Mutant of Vesicular Stomatitis Virus Which Allows Deoxyribonucleic Acid Synthesis and Division in Cells Synthesizing Viral Ribonucleic Acid

Some temperature-sensitive mutants of vesicular stomatitis virus were tested for their ability to block the initiation of deoxyribonucleic acid (DNA) synthesis and division in serum-stimulated hamster embryo fibroblasts at the nonpermissive temperature. Although the parental strain blocked these processes, one particular mutant allowed essentially normal DNA synthesis and division. By autoradiography, it was shown that individual cells infected with this mutant could synthesize viral ribonucleic acid and at the same time initiate DNA synthesis and divide. Cells infected with such conditional defective mutants appear to be suitable for studies on the effects of persistent viral infections on molecular and cellular functions in proliferating cell populations.     

Division Cycle of Myxococcus xanthus III. Kinetics of Cell Growth and Protein Synthesis

The kinetics of cell growth and protein synthesis during the division cycle of Myxococcus xanthus was determined. The distribution of cell size for both septated and nonseptated bacteria was obtained by direct measurement of the lengths of 8,000 cells. The Collins-Richmond equation was modified to consider bacterial growth in two phases: growth and division. From the derived equation, the growth rate of individual cells was computed as a function of size. Nondividing cells (growth phase) comprised 91% of the population and took up 87% of the time of the division cycle. The absolute and specific growth rates of nondividing cells were observed to increase continually throughout the growth phase; the growth rate of dividing cells could not be determined accurately by this technique because of changes in the geometry of cells between the time of septation and physical separation. The rate of protein synthesis during the division cycle was measured by pulselabeling an exponential-phase culture with radio-active valine or arginine and then preparing the cells for quantitative autoradiography. By measuring the size of individual cells as well as the number of grains, the rate of protein synthesis as a function of cell size was obtained. Nondividing cells showed an increase in both the absolute and specific rates of protein synthesis throughout the growth phase; the specific rate of protein synthesis for dividing cells was low when compared to growthphase cells. Cell growth and protein synthesis are compared to the previously reported kinetics of deoxyribonucleic acid and ribonucleic acid synthesis during the division cycle.     

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.     

Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides

The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (3H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (3H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.     

Electron Microscopy of Adenovirus 12 Replication II. High-Resolution Autoradiography of Infected KB Cells Labeled with Tritiated Thymidine

Incorporation of tritiated thymidine by KB cells infected with oncogenic adenovirus 12 was studied by means of high-resolution electron microscopic autoradiography. After a 1-hr pulse with tritiated thymidine, infected and control cultures were fixed at 8, 16, 24, 30, and 36 hr. Infected cultures showed a higher percentage of labeled cells. During early stages, the frequency of silver grains in the nucleus and in the nucleolus was higher in infected material. From 24 hr on, there was an inhibition of nuclear and nucleolar deoxyribonucleic acid (DNA) synthesis. At late stages, one-third of the label was located over nuclear inclusions, type II and IV, previously shown to be composed of DNA and protein, while a large majority of the remaining grains were located over the nucleoplasm. The possibility is considered, that the early increase in nuclear and nucleolar DNA synthesis produced by adeno 12 replication could in part be due to newly synthesized cellular DNA, as has been reported by others with respect to other oncogenic DNA viruses.     

Mating Reaction in Saccharomyces cerevisiae. IV. Retardation of Deoxyribonucleic Acid Synthesis

When a and a type haploid cells of Saccharomyces cere-visiae were mixed and cultured, deoxyribonucleic acid synthesis was retarded but ribonucleic acid and protein syntheses were not. It was found that culture filtrate of a type cells inhibited deoxyribonucleic acid synthesis of a type cells and that of a type cells inhibited that of a type cells. Thus, sex-specific diffusible substances secreted by opposite mating type cells are thought, at least partly, to be responsible for the retardation of deoxyribonucleic acid synthesis.     

Macromolecular Synthesis During Microcycle Sporogenesis of Bacillus cereus T

Microcycle sporogenesis induced in Bacillus cereus T by phosphate limitation occurs over a narrow range of phosphate to spore inoculum ratios. Sufficient phosphate is required to satisfy the demands for a twofold increase in deoxyribonucleic acid; net ribonucleic acid synthesis is not required. The total ribonucleic acid content of the culture was variable, and deoxyribonucleic acid synthesis was restricted to a twofold increase. Developmental changes during outgrowth occurred synchronously, whereas enzyme synthesis was periodic. The timing of the synthesis of tricarboxylic cycle enzymes, extracellular protease, arginase, histidase, and alkaline phosphatase was measured. Histidase could be induced after 2.5 hr throughout microcycle sporogenesis. Several other features of macromolecular synthesis during microcycle sporogenesis are described. Differences between this pattern and those observed during outgrowth leading to cell division are discussed. A technique for accurately estimating the levels and time of synthesis of incompletely extractable, labile enzymes is also presented.     

