I. Quantitative Determination of the Amount of Dna Per Nucleus by Interference Microscopy

Abstract

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment . The values found are slightly low, because, as indicated by biochemical determinations, consistently 5 % to 8 % of DNA is not extracted by DNase under these conditions . The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7 .7 and 15 .6 pg (10 -12 g), respectively .

Dry mass distribution among interphase nuclei of Tradescantia as determined by X-ray microradiography and microinterferometry.

“…Then the total dry mass (M) of the nucleus in the fixed state is given by M= where A is the projected area of the nucleus, x a centuple quantity of specific refractive increment, ~cp the refractive index of the fixed nucleus, and µw and µm the refractive indices of water and the medium used respectively (12) . The value of 0.18 was used for x, because this value has been reported to be applicable to the fixed nuclei (3,11,18) . The determinations of µ~, µw and µm values were carried out by refractometry with an Abbe's refractometer (Erma Optics Co., Tokyo) as referred to the D line of Fraunhofer.…”

Ii. Differences in Adrenal Medulla Nuclear Dna Content Among Rats of Different Strains Following Intermittent Exposure to Cold

“…The experimental groups of rats were kept at +2 -4°C for 15 hr a day and at +20 -22°C for the remaining 9 hr of the day, over a total period of 20 days (300 hr of total exposure to cold) ; the control groups of rats, made up of littermates of the animals belonging to the experimental groups, were kept at room temperature (+20 -22°C) for a period of 20 days. At the end of 300 hr all rats were sacrificed, and the amount of DNA per nucleus of the adrenal medulla cells was determined by means of a microinterferometric technique (8) .…”

“…When the light intensities of the fields are matched again by an adequate compensation with the goniometer analyzer, the measure of the OPD of the object integrated over the area of the measuring field is obtained . The error affecting the total OPD measurements is small if the maximum OPD of any point of the object does not exceed 90°( 12) ; in fact, it can be calculated that the error involved in measuring the optical retardation of the nuclei examined is about f2% (8,12) .…”

“…The amount of DNA per nucleus was calculated by measuring the difference between the total dry mass of the nuclei (as determined with the microinterferometric method) before and after DNase digestion (8) . The adrenal medulla was carefully separated from the cortical tissue (2), homogenized in Barnes, Esnouf, and Stocken's solution (9) with a glass microhomogenizer (Potter-Elvehjem, A .…”

Micro-interferometry of Insect Polyploid Nuclei

“…Micro interferometry procedures for determination of dry mass in polyploid somatic nuclei had been only used in the study of neoplasic cells in mouse tumours (Mellors et al 1955, Mellors 1957) and of isolated diploid and tetraploid nuclei of adult rat liver (Bibbiani et al 1969) and were considered of high precision, when com parisons were made with cytophotometric data for DNA.…”