of the cytoplasmic tail of the alga Poteriochromonas after attachment to a substrate. The ultrastructure of native and extracted stalk fibrils was studied with electron microscope methods. In addition, the structural polysaccharide was characterized by hydrolyses, separation of the monomers by thin-layer chromatography, gas-liquid chromatography and amino acid analysis, and by X-ray diffraction. The alkali-resistant fibrils yielded mostly glucosamine upon extensive hydrolysis, and showed X-ray diffraction patterns similar to those of fugal chitin. It is concluded that the resistant core of the fibrils is chitinous.

Isolation, ultrastructure, and protein composition of the flagellar rootlet of Naegleria gruberi.

Attached to the basal bodies of Naegleria gruberi flagellates is a striated rootlet or rhizoplast. The rootlet-basal body complex has been isolated by Triton X-100 lysis of deflagellated cells and differential centrifugation through a 25% glycerol medium. Rootlets isolated from mature flagellates are approximately 13 micrometers long but vary from 8 to 15 micrometers in length: they taper at both ends from a maximum width of approximately 0.25 micrometers in the vicinity of the basal bodies. They are highly stable during isolation but can be solubilized by urea, high salt, low pH, or detergent (Sarkosyl). Partial dissociation of rootlets with 1 M urea reveals that they are composed of filaments, approximately 5 nm diameter, associated in a linear fashion to yield the characteristic 21-nm cross-banded appearance. Differential solubilization of rootlets and their associated contaminants allowed identification of a major rootlet protein, comprising at least 50% of any purified rootlet preparation, with an apparent subunit molecular weight of 170,000. The localization of rootlets in situ by indirect immunofluorescence using a specific antibody directed against the purified rootlet protein demonstrated unequivocally that this 170,000-dalton protein is an organelle component.

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of Polytomella.

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the beta-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two alpha-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant alpha-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic alpha-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the alpha-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.

Localization of phycoerythrin at the lumenal surface of the thylakoid membrane in Rhodomonas lens.

The thylakoids of cryptomonads are unique in that their lumens are filled with an electron-dense substance postulated to be phycobiliprotein. In this study, we used an antiserum against phycoerythrin (PE) 545 of Rhodomonas lens (gift of R. MacColl, New York State Department of Health, Albany, NY) and protein A-gold immunoelectron microscopy to localize this light-harvesting protein in cryptomonad cells. In sections of whole cells of R. lens labeled with anti-PE 545, the gold particles were not uniformly distributed over the dense thylakoid lumens as expected, but instead were preferentially localized either over or adjacent to the thylakoid membranes. A similar pattern of labeling was observed in cell sections labeled with two different antisera against PE 566 from Cryptomonas ovata. To determine whether PE is localized on the outer or inner side of the membrane, chloroplast fragments were isolated from cells fixed in dilute glutaraldehyde and labeled in vitro with anti-PE 545 followed by protein A-small gold. These thylakoid preparations were then fixed in glutaraldehyde followed by osmium tetroxide, embedded in Spurr, and sections were labeled with anti-PE 545 followed by protein A-large gold. Small gold particles were found only at the broken edges of the thylakoids, associated with the dense material on the lumenal surface of the membrane, whereas large gold particles were distributed along the entire length of the thylakoid membrane. We conclude that PE is located inside the thylakoids of R. lens in close association with the lumenal surface of the thylakoid membrane.

Autoradiographic evidence for many segregating DNA molecules in the chloroplast of Ochromonas danica.

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-(3)H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi.

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.

RNA synthesis and turnover during density-inhibited growth and encystment of Acanthamoeba castellanii.