Electron Microscope Studies of Deoxyribonucleic Acid Synthesis in Escherichia coli: Localization of [3H]Thymidine in Deoxyribonucleic Acid-Membrane Complexes

The attachment of deoxyribonucleic acid to the membrane in Escherichia coli 15 T(-) cells incubated with [(3)H]thymidine was studied by electron microscopy. Isolated deoxyribonucleic acid-membrane complexes were prepared from synchronized and unsynchronized cells during the exponential or stationary phase of growth and were examined by autoradiography. After short pulses with [(3)H]thymidine, a specific enrichment in radioactivity was observed in areas of membranous structures in exponentially growing cells. In contrast, the grain tracks produced in autoradiographs of chromosomes from cells in stationary phase were randomly distributed. The autoradiographic patterns are, therefore, evidence that deoxyribonucleic acid replication is closely related to the bacterial membrane.     

Correction of autoradiographic grain count in respect to precisely calculated background

Summary It was endeavored to find a criterion for significantly labeled cells in quantitative autoradiography. Measurements of the autoradiographic background were performed and it was found that: 1. the value of the background over the non-proliferating epithelial cells from an animal injected with ³H-thymidine is higher than over the same cells from animals not injected with an isotope, 2. the value of the background in emulsion over the tissue specimen is higher than away from the specimen. Therefore, one should take into account the background over the tissue. Nomograms are shown for quick evaluation of the percentage of cells labeled with 1, 2, 3 or 4 grains, which should be disregarded as due to the background. To obtain this percentage for a given experiment its appropriate parameters: the labeling index, the mean grain count over the cell, the standard deviation of the grain count distribution and the background grain count distribution should be taken into account.     

Quantitative carbon-14 autoradiography at the cellular level: Principles and application for cell kinetic studies

Summary Amounts of radio-labelled substances as low as 10^–18 moles incorporated into individual cells can be measured by utilizing techniques of quantitative autoradiography. For this purpose, radioactive standard sources are processed with the labelled cells smeared to slides. Carbon-14 is a favourable isotope with regard to minimal loss of -disintegrations due to self-absorption, and to limited cross-fire effects complicating the attribution of silver grains to individual cells. Silver grain densities can be counted by automated microphotometry allowing on-line data processing by an interfaced computer.Rate measurements of¹⁴C-thymidine incorporation into individual cells yield values of the DNA synthesis rate provided that the endogenous pathway of thymidine-phosphate formation has been previously blocked. From the rate values of individual cells the DNA synthesis time of a cell compartment is derived. This is an essential time parameter for the evaluation of kinetic events in proliferating cell populations. This method is applicable to human cells without radiation hazard to man, and provides an optimal source of detailed information on the kinetics of normal and diseased human haematopoiesis. Examples of application consist of thalassaemia, malaria infection, iron deficiency anaemia and acute myelogenous leukaemia.     

Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides

Summary The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (³H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (³H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.     

Bacterial Cell Division Regulation: Characterization of the dnaH Locus of Escherichia coli

The dnaH locus is the fourth gene to be identified as required for deoxyribonucleic acid polymerization in Escherichia coli. A temperature-sensitive mutant defective in this gene exhibited an abrupt decrease in rate of deoxyribonucleic acid synthesis when shifted to 42 C. The locus mapped in the proC-purE region of the chromosome by conjugation and was co-transducible with purE. dnaH(+) is carried on the F'(13) episome and is dominant over the dnaH(-) mutation.     

Reinitiation of deoxyribonucleic acid synthesis by deoxyribonucleic acid initiation mutants of Escherichia coli: role of ribonucleic acid synthesis, protein synthesis, and cell division.

The dnaA and dnaC genes are thought to code for two proteins required for the initiation of chromosomal deoxyribonucleic acid replication in Escherichia coli. When a strain carrying a mutation in either of these genes is shifted from a permissive to a restrictive temperature, chromosome replication ceases after a period of residual synthesis. When the strains are reincubated at the permissive temperature, replication again resumes after a short lag. This reinitiation does not require either protein synthesis (as measured by resistance to chloramphenicol) or ribonucleic acid synthesis (as measured by resistance to rifampin). Thus, if there is a requirement for the synthesis of a specific ribonucleic acid to initiate deoxyribonucleic acid replication, this ribonucleic acid can be synthesized prior to the time of initiation and is relatively stable. Furthermore, the synthesis of this hypothetical ribonucleic acid does not require either the dnaA of dnaC gene products. The buildup at the restrictive temperature of the potential to reinitiate deoxyribonucleic acid synthesis at the permissive temperature shows rather complex kinetics the buildup roughly parallels the rate of mass increase of the culture for at least the first mass doubling at the restrictive temperature. At later times there appears to be a gradual loss of initiation potential despite a continued increase in mass. Under optimal conditions the increase in initiation potential can equal, but not exceed, the increase in cell division at the restrictive temperature. These results are most easily interpreted according to models that postulate a relationship between the initiation of deoxyribonucleic acid synthesis and the processes leading to cell division.     

