Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.     

Isolation, Composition, and Structure of Membrane of Listeria monocytogenes

The plasma membrane of Listeria monocytogenes strain 42 was prepared by osmotic lysis of protoplasts with tris(hydroxymethyl)aminomethane (Tris) buffer, pH 8.2, containing MgCl₂ and glucose, followed by washing with NaCl and MgCl₂ in Tris buffer. Electron microscopy showed that the preparation was not contaminated with cytoplasmic material. The membrane preparation was composed of 55 to 60% protein, 1.5% ribonucleic acid, 0.1% deoxyribonucleic acid, 1.3 to 2.3% carbohydrate, 0.17 to 0.38% amino sugar, 0.2 to 0.4% rhamnose, 3.5 to 4.0% phosphorus, 10.5 to 12.0% nitrogen, and 30 to 35% lipid. Amino acid composition of the washed membrane showed some variation from that of the whole cells. Sulfur-containing amino acids were not present in the membrane hydrolysate. The membrane carbohydrate contained glucose, galactose, ribose, and arabinose. The membrane lipid was 80 to 85% phospholipid and 15 to 20% neutral lipid. The lipid contained 2.3 to 3.0% phosphorus, 2.5 to 3.0% carbohydrate, and a very small amount of nitrogen (0.2 to 0.3%). The phospholipid was of the phosphatidyl glycerol type. Electron micrographs of the washed membrane showed three layers. The outer and inner layers varied in thickness from 25 to 37 A and the middle layer from 20 to 25 A. The total thickness varied between 85 and 100 A. These preparations contained many vesicles which stained heavily with lead citrate. Some vesicles were also attached to the protoplast ghosts in the form of extrusions or intrusions, or both. Membrane preparations obtained by lysis of protoplasts in the absence of MgCl₂ were fragmented and contained less lipid (20 to 22%) and ribonucleic acid (0.3 to 0.5%) than preparations prepared with MgCl₂.     

Macromolecule Synthesis and Breakdown in Relation to Sporulation and Meiosis in Yeast

The time course of synthesis and breakdown of various macromolecules has been compared for sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast cells transferred to potassium acetate sporulation medium. Both types of cells incorporate label into ribonucleic acid and protein. The gel electrophoresis patterns of proteins synthesized in sporulation medium are identical for sporulating and nonsporulating diploids; both are different from electropherograms of vegetative cells. Sporulating and nonsporulating strains differ with respect to deoxyribonucleic acid synthesis; no deoxyribonucleic acid is synthesized in the latter case, whereas the deoxyribonucleic acid complement is doubled in the former. Glycogen breakdown occurs only in sporulating strains. Breakdown of preexisting vegetative ribonucleic acid and protein molecules occurs much more extensively in sporulating than in nonsporulating cells. A timetable of these data is presented.     

Fluorometric detection of deoxyribonucleic acid synthesis; possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry.

Fluorometric detection of the biosynthetic incorporation of 5-bromodeoxyuridine (BrdU) into deoxyribonucleic acid has permitted cytologic studies of chromosome structure, replication, and repair. Some of these phenomena, previously detected using BrdU-dye techniques on fixed microscopic preparations, should be particularly amenable to analogous experimentation in fluorescence flow systems. Problems involved in interfacing BrdU-dye methodology with flow fluorometry are discussed. The effects of certain chemical modifications of bisbenzimidazole dyes on their spectroscopic properties and potential use for detecting BrdU incorporation into unfixed cells are described. Data on the use and energy transfer characteristics of a pair of deoxyribonucleic acid-binding dyes (33258 Hoechst and ethidium bromide) capable of simultaneously providing information about BrdU substitution and total deoxyribonucleic acid content are presented.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Characteristics of cold-sensitive mutants of Escherichia coli K-12 defective in deoxyribonucleic acid replication.

Four cold-sensitive mutants of Escherichia coli have been isolated which show a reduced ability to synthesize deoxyribonucleic acid at low temperature. The mutants also have a reduced ability to incorporate nucleoside triphosphates into deoxyribonucleic acid at low temperature in cell preparations made permeable with toluene. All four mutations are located at or near the dnaA locus on the E. coli genetic map. They are recessive to the wild-type allele and two of them can be integratively suppressed by F episomes.     

