URIDINE-5-H3 RADIOAUTOGRAPHY OF THE HUMAN SEX CHROMATIN BODY

To obtain an estimate of the rate of RNA synthesis by the heterochromatic sex chromatin body, human female fibroblasts were labeled with uridine-5-H³ and radioautographed. The number of grains over the sex chromatin body was compared with the number of grains over a comparable area of euchromatin. The ratio was 0.37. When corrected for the higher content of DNA per unit area in heterochromatin, the maximum rate of RNA synthesis by the DNA of the sex chromatin body was approximately 18% of the rate of RNA synthesis by a comparable amount of euchromatin DNA. The rate of RNA synthesis by the sex chromatin body did not increase significantly with partial despiralization of this chromatin at prophase.     

A RADIOAUTOGRAPHIC STUDY WITH H3-THYMIDINE ON ADRENAL MEDULLA NUCLEI OF RATS INTERMITTENTLY EXPOSED TO COLD

A considerable decrease (24 to 40%) of DNA content per nucleus previously observed in the adrenal medulla of rats exposed intermittently to cold is followed by restoration to normal and supranormal values. This phenomenon has now been studied by use of H³-thymidine, which was given to normal rats, to rats exposed to cold, and to animals brought to room temperature after cold exposure. In the first two conditions, no significant labeling of nuclei was observed. In the third, labeling took place clearly in the 1st 3 days. The grain counts showed that the early labeled nuclei had more grains than those labeled later, indicating differences in the rate of DNA synthesis. A statistically significant correlation was found, on the same nuclei, between amount of Feulgen dye and number of grains. It is concluded that net synthesis of DNA takes place in the phase of recovery from cold. This fact is not related to cell division, as no mitoses could ever be detected, but rather to the cold-induced loss of DNA. Clear demonstration is thus given of a marked variation in the amount of DNA per nucleus in relation to the functional conditions of adrenal medulla cells.     

Cell-cycle dependent biosynthesis and localization of p53 protein in untransformed human cells

Summary— Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno-fluorescent microscopy and post-embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus. p53 in the nucleus was found associated with the non-chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti-p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent-resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin in the cells.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis

The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In addition, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.     

Suppression for the proliferation of fibroblasts by external DNA

Phase-contrast and fluorescence microscopic observation showed that DNA added in the cell-culture medium for fibroblasts localized just on the surface of fibroblasts. The DNA bound to fibroblasts was found to be eluted by treating with collagenase. The suppression for the proliferation of fibroblasts by external DNA was confirmed with microscopic observation for the cells cultured in the presence and absence of DNA. Proliferation of the cells decreased from 412 to 155% by the addition of DNA. These results indicate that DNA has an affinity for collagen, the most major extracellular-matrix produced by fibroblasts, and suppresses the growth of fibroblasts.     

Increased SCE inducibility by low doses of methylcholanthrene in lymphocytes obtained from patients with Down's disease

The inducibility of sister-chromatid exchanges (SCEs) following 3-methylcholanthrene (MC) treatment of normal human peripheral blood lymphocytes and of in vitro cultured normal human embryo fibroblasts, as well as peripheral blood lymphocytes of patients with Down's disease and of fibroblasts of an embryo with trisomy 21 has been investigated. 10(-6) M MC treatment increased the frequency of SCEs by 2.5 in the case of Down lymphocytes when compared to the healthy control. The fibroblasts with trisomy 21, however, did not show an increased sensitivity to MC treatment when compared with normal fibroblasts, expressed in the number of SCEs per nucleus found in the cells.     

Computerized measurement of the DNA content, areas, and autoradiographic grains of the same nuclei: demonstration that lightly (3H)thymidine-labeled bone marrow cells are predominantly in G0/G1 and G2

A computerized "flying spot" microdensitometer and scanning stage have been combined to measure the cellular DNA content, nuclear areas, and autoradiographic grain areas of the same cells. The slide positions of the Feulgen-stained, (3H)thymidine-labeled cells are mapped with the computerized stage, and nuclear DNA content and areas are then determined by integral absorbance measurements at 588 nm. Following autoradiograph preparation, the cells are relocated and the areas of the autoradiographic grains over each nucleus are measured at a light wavelength (625 nm) and an optical density setting (greater than 0.10) that do not detect the Feulgen stain. The microcomputer calculates the portion of each nucleus covered with autoradiographic grains (grain area proportion, GAP), and it links the GAP value to the DNA content of each nucleus in the computer file for subsequent sorting and analysis. By using this system in a study of mouse bone marrow cells labeled in vivo with (3H)thymidine, we found that all S-phase cells were clearly labeled after 8 or more days of autoradiographic exposure. Prolonged exposures (up to 64 days) led to detection of lightly labeled cells (0.1 less than GAP less than 0.8) with G1/G0 and G2 DNA content.     

Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [³H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylamino-fluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2,3 and 4 were similar, although shifted about 3–10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.     

