Use of ethidium bromide fluorescence enhancement to detect duplex DNA and DNA bacteriophages during zone sedimentation in sucrose gradients: molecular weight of DNA as a function of sedimentation rate

Duplex DNA molecules and DNA bacteriophages have been sedimented through 5--25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DNAs from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0.38 when 0.025--0.70 microgram of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide--sucrose gradients and detected by fluorescence enhancement.     

The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0·1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2×10⁶) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.     

Chemical and physical properties of mammalian mitochondrial aminoacyl-transfer RNAs. I. Molecular weights of mitochondrial leucyl- and methionyl-transfer RNAs.

The sedimentation and electrophoretic properties of Syrian hamster cytosolix and mitochondrial methionyl- and leucyl- +RNAs have been compared under denaturing conditions. Mitochondrial leucyl-tRNA could be separated into three species by chromatography on RPC-5. Their apparent molecule weights as determined by polyacrylamide slab gel elecltrophoresis were 23 000 for one species and 24 000 for the other two compared to the five cytosolic leucyl-tRNA species whose apparent molecular weights ranged from 26 000 to 28 000. Mitochondrial leucyl-tRNAs sedimented more slowly than their cytosolic counterparts, again indicating a lower molecular weight. The apparent molecular weights of the mitochondrial methionyl-tRNAs were identical or only slightly lower than their cytosolic counterparts as determined by polyacrylamide slab gel electrophoresis but both mitochondrial methionyl-tRNA and formylmethionyl-tRNA sedimented slightly more slowly than cytolsolic methionyl-tRNA. It is suggested that mitochondrial tRNAs fall into the size range of other t RNAs and might be uniform in size.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

Progesterone receptor in human endometrium of leiomyoma uteri

This study was designed to examine whether 8S protein as progesterone receptor exists in the human endometrium which has been primed with estrogen. The kinetic study showed that 8S-progesterone binding was specific with Kd of 2.0 X 10(-9) M. 5S-progesterone binding was inhibited competitively by cortisol. The study of ligand specificity also showed that progesterone and its related steroids had much stronger affinity for 8S component than for 5S component. Therefore, 5S protein may be CBG. Progesterone-8S protein binding was easily dissociated during the 5-20% sucrose gradient centrifugation, but such a protein from which progesterone had been dissociated could be sedimented at 8S region. Glycerol could stabilize progesterone-8S protein binding. These results indicate the existence of 8S protein as a progesterone receptor under the low salt medium in the estrogen primed human endometrium.     

Size of virus-specific RNA in B-34, a hamster tumor cell producing nucleic acids of type C viruses from three species.

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.     

Structural and metabolic study of the mycolic acids of Mycobacterium fortuitum

The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1-14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors. If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1-14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured. The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.     

INTRACELLULAR CENTRIFUGAL SEPARATION OF ORGANELLES IN PHYCOMYCES

Live sporangiophores of Phycomyces blakesleeanus were centrifuged at 35,000 rpm. The cell contents sedimented into distinct layers, and each layer was studied with an electron microscope and with cytochemical methods. The following layers were found (their volumes and their densities are shown in Fig. 3): 1. polyphosphates; 2. polyphosphates and protein crystals; 3. glycogen; 4. yellow layer with ferritin; 5. ribosomes; 6. protein crystals; 7. mitochondria; 8. mitochondria and fibrils; 9. nuclei; 10. endoplasmic reticulum; 11. vesicles, membranes, and reticulum; 12. vacuole; 13. lipoproteins, membranes; 14. fat droplet. The densities of the various layers were determined by the injection of droplets of inert oils of known density into the sporangiosphores before centrifugation. Sedimented cell organelles could be isolated. Centrifuged nuclei of a lycopene-producing mutant were injected into the intact sporangiophore of an albino host where they induced color formation. The ensuing spores, when plated, gave a mixture of white and colored colonies. It was concluded that cell organelles, sedimented by centrifugation of living sporangiophores, remain alive and can be used for biochemical studies. Microspectrophotometric examination of the layers indicated the presence of cytochromes and flavines in the mitochondria and of cytochromes in the nuclei. No pigments corresponding to the action spectrum for the light growth response were found.     