Mode of Action of Colicins of Types E1, E2, E3, and K

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).     

Quantitative 125-I-autoradiography of individual cells (author's transl)]

Iodine 125, an emitter of beta-radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures. The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.     

Autoradiographic measurements of protein synthesis in hippocampal slices from rats and guinea pigs.

Protein synthesis is an extremely important cell function and there is now good evidence that changes in synthesis play important roles both in neuronal cell damage from ischemic insults and in neural plasticity though the mechanisms of these effects are not at all clear. The brain slice, and particularly the hippocampal slice, is an excellent preparation for studying these effects although, as with all studies on slices, caution must be exercised in that regulation in the slice may be different from regulation in vivo. Studies on neural tissue need to take into account the heterogeneity of neural tissue as well as the very different compartments within neurons. Autoradiography at both the light and electron microscope levels is a very powerful method for doing this. Successful autoradiography depends on many factors. These include correct choice of precursor amino acid, mechanisms for estimating changes in the specific activity of the precursor amino acid pool, and reliable methods for quantitation of the autoradiographs. At a more technical level these factors include attention to detail in processing tissue sections so as to avoid light contamination during exposure and developing and, also, appropriate choices of the various parameters such as exposure time and section thickness. The power of autoradiography is illustrated here by its ability to discern effects of ischemia and of plasticity-related neural input on distinct cell types and also in distinct compartments of neurons. Ischemia inhibits protein synthesis in principal neurons but activates synthesis in other cell types of the brain slice. Plasticity-related neural input immediately enhances protein synthesis in dendrites but does not affect cell bodies.     

Quantitative autoradiography of neurochemicals

Several new methods have been developed that apply quantitative autoradiography to neurochemistry. These methods are derived from the 2-deoxyglucose (2DG) technique of Sokoloff (1), which uses quantitative autoradiography to measure the rate of glucose utilization in brain structures. The new methods allow the measurement of the rate of cerbral protein synthesis and the levels of particular neurotransmitter receptors by quantitative autoradiography. As with the 2DG method, the new techniques can measure molecular levels in micron-sized brain structures; and can be used in conjuction with computerized systems of image processing. It is possible that many neurochemical measurements could be made by computerized analysis of quantitative autoradiograms.     

Photometric methods in quantitation of light microscope radioautography

Photometric evaluation of autoradiographic grain densities can be performed according to various optical principles. In all instances the amount of light recorded should be a measure of the radioactive substance amount in the specimens. It is shown that grain densities suitable for light microscopic autoradiography are indeed linearly related to radiation exposure. Dependent on the photometric system used, there is a larger or smaller section of linear correlation of the photometric response with grain density. The advantages of incident light bright-field illumination for silver grain counting are discussed. Quantitation of substance amounts in autoradiographs depends on the use of radioactive standard sources. Two different approaches of quantitation are discussed. In 14C-autoradiography which can be applied for cell-kinetic studies, standard plates are used consisting of 14C-polymethylmethacrylate. Allowance has to be made for the different condition of radiation geometry of the cells and the standards. In 125I-autoradiography, 125I-labeled red cells are used as standards. This technique allows for quantitating the number of antibodies bound to individual cell surfaces.     

Genetic and phenotypic characterization of dnaC mutations.

The dna-1, dna-2, dna-7, and dna-28 mutations, all of which are located near min 89.5 on the E. coli linkage map, have been characterized further. As previously demonstrated for dna-2 and dna-28, neither the dna-1 nor dna-7 mutation affects the ability of a strain to produce bacteriophage lambda at temperatures non-permissive for the continued replication of the bacterial chromosome. The reported temperature-sensitive inhibition of lambda production in a strain carrying dna-7 is shown to be a consequence of a thermosensitive host specificity mutation in the hsm gene and not of the dna-7 mutation. The four dna mutations are recessive to the wild type and define a single dnaC cistron according to standard complementation criteria. Unlike other characterized dnaC mutants, however, strains carrying the dnaC1 or dnaC7 alleles exhibit an abrupt cessation of deoxyribonucleic acid synthesis at 42 C that appears to be more compatible with a defect in deoxyribonucleic acid chain elongation rather than in initiation. The possibility that the apparent elongation defect is actually a composite effect of residual synthesis and deoxyribonucleic acid degradation is raised by the net deoxyribonucleic acid degradation observed in the dnaC1 strain at 42 C. Several alternative possibilities for the function of the dnaC gene product are suggested.