Histochemical Observations on Mycoplasma after Staining with Acridine Orange

Mycoplasma colonies and Mycoplasma cells in preparations from infected milk and lymph nodes were observed for their fluorescent qualities after treatment with acridine orange. Mycoplasma colonies fluoresced brilliant red or red-orange. When treated after exposure to ribonuclease, the colonies fluoresced lime-green. There was no fluorescence when both ribonucleic acid and deoxyribonucleic acid were destroyed by perchloric acid. Detection of Mycoplasma in smears of mastitic milk or smears of infected lymph nodes was not definitive because of the large amount of nonspecific ribonucleic acid-rich material present during inflammatory reactions.     

Transport of DNA from Lymphocytes to Fibroblasts in Culture

The exchange of radioactivity between lymphocytes, labelled with (³H) thymidine after stimulation with Concanavalin A, and recipient V79 fibroblasts in culture was studied. The radioactive material involved in this exchange was macromolecular deoxyribonucleic acid as well as its breakdown products. This deoxyribonucleic acid from lymphocytes localised in the nuclei of the host cells soon after contact between donor and recipients. This occurred even when the V79 fibroblasts were confluent at high cell density, and thus in a steady, non-growing state with respect to cell numbers.
The fate of the radioactive donor lymphocyte deoxyribonucleic acid, substituted with bromodeoxyuridine, was followed in the recipient cells by analysing its buoyant density in caesium chloride gradients. This deoxyribonucleic acid was found to become associated with the nuclear deoxyribonucleic acid of the host cells, involving both retention of relatively intact donor deoxyribonucleic acid as well as its breakdown and re-utilisation for host cell deoxyribonucleic acid synthesis. Nongrowing recipient cells were found to retain the donor deoxyribonucleic acid in relatively intact form for much longer periods than when the same cells were in logarithmic growth phase.     

Effects of acetate and other short-chain fatty acids on sugar and amino acid uptake of Bacillus subtilis

Acetate and other short chain n-fatty acids (C(1)-C(6)) inhibit strongly the uptake of l-serine or other l-amino acids but inhibit only weakly that of alpha-methylglucoside or fructose, whether measured in whole cells of Bacillus subtilis or in membrane vesicles that have been energized with reduced nicotinamide adenine dinucleotide (NADH), l-alpha-glycerol phosphate, or ascorbate plus phenazine methosulfate. The acetate inhibition is noncompetitive, as was shown for l-alpha-aminoisobutyric acid uptake by whole cells and for l-serine uptake by membrane vesicles. In membrane preparations, neither NADH oxidation nor the reduction of cytochromes by NADH are affected by fatty acids. All of these effects are similar to those of 2, 4-dinitrophenol. It is concluded that the fatty acids "uncouple" the amino acid carrier proteins from the cytochrome-linked electron transport system (to which they may be coupled via protein interaction or via a cation gradient).     

Incorporation and excision of 5-fluorouracil from deoxyribonucleic acid in Escherichia coli.

When Escherichia coli are grown in the presence of 5-fluorouracil, the 5-fluorouracil is incorporated almost exclusively into ribonucleic acid as fluorouridylate. In this study, small but detectable amounts were incorporated into ribonucleic acid as fluorocytidylate and into deoxyribonucleic acid as fluorodeoxyuridylate and fluorodeoxycytidylate. The amount of 5-fluorouracil found in deoxyribonucleic acid as fluorodeoxyuridylate increased 50-fold when the cells were deficient in both deoxyuridine triphosphatase and uracil-deoxyribonucleic acid glycosylase activities. Therefore, the same mechanisms which excluded uracil from deoxyribonucleic acid in vivo also excluded 5-fluorouracil. Even though purified uracil-deoxyribonucleic acid glycosylase excised 5-fluorouracil from deoxyribonucleic acid at only 5% the rate with which it excised uracil, most of the 5-fluorouracil excised from deoxyribonucleic acid in vivo was apparently excised directly by uracil-deoxyribonucleic acid glycosylase rather than by repair initiated by excision of uracil.     

Use of actinomycin D for the specific quenching of fluorescence of deoxyribonucleic acid in cells stained with acridine aminoderivatives.

Actinomycin D specifically quenches the fluorescence of acridine orange and quinacrine bound to deoxyribonucleic acid in cytologic preparations, but does not change the fluorescence of these fluorochromes bound to RNA. The following fluorescence-cytochemical applications of techniques based on these findings can be suggested: (a) distinction between deoxyribonucleic acid and ribonucleic acid; (b) detection of double-stranded virus ribonucleic acid; (c) approximate estimation of the lengths of A-T sequences in deoxyribonucleic acid molecules.     

Purification and properties of a manganese-stimulated endonuclease from Bacillus subtilis.