The transmission of European wheat striate mosaic virus by Javesella pellucida (Fabr.) injected with extracts of plants and plant-hoppers

Virus-free individuals of the plant-hopper Javesella pellucida (Fabr.) infected plants with European wheat striate mosaic virus (EWSMV) after being injected at 5° C. with extracts of either plants or hoppers, but extracts of hoppers provided a better inoculum. Hoppers were unable to infect plants until at least 8 days at 20–25° C. after they were injected, and nymphs fed on infected plants similarly required 8 days before they gave infective extracts. Few hoppers survived more than a week after injection with untreated extracts of hoppers or with material sedimented from them by centrifuging the extracts at 8000g, but 60–70% survived injection with purer virus preparations. Injection of the virus seemed harmless, because as many hoppers survived CO₂ anaesthesis + injection, whether or not they later infected plants, as survived anaesthesis without injection. Attempts to determine the properties of the virus in vitro gave inconsistent results, but virus from hoppers was still infective after 10 min. at 30° C, 36 hr. at 5° C, precipitation at pH 4.0, storage for several months at -15° C, or at a dilution equivalent to 0.0014 g. hopper/ml. The best extraction medium contained 0.2 M-Na₂HPO₄+ ascorbic acid + 0.01 M-DIECA at pH 7.0–7.3. In sucrose density-gradients, EWSMV sedimented more slowly than tobacco mosaic virus. No specific particle with which infectivity could be correlated was seen by electron microscopy.     

Comparison of Undefined Medium and Its Dialyzable Fraction for Growth of Mycoplasma

In examining the medium used in cultivation of Mycoplasma for deoxyribonucleic acid isolation, it was found that an aggregate was present which sedimented with the organisms and which was ethyl alcohol-precipitated during deoxyribonucleic acid purification. To eliminate the contaminating material, a method was devised to obtain only the dialyzable constituents of the medium. This report describes the preparation of a dialysate of soy peptone-yeast extract. The medium, obtained by immersion of the encased dehydrated ingredients in sodium chloride solution for 5.5 hr at approximately 80 C, has been employed as the basal medium for cultivation of a number of Mycoplasma species. Comparative growth curves of two saprophytic strains and two parasitic species indicated that multiplication in dialysate, with suitable supplement, followed the pattern typical of the common eubacteria. Thus, by elimination of the sediment which occurred in nondialyzed medium, Mycoplasma could be concentrated without concomitant accumulation of contaminating macromolecules.     

Ultrastructural localization of cell wall teichoic acids in Streptococcus faecalis by means of concanavalin A

Some teichoic acids are known to be partially substituted by α-D-glucopyranosyl residues such as the teichoic acids of Streptococcus faecalis NCIB 8191. They will, therefore, bind specifically the phytohemagglutinin concanavalin A. Concanavalin A labelled with mercury or colloidal gold coated with concanavalin A has been used to mark isolated cell walls in order to localize the teichoic acids at the ultrastructural level. Besides these two direct marking techniques, the indirect concanavalin A-peroxidase technique (localization of peroxidase by the diaminobenzidine method followed by postosmication) has been applied to thin sections of premarked cells. All three methods gave almost identical results, namely, a dense and homogeneous distribution of the cell wall teichoic acids. In control experiments total inhibition was achieved in the presence of methyl-α-D-mannopyranoside. After trichloroacetic acid or alkali extraction of the teichoic acids from isolated walls no marking could be detected.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Gravity-regulated formation of the peg in developing cucumber seedlings

It has been proposed that peg formation in the vascular transition region (TR zone) between the hypocotyl and the root in Cucurbitaceae seedlings is a gravimorphogenetic phenomenon. Initiation of the peg became visible 36 h after imbibition when cucumber (Cucumis sativus L. cv. Burpee Hybrid II) seeds were germinated in a horizontal position at 24°C in the dark. Simultaneously, sedimented amyloplasts (putative statoliths) were apparent in the sheath cells surrounding the vascular strands, and in the cortical cells immediately adjacent to them, in the TR zone. In contrast, the other cortical cells, some of which were destined to develop into the peg, contained amyloplasts which were not sedimented. These results suggest that the graviperception mechanism for peg formation may be like that of statoliths in shoot gravitropism. By 48 h following imbibition, the cells of the TR zone still had sedimented amyloplasts but had lost their sensitivity to gravity, possibly because of their maturation.     

Comparison of two direct methods for the determination of fatty acids in infant feces

We have validated and compared two direct methods for the determination of fatty acids in feces by capillary gas chromatography. Method I consisted of esterification of fatty acids using acetyl chloride. Method II used boron trifluoride-methanol as esterification reagent. The two methods were assayed with and without previous freeze-drying of the fecal sample. We found that the two methods could be carried out without sample freeze-drying. Precision and recovery rates were determined and the results were satisfactory. Both methods gave similar results, but Method II has certain advantages over Method I, such as speed, safety, and better recovery rates.     