Biological and morphological characterization of human neonatal fibroblast cell culture B-HNF-1

In the present study, human neonatal fibroblasts were isolated from a two-month-old human male. The purpose of the present investigation was the analysis of the morphology (light and transmission electron microscopy), karyotype and growth characteristics of the human neonatal fibroblast cell culture B-HNF-1. Moreover, STR typing and mitochondrial DNA amplification and sequencing was also performed. Analysis of chromosomes count showed that B-HNF-1 cell culture is diploid and has normal male karyotype 46, XY, which was stable during cultivation. The transmission electron microscopy demonstrated the ultra-structure of the B-HNF-1 cells; they have typical morphological features of proteosynthesis-active cells. Large number of fibroblasts bearing different shapes and surface characteristics adhered to the substrate with microvilli and filopodia. Our in vitro expanded fibroblasts have a large and irregular nucleus with prevalence of euchromatin. One to three nucleoli are present in each nucleus. The cytoplasm contains a richly developed rough endoplasmic reticulum and prominent Golgi apparatus cisterns. The result of the fragment analysis is a DNA profile defined as multiplex of STR markers and sex determining system. Sequencing analysis of hypervariable segments of control region of mitochondrial DNA showed haplotype that belongs to haplogroup W. In this study, we complexly characterized a new cell culture of human neonatal fibroblasts B-HNF-1 from different points of view. This culture should be used for further biomedical experiments.     

Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

Characteristics of Starch Grains Isolated from Mature Pepper Leaves Grown under Different Irradiances and Daylengths

The size, shape and number of starch grains have been determinedin mature pepper leaves taken from plants grown under definedconditions of daylength and irradiance. Starch grains were 0.2–7.0 µm diameter and 02–1.5µm in thickness. Grain diameter was positively relatedto daylength and the number of grains per unit leaf area inverselyrelated to daylength. Mean grain diameter was also positivelyrelated to leaf area. Analysis of starch grains from leaves having a wide range ofstarch contents showed that grain diameter was linearly relatedto leaf starch content. However, mean diameter only doubledwith a 10-fold increase in starch content. The number of grainsincreased from approximately 5 ? 10¹⁰ m^–2 of leaf to over200 ? 10¹⁰ m^–2 with increasing starch content. The totalsurface area of grains increased from less than 1.0 m² m^–2leaf to over 20 m² m^–2 leaf. Leaf starch grain shape and size are compatible with both efficientstorage as disc-shaped chloroplasts and the maintenance of hightotal grain surface area by increasing grain number more thandiameter. Possible mechanisms for the control of grain initiation,growth and degradation are suggested. Key words: Starch grains, size, shape, pepper leaves     

DNA repair synthesis induced by N-hydroxyurea, acetohydroxamic acid, and N-hydroxyurethane in primary rat hepatocyte cultures: comparative evaluation using the autoradiographic and the bromodeoxyuridine density-shift method

N-Hydroxyurea and two structurally related compounds, acetohydroxamic acid and N-hydroxyurethane, were investigated for their potential to induce DNA repair synthesis in primary rat hepatocyte cultures. Repair was determined as repair replication by means of the bromodeoxyuridine density-shift method and, in the same cell preparations, as unscheduled DNA synthesis (UDS) by autoradiography. For all 3 compounds, a clear concentration-dependent induction of DNA repair replication could be demonstrated. Interpretation of the UDS data, however, depended on the mode whereby the results were evaluated. Expression of the results as net grains per nucleus after subtraction of cytoplasmic from nuclear grain counts yielded statistically significant increases over the control values for all compounds. In contrast, no significant changes of the nuclear labeling were obtained when nuclear and cytoplasmic grain counts were plotted separately. These findings demonstrate that the two modes to present UDS data may lead to different conclusions, a consequence of the uncertainty regarding the origin and importance of the cytoplasmic background. The observation that both hydroxyurea and the structurally related compounds acetohydroxamic acid and N-hydroxyurethane induce DNA repair in primary hepatocyte cultures suggests that metabolism-dependent genotoxicity may be a common property of aliphatic hydroxamic acids.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size,Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

Summary A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 x oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r=0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r=0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r=0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30–100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm× 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

THE ROLE OF THE NUCLEOLUS : I. Tritiated Cytidine Activity in Liver Parenchymal Cells of Thioacetamide-Treated Rats

Male rats of the Sherman strain were fed for 2 weeks a diet of ground purina rat chow containing 0.04 per cent thioacetamide. Animals were injected intraperitoneally with tritiated cytidine, 200µc/100 gm body weight, and sacrificed in pairs, a control and a thioacetamide-treated rat, at prescribed intervals. Liver tissues were preserved with the freeze-substitution method and postfixed in anhydrous OsO₄. Other samples were fixed directly with an acetic acid-ethanol mixture (1:3). AR-10 stripping film was applied to 2- and 4-µ sections and exposed for appropriate lengths of time. Nuclear and nucleolar volumes were obtained by direct measurement. Cytoplasmic volumes were obtained with the aid of Chalkley ratios. Nucleolar and cytoplasmic RNA concentrations were calculated from cytophotometric extinction (E_{540 mµ}) measurements. Data were expressed as grains/unit area, grains/unit area/concentration (or specific activity) and grains/total structure. In the liver parenchymal cells of thioacetamide-treated rats, the nucleolus shows vast increases in volume, RNA content, and grain count/total structure, 14-fold, 25-fold, and over 30-fold, respectively. The nucleus increases 2-fold in volume and about 3-fold in total grain count. Cytoplasmic volume increases only 20 per cent and displays a total grain count about equal to that in the control. The time course of incorporation curves for nucleolus and non-nucleolar nucleus (NNN) contain 2 distinct turnover fractions, rapid and slow. Both fractions were increased after thioacetamide treatment but remained proportional to those of controls. The unique stimulated RNA turnover in the nucleus and nucleolus, coupled to a "normal" turnover in the cytoplasm, suggests that this nuclear-nucleolar loss of label does not represent an exclusive passage of formed nuclear RNA to the cytoplasm.     

