Hewson Swift

Quantitative Aspects of Nuclear Nucleoproteins

Publisher Summary The nucleus is usually considered as playing a dual role in the cell. It is the carrier of genetic specificity and the organization center for cell function. Techniques of importance in the study of nucleoproteins include methods for determining the natural ultraviolet absorption of living and fixed cells and also the absorption in visible light of cells fixed and stained with acid or basic dyes and various cytochemical reactions. The photometric methods are applied to cells, including two recent symposia. Much of the modern quantitative cytology depends upon the validity of these techniques. Cytophotometric methods are simple in principle; and yet the possible sources of error are so numerous and difficult to estimate under practical measuring conditions that there are few adequate studies of the accuracy and reproducibility available. This chapter presents the tests devised to check the actual performance of measuring instruments. Obstacles, such as inhomogeneity, which has been estimated on theoretical grounds to give very large errors in measurements on nuclei, are discussed.

CYTOCHEMICAL STUDIES ON NUCLEAR FINE STRUCTURE.

Simple cytochemical methods, employing formalin fixation, nuclease extraction, and uranyl staining, have been applied to the localization of nucleic acids in electron micrographs of Ehrlich ascites tumor cells and Drosophila salivary glands. Two RNA-containing particulates were found in ascites tumor nuclei: particles about 14 mμ in diameter in nucleoli, and 20–50 mμ in the nucleoplasm. Numerous small, irregular particles about 10 mμ in diameter also bound uranyl ions, but were RNase-resistant. Clusters of these particles were lost from nuclei during mitosis, and remained in the cytoplasm during early interphase. In Drosophila polytene chromosomes, RNA-containing particulates of different sizes occurred in different puffs.

URICASE: LOCALIZATION IN HEPATIC MICROBODIES.

Microbodies of rat hepatic cells and hepatomas contain a crystalloid with the same structural characteristics as crystals of purified uricase, when viewed by electron microscopy. We conclude that crystalline uricase is located within hepatic microbodies.

Altered structure and composition of retinal cells in dark-reared mammals☆

Abstract Retinas of cats, hooded rats, and chimpanzees reared in total darkness from birth were compared with those from controls matched for age and reared in light with normal day and night variation. Microphotometric measurement of azure B binding was used to estimate ribonucleic acid (RNA) concentration in individual ganglion cells. Relative protein levels were judged visually after staining with fast green or the Millon reaction. There was a marked lowering of cytoplasmic and nucleolar RNA levels in neurons from the multipolar, bipolar, and receptor cell layers of retinas from dark-reared cats. Protein levels were also appreciably lowered in these subjects, and a significant reduction of mean nucleolar volume and in cytoplasmic cross-sectional area of ganglion cells was found. The inner plexiform layer was consistently thinner in retinas from cats reared in darkness, although there were no apparent differences in thickness of cell body layers, nor in frequencies of the several cell types within any one particular layer. Comparable but less extensive data are presented for hooded rats. Markedly depressed RNA and protein levels were also found in retinal ganglion cells of two dark-reared chimpanzees, associated with degeneration of the ganglion cell layer. The data suggest that adequate light stimulation is a major variable controlling the development of normal ribonucleoprotein levels in cells of the mammalian retina.

Synthesis of mitochondrial and nuclear DNA in anerobically grown yeast during the development of mitochondrial function in response to oxygen

Abstract When Saccharomyces cerevisiae is grown anerobically in the presence of a fermentable substrate, oxidative mitochondrial function disappears. When such anerobic cells are exposed to oxygen, even in a medium which does not support growth for lack of an assimilable nitrogen source, mitochondrial functional development proceeds rapidly. Tritium-labeled adenine was rapidly incorporated into mitochondrial DNA during respiratory oxygen adaptation. The specific activity of mitochondrial DNA was 6 to 30 times greater than that of nuclear DNA. Incorporation of radioactivity into mitochondrial DNA was maximal during the first 30 minutes after exposure to oxygen, declining rapidly thereafter. DNA synthesis preceded the development of respiratory enzyme activity which continued to rise for at least nine hours. The early DNA synthesis persisted during a two-hour pre-incubation period in adaptation medium in a nitrogen atmosphere, but an additional burst of DNA synthesis appeared after exposure to air. High glucose concentration in the oxygen adaptation medium inhibited both mitochondrial DNA synthesis and the development of respiratory activity. We estimate that 10 to 35% of the mitochondrial DNA replicates during oxygen adaptation. A respiratory-deficient mutant showed a similar response to oxygen and glucose with respect to DNA synthesis although oxidative enzyme activity was absent. Cycloheximide and chloramphenicol blocked the development of respiratory activity in response to oxygen. Cycloheximide did not inhibit mitochondrial DNA synthesis. Chloramphenicol had either no effect or inhibited both mitochondrial and nuclear DNA synthesis symmetrically.

Scaffold attachment regions in centromere-associated DNA.