Alterations in transcription that precede and accompany encystment (E) of suspension grown A. castellanii have been investigated. Comparative studies were performed on cells undergoing spontaneous E in high density stationary phase cultures or after experimental induction of E at low cell densities by deprivation of nutrients in exponential growth. Onset of growth deceleration at high cell densities was accompanied by an increase in the cellular RNA. The maximum RNA content occurred in cells at stationary phase and subsequently declined with the appearance of cysts in the cultures. On the contrary, the RNA content in cells whose growth was immediately terminated by experimental E induction remained at a constant exponential level through 5 h postinduction and then began to decline shortly before the appearance of cysts. The mature cyst formed in stationary phase cultures and after experimental E induction contained an equivalent amount of RNA ( approximately 50% of the exponential value). Comparison of the kinetics of [(3)H]uridine incorporation demonstrated that there was an abrupt reduction in the rate of uridine incorporation into RNA with onset of growth deceleration or after growth termination in experimental E induction. The reduced incorporation of uridine into RNA could not be attributed to to a reduced uptake of the isotope by the cells or an altered capacity of the cells to phosphorylate uridine. Uridine continued to be incorporated into RNA at a reduced rate in cells throughout growth deceleration, in stationary phase, and up to 12 h postexperimental induction. Considered together, these results indicate that a buildup in RNA is not necessary for induction of encystment in acanthamoeba. The accumulated RNA in stationary phase cells appears to be due to the greater reduction in the growth rate than in transcription and the absence of RNA turnover in cells during growth deceleration. Initiation of RNA turnover appears to accompany growth termination and induction of E. The results further demonstrate that the regulation of the rate of transcription is closely coordinated with the control of growth and encystment in acanthamoeba.

Exocytosis of latex beads during the encystment of Acanthamoeba.

Cells of Acanthamoeba castellanii (Neff) are known to form mature cysts characterized by a cellulose-containing cell wall when transferred to a nonnutrient medium. Amebas which engulfed latex beads before encystment formed mature cysts essentially devoid of bead material. The encystment of bead-containing cells appeared to be similar to that of control cells since no important differences between the two were observed with respect to cellular levels of glycogen or protein, cellulose synthetase activity, the amount of cyst wall polysaccharide formed, or the percentage of cysts formed. Actinomycin D and cycloheximide inhibited encystment as well as bead expulsion. Ultrastructural analysis revealed that the beads, which initially were contained in phagocytic vesicles, were released from the cell by fusion of vesicular membranes with the plasma membrane. Exocytosis was observed in cells after 3 hr of encystment, with most of the beads being lost before cyst wall formation. Each bead-containing vesicle involved in expulsion was conspicuously demarcated by an area of concentrated cytoplasm, which was more homogeneously granular than the surrounding cytoplasm. Beads were not observed in the cytoplasm of mature cysts but were occasionally found in the cyst wall.

Oxygen-isotope analysis of recent tropical Pacific benthonic Foraminifera.

Analysis by the oxygen-isotope method of samples of benthonic Foraminifera, collected at different depths on the continental shelf and slope of western Cenitral America, yielded isotopic temperatures agreeing closely with the temperatures measured in the field. The validity of the oxygen-isotope method as a means of analysis of paleotemperatures is further supported.

Olefins of high molecular weight in two microscopic algae.

The hydrocarbon composition of two algae, a golden-brown (Bot-ryococcus braunii) and a blue-green (Anacystis montana), has been investigated by gas chromatography-mass spectrometry. Both show distributions of aliphatic hydrocarbons of odd carbon numbers in the medium and high ranges of molecular weight, with maxima at n-C(17) and n-C(29) for B. braunii and n-C(17) and n-C(29) for A. montana. With the exception of the n-heptadecane of A. montana all the hydrocarbons are monoenes, dienes, or trienes. Since certain continental sediments and oils show similar distributions of alkanes with respect to carbon number, these organisms may be the precursors of the hydrocarbons in these formations.

Bromophenols from red algae.

3,5-Dibromo-p-hydroxybenzyl alcohol is reported as a natural constituent of Odonthalia dentata and Rhodomela confervoides. The amounts isolated, based on the fresh weight of the tissue, were 0.024 and 0.003 percent, respectively. A major phenolic compound in both algae was 2,3-dibromo-4,5-dihydroxybenzyl alcohol.

