Effects of photosynthetic inhibitors and light-dark regimes on the replication of cyanophage SM-2

The effects of photosynthetic inhibitors and light-dark regimes on the replication of cyanophage SM-2 in its host cyanobacteria (Synechococcus elongatus UTEX 563 and Microcystis aeruginosa NRC-1, Synechococcus NRC-1 UTEX 1937) have been investigated. Photoassimilation of CO₂ by infected cells was enhanced and remained elevated until late in the infection cycle. Photosynthetic inhibitors and the removal of light suppressed viral replication. SM-2, like other cyanophage of unicellular cyanobacteria, is highly dependent on host photosynthetic metabolism for the energy required in replication.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - CCCP carbonyl-cyanide m-chlorophenyl hydrazone - MMH Modified Modified Hughes Medium     

Utilization of enzyme markers to determine the location of plasma membrane from Pisum epicotyls on sucrose gradients

Using linear sucrose gradients, particulates derived from pea (Pisum sativum L. cv. Alaska) epicotyls have been fractionated and examined for marker enzyme activity. The coincidence of three reputed plasma-membrane markers [cellulase (EC 3.2.1.4), K⁺-stimulated Mg²⁺-ATPase, and glucan synthetase] at the same position on sucrose density gradients, in combination with electron microscopic evidence reported by G. Shore and G. Maclachlan (J. Cell Biol. 64, 557–571; 1975), indicates that plasma membrane of pea epicotyl has a buoyant density of about 1.13 g/cm³. This density disagrees with those usually reported for plant plasma membranes and also with recent reports for Pisum. It is, however, shown to be distinct from the equilibrium densities of enzymic markers for particulate components derived from Pisum endoplasmic reticulum (1.10–1.11 g/cm³), Golgi (1.12 g/cm³) and mitochondria (1.18 g/cm³). Furthermore, other recent literature indicates that the 1.13 g/cm³ buoyant density may be characteristic of the plasma membrane of many members of the Leguminosae. Our data indicate that the conditions of differential centrifugation (time, centrifugal force), coupled with the amount of protein utilized, affect the resolution and interpretation of profiles of marker enzymes on sucrose gradients (e.g. glucan synthetase and K⁺-stimulated Mg²⁺-ATPase were sometimes found to be associated not only with particles of 1.13 g/cm³ density, but with particles of higher densities as well). Particulate cellulase was found to be associated only with particles with equilibrium densities of about 1.13 g/cm³. Cellulase thus proved to be the most useful marker for establishing a differential centrifugation regime which would permit examination of the 1.13 g/cm³ particulate components with minimal contamination by particles of higher densities.     

Chromatographic resolution of nucleic acids extracted from Penicillium chrysogenum

Summary High molecular weight DNA extracted from Penicillium chrysogenum has been fractionated using RPC-5 Analog, into three distinct types designated 1, 2 and 3. Types 1 and 2 have the same buoyant density of 1.710 g/cm³ and together appear to comprise the nuclear DNA. Type 1 is enriched for repeated sequences which are normally observed in restriction digests of P. chrysogenum total DNA. Conversely, type 2 appears to be composed entirely of non-repetitive sequences. Type 3 has been identified as mitochondrial DNA, having a buoyant density of 1.695 g/cm³ and an estimated molecular weight of 31.6×10⁶ Daltons.     

Particle properties of parsnip yellow fleck virus

Particle preparations of parsnip yellow fleck virus (PYFV) isolates A-421 and P-121, representing the two major serotypes, were made by clarifying leal extracts with ether or butan-1-ol and concentrating the virus particles by precipitation with polyethylene glycol and differential centrifugation. The preparations contained c. 31 nm-diameter particles comprising two sedimenting components. Top component (T) consisted of stain-penetrable protein shells with A₂₆₀/A₂₈₀= 0.8–0.9, sedimentation coefficient (S₂₀) = 56 S (A-421) or 60 S (P-121), and buoyant density = 1.297 g/cm³. Bottom component (B) consisted of nucleoprotein particles, not penetrable by negative stain, with A₂₆₀/A₂₈₀= 1.9, sedimentation coefficient (S⁰_20.w) = 148 S (A-421) or 153 S (P-121), and buoyant density = 1.520 g/cm³ (A-421) or 1.490 g/cm³ (P-121). Yields of B component particles were up to c. 1 mg/100 g leaf tissue (both isolates); yields of T component particles were up to c. 0.6 mg (A-421) or 5.5 mg (P-121) per 100 g leaf tissue. PYFV particles were found to contain a single RNA species (mol. wt c. 3.4 × 10⁶, c. 9800 nucleotides), constituting 40% of the particle weight, and three polypeptide species, of mol. wt (× 10 ³) 30 , 26 and 24 (A-421) or 31 , 26 and 23 (P-121).     