Due to indications that kinetochore proteins are an integral part of the protein scaffold component of the chromosome (Earnshaw et al. 1984), we chose to map the distribution of scaffold attachment regions (SARs) at centromeres. Using the SAR mapping assay of Mirkovitch et al., Southern blots were prepared and probed with32P-labeled fragments from the human 1.9 kb centromeric α-satellite repeat unit of chromosome 1 or the 1.7 kb centromeric α-satellite repeat unit of chromosome 16. Our results demonstrated the presence of one SAR site per 1.9 kb repeat unit in chromosome 1, and every 1.7 kb repeat unit in chromosome 16, separated by regions of small DNA loops over the length of the α-satellite regions. We also identified several in vitro vertebrate topoisomerase II and cenP-B consensus sequences throughout the chromosome 1 α-satellite region using computer and base ratio analysis, to address the question as to why some α-satellite regions are SAR related and others are not. To provide in situ indications of SAR localization in the human genome, SAR DNA and non-SAR DNA were prepared following lithium 3,5-diiodosalicylate extraction. Sequences protected from DNAse I digestion by SAR proteins, as compared with unprotected DNA that was digested by the enzyme, was labeled with biotin-UTP, hybridized to chromosomal DNA in situ, and then detected with fluorescein-avidin-DCS. Both SAR and non-SAR DNA selectively labeled virtually all centromeric regions of the human metaphase karyotype. Chromosomal arms were less strongly bound by SAR DNA, with a pattern that followed the chromosomal axis. In the more condensed chromosomes an R-banding pattern was evident. In general, labeling patterns produced by both SAR and non-SAR fractions were similar, as expected from the indications that SAR DNAs are heterogenous in sequence and do not form a specific class of sequences. We conclude that centromeric regions of several, possibly all, human metaphase chromosomes are also regions where the chromosomal axis contains loops, smaller in size than in the arms and where attachment sites are concentrated. This clustering of SARs may be responsible in part for the tight chromatin packing associated with the primary constriction of the centromeric region.

Analog-induced inclusions in pancreatic acinar cells.

Feeding of β -3-thienyl- dl -alanine in semisynthetic diets devoid of phenylalanine produces characteristic focal areas of cytoplasmic necrosis. The cytoplasmic components within sequestered areas give rise to myeloid structures and small granules within complex dense bodies. Further alteration of the contents of complex dense bodies results in appearance of homogeneous bodies. This process appears to be related to a disturbed protein synthesis and to formation of abnormal proteins; it may represent a mechanism by which the cell eliminates abnormal proteins of endogenous and exogenous origin.

Monolayers of valinomycin and its equimolar mixtures with cholesterol and with stearic acid

Abstract The properties of mixed films of horizontally and vertically oriented molecules bear on many problems in surface chemistry and on the development of models for biological membranes. Pressure-area isotherms for valinomycin, a cyclic dodecadepsipeptide antibiotic, give an extrapolated molecular area of 370 A 2 and an approximate film thickness of 5 A. Horizontal orientation is thus established. Monolayer compressibility is 0.0147 cm/dyn and the collapse pressure is 23 dyn/cm. Cholesterol has a corresponding molecular area of 39 A 2 , a much lower compressibility, 0.0012 cm/dyn, and a higher collapse pressure, 43 dyn/cm. An equimolar mixture of valinomycin and cholesterol gives an isotherm contour that indicates not only the presence of the two components but considerable condensation. A mixed monolayer of valinomycin and stearic acid shows a greater condensation. Electron micrographs of collapsed monolayers of valinomycin and cholesterol demonstrate further the mixing and possible interaction of the two components in the high-pressure region.

CYANOBACTERIAL EVOLUTION AND PROCHLOROPHYTE DIVERSITY AS SEEN IN DNA‐DEPENDENT RNA POLYMERASE GENE SEQUENCES1

Nucleotide sequence data from DNA‐dependent RNA polymerase (rpoC) genes were used to examine the phylogenetic relationships among the phycobiliprotein‐ and three known chlorophyll b‐containing (prochlorophyte) cyanobacteria. The phylogenetic trees obtained confirm the polyphyletic nature of the prochlorophytes. Data from Prochloron cells obtained from six different tunicate host species suggest that at least two closely related groups of Prochloron exist in the same area in Palau, West Caroline Islands. Overall, however, the genetic diversity within the analyzed samples was much smaller than within the nonsymbiotic Prochlorococcus.

Microphotometric analysis of the cytochemical Millon reaction.

The cytochemical Millon reaction has been studied with test protein systems and with several plant and animal tissues. Absorption curves of the Millon chromophores have been analyzed and compared with natural ultraviolet protein absorption. The findings indicate the validity of applying the Millon reaction as a quantitative cytochemical method in studies of the nucleoprotein complexes of cells, but only when proper corrections for non-specific light loss and other variables have been applied. Under these conditions, a roughly linear relationship was found between natural ultraviolet protein absorption at 280 mµ and the intensity of the Millon chromophore at 500 mµ. A scheme of intermediate products and competing reactions coincident to formation of Millon chromophores has been proposed, based on changes in absorption spectra during color development in test protein systems.