An endonuclease stimulated by manganese or calcium ions was isolated from Bacillus subtilis. This enzyme attacked double- or single-stranded deoxyribonucleic acid from a variety of sources, including B. subtilis, and was purified from the material released into the medium during protoplast formation. The enzyme appeared as a single peak after glycerol gradient centrifugation and comprised approximately 30 to 35% of the protein in the most purified preparations, as estimated by gel electrophoresis. It had a molecular weight of about 46,000. The mode of action of the enzyme was endonucleolytic, and circular deoxyribonucleic acid was readily cleaved. The enzyme introduced a limited number of both double- and single-strand breaks into native deoxyribonucleic acid, generally yielding products of 1 X 10(6) daltons or more in size. The reasons for this limitation of cleavage were not clear. The activity of the enzyme was inhibited by low levels of Cu2+, Co2+, Hg2+, and Zn2+. It was also inhibited by high concentrations of NaCl. A role for this enzyme in bacterial transormation is suggested.     

Mode of action of pesticin: N-acetylglucosaminidase activity.

Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli phi nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label ([14C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with [14C]diaminopimelic acid in the murein peptide or with [14C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticin-dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption.     

Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein.

The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322. These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long. An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500. Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein. Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane. Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane. This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.     

Host cell and ultraviolet reactivation of ultraviolet-irradiated Mycoplasmaviruses.

The mycoplasma Acholeplasma laidlawii was shown to have mechanisms for both host cell and ultraviolet (UV) reactivation of UV-irradiated mycoplasmaviruses. Host cell reactivation was examined by comparing the survival abilities of UV-irradiated double-stranded deoxyribonucleic acid mycoplasmavirus plated on both untreated and on acriflavine-treated cells. Acriflavine treatment inhibited cell exision repair. Decreased survival on the acriflavine-treated cells demonstrated host cell reactivation. UV reactivation was studied by comparing the survival of UV-irradiated virus plated on untreated cells with its survival on cells that received a small UV dose before plating. The UV-irradiated cells gave increased virus survival, showing UV reactivation. Similar experiments with a single-stranded deoxyribonucleic acid mycoplasmavirus showed that this virus could be UV reactivated, but not host cell reactivated.     

Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles

Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K(m) values for NADH. The higher K(m) corresponds to the only K(m) observed for NADH oxidation by whole cells, whereas the lower K(m) corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K(m) reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K(m) reaction. In contrast, the oxidation of l-alpha-glycerol-phosphate (glycerol-P) by membrane preparations shows only one K(m) that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K(m), this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K(m) for amino acid uptake by vesicles in the presence of NADH corresponds to the high K(m) for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of l-alpha-glycerol-P.     

Plasma membrane vesicles isolated from epimastigote forms of Trypanosoma cruzi.

Membrane vesicles can be obtained from epimastigote forms of Trypansoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5--10 times richer in protein-bound iodine when they are prepared from cells previously labeled with 131I by the lactoperoxidase catalyzed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane. Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein=1.5--2.0; orcinol : protein-0.07 and absence of diphenylamine reaction. Vesicles contain 0.2--0.5% and 0.3--1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and 30--50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

Plasma membrane vesicles isolated from epimastigote forms of trypanosoma cruzi

Membrane vesicles can be obtained from epimastigote forms of Trypanosoma cruzi by incubating cells with either cross-linking reagents or acid pH. Acetate, phtalate or citrate, at pH 4.0, but not at higher pH values, were able to induce plasma membrane vesiculation. Vesicles have been purified by sucrose density centrifugation and their membrane origin was demonstrated by the following criteria: (a) Vesicles are 5–10 times richer in protein-bound iodine when they are prepared from cells previously labeled with ¹³¹I by the lactoperoxidase catalysed reaction. (b) Electron microscopy of vesiculating cells shows physical continuity between cell plasma membrane and vesicle membrane. (c) Antibodies prepared against purified vesicles are able to agglutinate epimastigote forms of T. cruzi with sera dilutions up to 1 : 256 to 1 : 512. (d) Freeze-fracture studies of the purified vesicles have shown images of faces P and E compatible with known images of the intact cell plasma membrane.Typical preparations of acetate vesicles present the following characteristics: total carbohydrate : protein = 1.5–2.0; orcinol : protein = 0.07 and absence of diphenylamine reaction. Vesicles contain 0.2–0.5% and 0.3–1.0% of the total homogenate protein and carbohydrate, respectively. The presence of 10 major protein bands and a 30–50-fold enrichment of the four sugar-containing macromolecules present in epimastigote forms of T. cruzi have been demonstrated in these preparations.     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic dnaA mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in dnaA mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or dnaA mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of dnaA mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or dnaA membrane preparations. Thus, the dnaA mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.