Role of lysine in the replication of reovirus: I. Synthesis of complete and empty virions

Lysine is essential for the replication of infectious reovirus. Omission of lysine from the extracellular medium not only permitted the continued synthesis of structural viral proteins and viral double-stranded ribonucleic acid (RNA), but also caused an enhanced formation of viral structures which were separable by isopycnic sedimentation of CsCl into a top band consisting of empty particles with a buoyant density of 1.29 g/cm³ and essentially free of viral RNA, and two lower bands which were difficult to resolve and had an average buoyant density of 1.37 g/cm³. The lower bands contained most of the viral nucleic acid. The above effects were reversed when lysine was restored early after infection. In contrast, a single band with a buoyant density of 1.38 g/cm³ was obtained from lysine-plus infected cells.     

Separation of B and C Type Virions by Centrifugation in Gentle Density Gradients

Murine type B particles were separated from type C (Rauscher leukemia virus) by means of gentle (low-increment rate) density gradients. The best separation was obtained when the density ranged from 1.13 to 1.20 g/cm³ when sucrose was used and from 1.12 to 1.28 g/cm³ with CsCl. The buoyant densities of the B and C particle bands in sucrose were 1.18 and 1.16 g/cm³, respectively. The CsCl gradient gave a better separation with the B particles banding at a density of 1.20 g/cm³ and with the C particle density little different from its value in sucrose.     

Isolation and preliminary characterization of cryptic satellite DNA in pea

Optimum conditions have been established for isolation of ‘cryptic’ satellite DNA from the genome of pea (Pisum sativum), using gradients of CS₂SO₄ containing silver ions. At an Ag⁺ :DNA-P ratio (R) of 0.1, and at alkaline pH, four fractions are obtained: mainband (buoyant density 1.437 g cm³; 67% of total DNA), satellite I (buoyant density 1.582 g/cm³; 7% of total DNA), satellite II (buoyant density 1.520 g/cm³, 11% of total) and satellite III (buoyant density variable between 1.45 and 1.51 g/cm³; 15% of total). The reiterated DNA content of these four fractions has been investigated by reassociation experiments conducted over a Cot range of 1 × 10^?5 to 2.0. All four fractions contain at least two kinetic components; each fraction, including the mainband, consists at least partly of highly reiterated DNA. Ribosomal RNA hybridizes only to the mainband.     

Isolation and partial characterization of a mitochondrial deoxyribonucleic acid-protein complex from sea urchin embryos

Summary Lysis of mitochondria from sea urchin embryos with Triton X-100 led to a complete conversion of DNA-containing mitochondrial residues into protein-DNA complex with a density higher than 1.22 g/cm³ in sucrose solutions. This complex banded isopycnically in metrizamide gradients at a density of 1..26 g/cm³. Exposure to mixtures of Triton X-100 with Tween 80 resulted in progressively less delipitated and disorganized mitochondria over Tween/Triton weight ratios from 1 to 2, with the retention of the starting buoyant density in sucrose of approximately 1.16 g/cm³ at Tween/Triton ratios above 2.5. The DNA-internal protein complex sedimented with the bulk of the surviving mitochondrial structure under all conditions studied. No free DNA could be detected under any conditions of membrane removal.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by arginine and biotin: a two-factor analysis

Summary The distribution of glyoxylate-cycle enzymes between microbodies and mitochondria was examined in ethanol-grown Aspergillus tamarii Kita. Particulate activities of catalase and the two glyoxylate by-pass enzymes, malate synthase and isocitrate lyase, were localized in the microbodies. The microbodies had a buoyant density of about 1.23 g cm^-3 after isopycnic centrifugation in linear sucrose gradients. Particulate activities of the other two glyoxycitrate synthase, together with that of succinate dehydrogenase were restricted to the mitochondria, which had a buoyant density of about 1.20 g cm^-3. Catalase also appeared to be localized in a second particle, perhaps the microbody inclusions or the Woronin bodies, having a buoyant density of about 1.26 g cm^-3.     