Different forms of simian virus 40 large tumor antigen varying in their affinities for DNA

In various permissive monkey cell lines infected with simian virus 40 there are two major forms of large T antigen which differ in their rate of sedimentation through sucrose gradients. The lighter (5 to 7S) form sedimented slightly more rapidly than the 4S tRNA marker, whereas the heavier (16S) form sedimented slightly more slowly than the 18S rRNA marker. The small t antigen did not form complexes which sedimented as rapidly as those formed by the large T antigen. The 16S T antigen form was converted to the slowly sedimenting 5 to 7S form in the presence of 1.0 M NaCl. The majority of large T antigen synthesized in cell-free protein-synthesizing systems primed by mRNA isolated from infected cells sedimented as the 5 to 7S form even when premixed with excess quantities of cellular T antigen. The formation of the 16S form in infected cells did not require ongoing viral or cellular DNA replication because considerable quantities of this T antigen class were produced in the presence of DNA synthesis inhibitors, such as cytosine arabinoside. Both 5 to 7S and 16S forms could be isolated separately and, therefore, each could be analyzed as to its individual properties. The 5 to 7S T antigen form bound more efficiently and tightly to DNA and had specific affinity for sequences at the viral origin of replication, whereas the 16S form bound less efficiently to DNA and exhibited very little specificity for origin-containing DNA sequences. It is therefore likely that the active DNA-binding species of T antigen isolated from infected cells is the 5 to 7S form.     

Synthesis of fatty acids by yeast particles

Klein, Harold P. (Ames Research Center, Moffett Field, Calif.). Synthesis of fatty acids by yeast particles. J. Bacteriol. 92:130-135. 1966.-When a mitochondria-free homogenate of Saccharomyces cerevisiae was centrifuged at 100,000 x g for 60 min, the sedimented crude particles incorporated acetate into fatty acids, but not into nonsaponifiable lipids. Degradation of the fatty acids formed indicated this to be de novo synthesis rather than chain elongation. Subfractions of the crude particles were obtained. The "ribosomal" fraction was unable to synthesize fatty acids, but had properties indicating the presence of acetokinase, fatty acid desaturase, and, probably, acetyl-coenzyme A carboxylase. A "light" particle fraction with a high specific activity of fatty acid synthetase was also obtained. Fatty acid synthesis by the "soluble" supernatant fluid appeared to be the result of contamination by the "light" particles. The data suggested the presence of several particulate entities in mitochondria-free homogenates.     

High-performance liquid chromatography of proteins on deformed nonporous agarose beads: immuno-affinity chromatography, exemplified with human growth hormone as ligand and a combination of ethylene glycol and salt for desorption of the antibodies

An affinity chromatography column packed with nonporous agarose beads derivatized with human growth hormone via carbonyldiimidazol was used for the purification of antibodies against human growth hormone from antiserum. Desorption with 1 M sodium chloride in 60% ethylene glycol at pH 9.8 gave 100% total recovery of the antibodies, as measured by radioimmunoassay. The adsorption/desorption process is discussed in terms of hydrophobic and electrostatic interaction (these interactions may be involved in the bond between antibody and antigen in a cooperative fashion). The binding capacity of the column was estimated at about 50 micrograms of antibodies per gram sedimented agarose beads.     

Non-equilibrium isotopic compositions of shells of planktonic foraminifera in the Mediterranean sea

The deviation from isotopic equilibrium has been evaluated for eight foraminiferal species from the Mediterranean Basin. A detailed study of some forms presenting an outer calcitic crust in late ontogenetic stages and in sedimented specimens allows us to propose another hypothesis for the biological fractionation: it should be a function of metabolic processes which condition the secretion of the tests.     

A water-extractable Ca2+-atpase from erythrocyte membranes

The Ca2+- and Mg2+-stimulated ATPase activities present in low ionic strength extracts of erythrocyte membranes have been separated from each other. The Ca2+-ATPase appears to be associated with particulate meterial which could be sedimented by high-speed centrifugation. The pellet obtained was composed mainly of components 1, 2, 4.5, 5 and 7. A soluble protein from the band 3 region, known to be responsible for the Mg2+-ATPase activity, was not detected in this pellet.     

Polymorphism and immunochemical cross-reactivity of acetylcholinesterases from the brains of human, dog, hog, bovine and horse

In the caudate nucleus of the species tested about 20% of the acetylcholinesterase was salt soluble and sedimented in sucrose density gradient centrifugation as monomeric 5 S and tetrameric 10 S enzyme. About 80% was solubilized by micellar concentrations of Triton X-100 and sedimented as a tetrameric 10 S species in the presence of detergent but formed aggregates in the absence thereof. All the enzyme displayed poor cross-reactivity with a precipitating assay (Ouchterlony) but in a solid phase non-precipitating assay the cross-reactivity could be quantified and ranged from 96 to less than 1% depending on the species.