Molecular Biology Reports 25: 237–244, 1998. © 1998 Kluwer Academic Publishers. Printed in The Netherlands. 237 Effects of polyamines on higher-order folding of in situ chromatin Laura Vergani, Giancarlo Mascetti & Claudio Nicolini Institute of Biophysics, School of Medicine, University of Genoa, Corso Europa 30, 16132 GENOVA, Italy (Phone 39-10-3538404; Fax: 39-10-3538346; E-mail: vergani@ibf.unige.it) Accepted 22 May 1998 Abstract Modifications of the higher-order chromatin structure induced by polyamines have been quantitatively investigated in situ through a non-invasive biophysical approach using Differential Scanning Calorimetry and Quantitative Fluorescence Microscopy. Calorimetric and intensitometric profiles have been acquired for samples of native thymocytes, alternatively suspended in buffers, with or without natural polyamines (spermine and spermidine). The results here reported show that the structure and distribution of nuclear chromatin in situ considerably change upon the ionic composition of the environment. A quantitative analysis of this data and a comparison with previous results obtained from isolated chromatin fibers was carried out. Finally, an inverse relationship between chromatin condensation and nuclear volume was observed. Introduction Inside the interphase nucleus the eukaryotic chro- matin exhibits different levels of condensation which have been roughly correlated with the transcriptional activity. It has been proved that the mechanism of chromatin folding is essentially electrostatic [1]: iso- lated chromatin fibers are in fact very sensitive to ionic conditions and this sensitivity increases by increasing the level of chromatin condensation [2–4]. It has been shown that the phenomena of folding/unfolding be- tween nucleofilament (diameter of 10 nm) and fibers with higher folding (diameter of 30 nm or more) can also occur with few changes of ionic conditions. The way in which polyvalent cations significantly influ- ence the chromatin folding [5, 6] is in accordance with the predictions of the polyelectrolytes theory. This the- ory was introduced some time ago [7, 8] to quantita- tively describe the binding of cations with different va- lencies to a DNA double helix that in this way reduces its negative charge density. The binding of cations with a higher valency to DNA is favoured with respect to monovalent ones because less of them are required to obtain similar charge neutralization. In fact when polyvalent cations (Mg 2+ , spermidine 3+ , spermine 4+ or histones) are added to a DNA solution containing NaCl they displace Na + ions from the DNA to the bulk solution; upon neutralization the DNA double helix becomes more flexible and it can easily bend [9, 10]. In order to confirm these predictions, numerous stud- ies have been performed on isolated chromatin fibers using many biophysical and biochemical techniques such as small angle X-ray scattering [11–16], electron microscopy [17–20], dichroism [2–23], sedimentation velocity [24–25], viscosity [26], atomic force mi- croscopy [27], electrophoresis [28], calorimetry [29, 30] etc. The drawback to these studies is that during isolation the compact fiber morphology is known to be lost (31). Several authors in fact observed signifi- cant differences when chromatin is studied inside the nucleus or after isolation [32, 33]. For this reason, in spite of the common difficulties in carrying out a quantitative analysis of chromatin di- rectly inside the nucleus, it is very important to verify the effects of polycations on chromatin in situ [34]: this is the aim of our work. To this end Quantitative Fluorescence Microscopy is one of the most potent techniques for studying the nuclear chromatin in situ. It has been proved that fluorescence intensity of nuclei stained with DNA- selective fluorochromes is related to both the DNA content and DNA-chromatin structure. The DNA ac- cessibility is in fact strictly dependent on its con- densation [35]. Therefore this technique allows us