Oxygen-18 studies of recent planktonic Foraminifera: comparisons of phenotypes and of test parts.

Oxygen isotopic comparisons of phenotypes of Recent Planktonic Foraminifera with both normal and diminutive final chambers are compatible with a model in which the latter develop as a response to environmental stress. Isotopic evidence shows that Spheroidinella dehiscens is probably not a late-stage, aberrant form of Globogerinoides sacculifer.

Fatty acids in blue-green algae: possible relation to phylogenetic position.

Analyses of the lipids in five species of blue-green algae show that the fatty acids are largely the C(16) and C(18) acids. The only alga that could be grown heterotrophically, Chlorogloea, formed the triply unsaturated C(18) acid in the light but only the doubly unsaturated C(18) acid in the dark. Examination of these results and the results of others suggest that, except for one species, the more highly unsaturated acids are found in the morphologically more complex algae. The fatty acid compositions of blue-green algae are different from the fatty acid composition of the other prokaryotic organisms, the bacteria. It is speculated that the diversity of the patterns of fatty acid composition among the blue-green algae could be of phylogenetic significance.

Toxins of a blue-green alga: similarity to saxitoxin.

Toxins were isolated from the freshwater blue-green alga Aphanizomenon flos-aquae. The toxic fractions were characterized by paper and thin-layer chromatography, isolation characteristics, infrared spectra, physiological activity, and reactivity with specific color reagents. The toxic fractions appear to be similar, if not identical, to saxitoxin (paralytic shellfish toxin), which is produced by the marine dinoflagellate Gonyaulax catenella.

Sterols: isolation from a blue-green alga.

A crystalline mixture of sterols was isolated from the filamentous blue-green alga Phormidium luridum. The mixture consisted of unsaturated 24-ethylcholesterols possessing Delta(7)-, Delta(5,7)-, and Delta(5)-bonds together with their Delta(22)-derivatives and a small amount of cholesterol. The major component was 24-ethyl-Delta(7)-cholestenol. Squalene and phytol were also evident.

Algae: amounts of DNA and organic carbon in single cells.

An analysis of ten different unicellular algae, varying in size and containing from 10 to 6000 picograms of carbon per cell, indicates that the amount of DNA per cell is in direct proportion to cell size. The content of DNA is equal to approximately 1 to 3 percent af the cellular organic carbon.

Chromosomal proteins in the dinoflagellate alga Gyrodinium cohnii.

Chromatin has been prepared from nuclei isolated from the dinoflagellate alga Gyrodinium cohnii. This chromatin contains RNA, acid-insoluble proteins, and acid-soluble proteins; the respective ratios to amount of DNA are about 0.09, 0.48, 0.08 (by weight). Not only is the amount of acid-soluble protein associated with the DNA much less than it is in the typical eukaryote, but polyacrylamide gel electrophoresis in urea at pH 3.2 produces a banding pattern different from that of typical histones. There is one predominant band that migrates about as fast as does histone IV from corn. These findings are of interest, because the nuclear organization in the dinoflagellates appears to be intermediate between the prokaryotes and the eukaryotes.

Hydroxymethyluracil in eukaryote DNA: a natural feature of the pyrrophyta (dinoflagellates).

Analysis of the DNA of several diverse dinoflagellates and other algae has revealed that the presence of the base hydroxymethyluracil (HOMeU) is a feature common among the DNA's of dinoflagellates; this base has not been found in any other group of eukaryotes that has been examined. Among examined members of the dinoflagellates, the ratio of the base pairs HOMeU-A to T-A, where A is adenine and T is thymine, ranges from 0.14 to 2.13. In addition to hydroxymethyluracil, the DNA of one dinoflagellate contains methylcytosine, and that of another contains methyladenine, while the DNA of other dinoflagellates contains no detectable amounts of either of these two bases.