High-speed autoradiography of 3H-thymidine-labelled nuclei

Summary High-speed autoradiography with stripping film of ³H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20°C, (b) exposure of cell preparations and blanks for various times at either –70°C or +20°C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time 1 h at either temperature and relative humidity 20–30%. A linear relationship between autoradiographic efficiency and exposure time of 1–7 h was found at either temperature, although the efficiency of autoradiographs exposed at –70°C was by approximately 30% higher than that of autoradiographs exposed at +20°C. Background values of autoradiographs dried with a fan and exposed for 1/4–7h at either –70°C or +20°C were 0.6–0.8 grain/100 m². Theoretical calculations and experimental data showed that high-speed autoradiographs are 30–50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.This investigation was supported in part by MR II.1 grant. The technical assistance of Mrs. S. Bie is gratefully acknowledged     

Fluorescent feulgen staining of fungal nuclei

A rapid and alternative method for staining nuclei and chromosomes in fungal cells is reported. The method involves use of fluorescent dyes, specifically, auramin-O and acrinol as Schiff reagents. Micrographs depicting the distribution and division of nuclei in cells of four fungi are presented. These micrographs illustrate the potential of fluorescence microscopy for karyological investigations in fungi.     

A study of DNA depolymerisation during feulgen acid hydrolysis

Summary The binding of Schiff dye molecules after acid hydrolysis (1 M HCl) for varying lengths of time was studied in ascites tumour cells. The amount of dye bound to the tumour cells closely followed the number of aldehyde groups, calculated from the extraction of radioactive nucleotides. This constant dye to aldehyde ratio did not change when the hydrolysis was performed at a lower acid concentration (0.3 M HCl). The conclusion drawn is that Feulgen dye measurements represent, in a constant way, the number of aldehydes on DNA at any given time during hydrolysis. The alteration of the hydrolysis pattern of chromatin fixed in formalin was found to be due to a slower extraction of DNA depolymerisation products, the purine liberation being unaffected. A similar explanation is offered for the extreme pattern obtained from hydrolysis of bull spermatozoa chromatin.     

Quantitative autoradiography of 3H-thymidine-labelled nuclei: Reduction of grain count caused by washing in water

Summary Ehrlich ascites tumour cells and epithelial cells of intestinal crypts were labelled in vitro and in vivo with the pulse of either tritium or carbon-14-thymidine. Fixed cells were washed in either distilled or tap running water for 1/6–24 hr and autoradiographed. Quantitation of autoradiographs showed that the washing of cells labelled with tritiated thymidine caused remarkable diminution of grain count. The cells labelled with carbon-14-thymidine were not affected by washing. The strict control of washing in water of tritium-labelled specimens is recommended.     

Analysis of DNA isolated from intracellular yolk granules in the early chick blastoderm

DNA isolated from intracellular yolk granules of early chick blastoderms was analysed in the electron microscope and in the analytical ultracentrifuge. The yolk DNA molecules were found to be linear and comparatively short, with a buoyant density identical to that of nuclear DNA.     

Histones from chick embryo, chick and chicken liver nuclei

Histones were prepared and purified from chick embryo, chick and chicken liver nuclei. The comparative analysis of these histone preparations, fractionated by polyacrylamide gel electrophoresis, indicates that histone fractions of chick embryo, chick and chicken livers are respectively identical and they comigrate with calf thymus histones.     

A cytochemical kinetic investigation of the feulgen hydrolysis of fixed chromatin

Summary Measurements of the initial slope of the Feulgen hydrolysis curve under various conditions of preparation, temperature, and acid concentration demonstrate that this slope has the kinetic properties of the reaction rate of a simple hydrolytic reaction. This result allows a considerable refinement in both the kinetic analysis of chromatin fractions and the cytophotometric measurement of DNA amounts.     

Calcium-independent adhesion of extra-embryonic endoderm cells from the early chick blastoderm is inhibited by the blastoderm beta-D-galactoside-binding lectin and by beta-galactosidase

Extra-embryonic endoderm cells from gastrulating chick embryos possess Ca2+-dependent and Ca2+-independent adhesive mechanisms. These cells also contain an endogenous beta-D-galactoside-binding lectin and cell surface receptors bearing galactose groups. The endogenous lectin inhibits cellular adhesion. To test whether the adhesive interactions involving lectin and galactose molecules are part of the Ca2+-independent or Ca2+-dependent adhesive mechanism, dissociated cells which were preincubated in beta-galactosidase were allowed to aggregate in the presence and absence of Ca2+ ions. Significant decreases in adhesion were observed in both cases. Cells were also allowed to aggregate in the presence and absence of Ca2+ ions when blastoderm lectin was present in the medium. Adhesion was decreased in both cases. The results suggest that cell surface galactose groups and the beta-D-galactoside-binding lectin are involved in Ca2+-independent adhesion.     

Effects of zinc deficiency on the chick embryo blastoderm

A technique which permits the in vitro study of zinc deficiency in early embryos of Gallus domesticus is described using dithizone as a chelating agent. Zinc deficiency produces specific and constant lesions which are more severe as the embryo is cultivated in more early stages. The most serious alterations affect growth in general and the differentiation of the nervous system and mesoderm.     

Influence of acid concentration and temperature on fixed chromatin during feulgen hydrolysis

Summary Labelled nucleic acid were extracted from fixed, air-dried smears of Ehrlich's ascites tumour cells by fractionated hydrolysis and measured by liquid scintillation. It was found that the rates of RNA and DNA depolymerisation and of DNA depurination depended on temperature in the same way. The DNA extraction patterns retained their form when the temperature was varied. When the hydrolysis was performed in decreasing acid concentrations, however, there was a concomitant change in the form of the depolymerisation pattern. This change affects the amount of aldehyde groups available for dye-binding with the Feulgen method after the optimal hydrolysis time. The alteration in shape of the Feulgen curve is discussed and supposed to be due to an increased interaction between DNA and other macromolecules. It is suggested that this interaction may be useful in detecting differences in chromatin stability between cells which differ in gene activity.     

Studies on the surface of chick blastoderm cells. 3. Calcium binding to ionogenic sites

The effect of increasing calcium concentrations on the electrophoretic mobility of cells obtained from early chick blastoderms at successive developmental stages was studied. Cells had zero mobilities at calcium concentrations in the range of 1 to 2 × 10^?2 M CaCl₂, and became positively charged when calcium concentrations between 2 and 5 × 10^?2 M CaCl₂ were used. Results using trypsin suggest that while some calcium binding sites disappear from the surface ionogenic layer, the majority of them are not removed by this enzyme. The calcium binding capacity did not vary appreciably in the stages studied. Charge reversal induced by calcium is reversible.