Purification and particle properties of groundnut rosette assistor virus and production of a specific antiserum*

Purified preparations of the luteovirus, groundnut rosette assistor virus (GRAV), were made by treatment of groundnut leaf extracts with cellulase, followed by sucrose density gradient centrifugation. Yields of virus particles were about 0·5-1·0 mg/kg leaf material. The preparations contained isometric particles c. 28 nm in diameter with a sedimentation coefficient (s_{20, w}) of 115 S, a buoyant density in Cs₂SO₄ of 1·34 g/cm³, and A₂₆₀/A₂₈₀ of 1·86. The particles contained a single species of nucleic acid (presumably RNA), of mol. wt 2·09 × 10⁶and with no detectable polyadenylate sequence, and a single protein species, of mol. wt 24 × 10³. An antiserum produced in a rabbit had a titre of 1/256 in gel diffusion tests and detected GRAV in leaf extracts by ELISA. GRAV particles reacted in F(ab')₂-ELISA and immunosorbent electron microscopy (ISEM) tests with antisera to bean leaf roll, potato leafroll and tobacco necrotic dwarf luteoviruses, but did not react with antisera to carrot red leaf luteovirus.     

Isolation and characterization of microbodies and symphyomicrobodies with different buoyant densities from the fungus Blastocladiella emersonii.

Microbodies isolated from sporangia of the aquatic fungus $\text{Blastocladiella emersonii}$ have a mean buoyant density of 1.222 g/cm³ after centrifugation through a linear sucrose gradient, and contain catalase, isocitrate lyase and malate synthase. Microbodies fuse to produce one symphyomicrobody per zoospore at the time of sporogenesis. An increase in density accompanies this process. The symphyomicrobody has a mean buoyant density of 1.292 g/cm³ while the spore's single mitochondrion has a buoyant density of 1.219 g/cm³. Statistical data are also provided for both starting levels and purification of symphyomicrobody and mitochondrial enzyme markers.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Isolation and composition of bacteriophage-like particles from kappa of killer Paramecia

Summary Phage-like particles from kappa of stock 562 of Paramecium aurelia have been isolated by CsCl density gradient centrifugation. Analyses show that the particles contain about 1.6×10¹⁶g DNA and 2.0×10^-16g protein. Their buoyant density is approximately 1.47. DNA from the particles has a buoyant density very close to that of whole kappa DNA. The presence of DNA in the particles has been confirmed by a cytochemical technique. The results support the conclusion that kappa contains a bacteriophage.     

Chromoplasts of Tropaeolum majus L.: Isolation and characterization of lipoprotein elements

Summary Chromoplasts of unfolding petals of Tropaeolum majus contain large amounts of filaments (which, in sections, appear as tubules), and unevenshaped, isodiametric to elongated bodies (IBs). These structural elements are the major sites of the chromoplast pigments. They were freed from isolated chromoplasts and subjected to sucrose density gradient centrifugation. At a density of 1.080 g cm^-3 a distinct orange band contained almost exclusively fine filaments of 15–20 nm in diameter as shown after negative staining. Other filaments and most of the IBs were heterogeneous in size, shape, and density and were collected in two fractions of buoyant densities of 1.025 and 1.055 g cm^-3. The three fractions thus obtained comprise 15–33% protein, large amounts of carotenoids and their esters, glyco- and phospholipids, as well as minor amounts of tocopherols. A higher buoyant density of particles is correlated with a higher relative content of protein and glyco- and phospholipids and a lower relative content of carotenoids. The polypeptide pattern, as shown by SDS-polyacrylamide gel electrophoresis, is very similar in all three fractions. There is one main polypeptide, with a MW of about 30,000, accounting for up to 80% of the protein of each fraction.Abbreviations IBs uneven-shaped, isodiametric to elongated bodies     

Purification and partial characterisation of beet mild yellowing virus and its serological detection in plants and aphids