Endogenous DNA damage clusters in human skin, 3-D model, and cultured skin cells

Clustered damages-two or more oxidized bases, abasic sites, or strand breaks on opposing DNA strands within a few helical turns-are formed in DNA by ionizing radiation. Clusters are difficult for cells to repair and thus pose significant challenges to genomic integrity. Although endogenous clusters were found in some permanent human cell lines, it was not known if clusters accumulated in human tissues or primary cells. Using high-sensitivity gel electrophoresis, electronic imaging, and number average length analysis, we determined endogenous cluster levels in DNA from human skin, a 3-D skin model, and primary cultured skin cells. DNA from dermis and epidermis of human skin contained extremely low levels of endogenous clusters (a few per gigabase). However, cultured skin fibroblasts and keratinocytes-whether in monolayer cultures or in 3-D model skin cultures-accumulated oxidized pyrimidine, oxidized purine, and abasic clusters. The levels of endogenous clusters were decreased by growing cells in the presence of selenium or by increasing cellular levels of Fpg protein, presumably by increasing processing of clustered damages. These results imply that the levels of endogenous clusters can be affected by the cells' external environment and their ability to deal with DNA damage.     

INCORPORATION OF H3-THYMIDINE IN LEAF NUCLEI OF XANTHIUM PENNSYLVANICUM

The basic kinetics and the pattern of incorporation of H³-thymidine was studied in the leaf lamina of Xanthium pennsylvanicum. A method of foliar absorption was used to incorporate the radioisotope into leaf nuclei. The autoradiographic techniques employed provided data on the amount of the isotope incorporated. It was determined that 10 μc/ml (sp. act. 6.7 c/mmole) of H³-thymidine with 1–8 hr of isotopic growth and 4 hr of postisotopic growth gave the most satisfactory results. The percent of labelled nuclei and the number of grains per nucleus were presented as functions of isotopic and postisotopic growth periods. Distribution of grains in the nuclei approximated the Poisson distribution at 1 hr of isotopic growth. Increased time of isotopic growth changed the pattern of grain distribution. No deleterious effects were observed using an 8-hr period of isotopic growth, but prolonged incubation time significantly decreased the proportion of mitotic figures in the lamina. The amount of incorporation of the DNA precursor expressed as percent of labelled nuclei was linear to about 16 hr of isotopic growth and thereafter decreased gradually. As indicated by the average number of grains per nucleus, H³-thymidine incorporation increased to about 16 hr, and soon after reached a saturation level. The percent of labelled nuclei and the number of grains per nucleus decreased as a function of the postisotopic growth period. However, they were significantly greater in the lamina near the vein than in the lamina region at some distance from the vein. The radioactive precursor was initially absorbed by the cells of the lamina and was subsequently translocated into the vascular system. There it was circulated and made available to the dividing cells near veins of the lamina. This region may be a metabolically distinct part of the lamina with significantly higher rates of incorporation and mitotic turnover.     

WIDE AND THICK GRAIN 1, which encodes an otubain‐like protease with deubiquitination activity,influences grain size and shape in rice

Grain size and shape are two crucial traits that influence grain yield and grain appearance in rice. Although several factors that affect grain size have been described in rice, the molecular mechanisms underlying the determination of grain size and shape are still elusive. In this study we report that WIDE AND THICK GRAIN 1 (WTG1) functions as an important factor determining grain size and shape in rice. The wtg1‐1 mutant exhibits wide, thick, short and heavy grains and also shows an increased number of grains per panicle. WTG1 determines grain size and shape mainly by influencing cell expansion. WTG1 encodes an otubain‐like protease, which shares similarity with human OTUB1. Biochemical analyses indicate that WTG1 is a functional deubiquitinating enzyme, and the mutant protein (wtg1‐1) loses this deubiquitinating activity. WTG1 is expressed in developing grains and panicles, and the GFP–WTG1 fusion protein is present in the nucleus and cytoplasm. Overexpression of WTG1 results in narrow, thin, long grains due to narrow and long cells, further supporting the role of WTG1 in determining grain size and shape. Thus, our findings identify the otubain‐like protease WTG1 to be an important factor that determines grain size and shape, suggesting that WTG1 has the potential to improve grain size and shape in rice.