Beet mild yellowing virus (BMW) was reversibly precipitated at temperatures below about 5°C and this property was used as a final step in a purification procedure which yielded about 1 mg virus/kg tissue. Purified virus was infective and had an A200/A280 ratio of about 1–8. BMW particles were isometric with a diameter of 26 nm, sedimented at 116 S, had a buoyant density in caesium chloride of 1.42 g/cm³ and a coat protein mol. wt of 25 400. An antiserum to BMW had a titre in immunodiffusion tests of 1/256 and was used in immunodiffusion tests, immunospecific electron microscopy (ISEM) and enzyme-linked immunosorbent assay to demonstrate a close serological relationship between BMW and beet western yellows virus. BMW was readily detected by ISEM in plants and also in aphid vectors after treatment of aphid extracts with a chloroform:butanol mixture.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Location of Satellite and Homogeneous DNA Sequences on Human Chromosomes

HUMAN DNA^1,2 contains at least two satellite fractions—satellite I (0.5% of the genome) which bands at a density of 1.687 g/cm³ in neutral CsCl and satellite II (2% of the genome) which bands at 1.693 g/cm³. The main band DNA has an average buoyant density between 1.670 and 1.720 g/cm³ and a light shoulder (constituting 15% of the genome) with a buoyant density of 1.696 g/cm³, referred to as homogeneous mainband. Satellite DNA has been observed in many higher organisms³, usually with an unknown function, notable exceptions being cistrons coding for ribosomal RNA⁴ and the DNA coding for histone messenger RNA⁵. To investigate possible functions of human repetitive DNA we have studied the annealing of complementary RNA fractions to chromosomes using in situ hybridization^6,7. We describe here preliminary observations using human satellite II and homogeneous mainband fractions.     

Circular DNA from the water mold Saprolegnia

Summary Circular DNA has been investigated in the water mold Saprolegnia. This organism was shown to contain two satellite DNA components at 1.685 and 1.707 g/cm³ in addition to main band DNA at a buoyant density of 1.717 g/cm³. The component banding at a density of 1.685 g/cm³ was found to occur as closed circles of length 14 m in the relaxed form. On the basis of sucrose gradient studies this DNA is thought to be mitochondrial in origin. No other class of circular molecules was observed.     

Changing proportions of DNA components in Euglena gracilis

The ratios of satellite deoxyribonucleic acid components to chromosomal deoxyribonucleic acid in Euglena gracilis Z were measured by analytical density gradient ultracentrifugation. Chloroplast deoxyribonucleic acid with a buoyant density of 1.685 g/cm³ exhibited a constant ratio to chromosomal deoxyribonucleic acid during exponential growth and increased twofold as the culture reached the end of the exponential growth phase. The quantity of a satellite deoxyribonucleic acid with a buoyant density of 1.691 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but increased to approximately equal the quantity of chloroplast deoxyribonucleic acid as the culture approached the end of the exponential growth phase. The quantity of a deoxyribonucleic acid component with a buoyant density of 1.700 g/cm³ was not sufficient to measure the ratio to chromosomal deoxyribonucleic acid during exponential growth but represented approximately one-third of the total deoxyribonucleic acid as the culture entered the stationary phase of growth.     

Buoyant Densities and Dry-Matter Contents of Microorganisms: Conversion of a Measured Biovolume into Biomass

Several isolates of bacteria and fungi from soil, together with cells released directly from soil, were studied with respect to buoyant density and dry weight. The specific volume (cubic centimeters per gram) of wet cells as measured in density gradients of colloidal silica was correlated with the percent dry weight of the cells and found to be in general agreement with calculations based on the partial specific volume of major cell components. The buoyant density of pure bacterial cultures ranged from 1.035 to 1.093 g/cm³, and their dry-matter content ranged from 12 to 33% (wt/wt). Average values proposed for the conversion of bacterial biovolume into biomass dry weight are 1.09 g/cm³ and 30% dry matter. Fungal hyphae had buoyant densities ranging from 1.08 to 1.11 g/cm³, and their dry-matter content ranged from 18 to 25% (wt/wt). Average values proposed for the conversion of hyphal biovolume into biomass dry weight are 1.09 g/cm³ and 21% dry matter. Three of the bacterial isolates were found to have cell capsules. The calculated buoyant density and percent dry weight of these capsules varied from 1.029 g/cm³ and 7% dry weight to 1.084 g/cm³ and 44% dry weight. The majority of the fungi were found to produce large amounts of extracellular material when grown in liquid cultures. This material was not produced when the fungi were grown on either sterile spruce needles or membrane filters on an agar surface. Fungal hyphae in litter were shown to be free from extracellular materials.