Comp. Biochem. Physiol. Vol. 10011, No. 3, pp. 627~30, 1991 0305-0491/91 $3.00+ 0.00 Printed in Great Britain © 1991PergamonPress pie OF NON-HISTONE NUCLEAR PROTEINS IN LIVER JAPANESE QUAIL DIVERGENTLY SELECTED FOR RELATIVE SUSCEPTIBILITY OR RESISTANCE TO STARVATION JAN PALYGA,* ANDRZEJ WITKOWSKI,~ANDRZEJ KOWALSKI,* MALGORZATASYLWESTRZAK* and J6ZEF Z~BAt *Department of Genetics, Educational University of Kielce, 25-518 Kielce, Warszawska 33, Poland; and tAgricultural Academy, 20-934 Lublin, Akademicka 13, Poland (Received 3 April 1991) Abstract--l. Nucleoplasmic, 0.35 M NaC1- and 5 M urea-soluble non-histone proteins were isolated from the liver of females of Japanese quail selected for the largest (a susceptible line) and the smallest (a resistant line) decreases in body weight at 12 weeks of age following a 4-day starvation. 2. While the resistant line showed a specific increase in the amount of 40- and 60-kDa non-histones soluble in 0.35 M NaCI, the line sensitive to starvation did not exhibit significant changes in protein composition in comparison with a control line mated at random. 3. After a 4-day starvation, the all genetic groups of birds developed similar quantitative changes among nueleoplasmic proteins. The 0.35 M NaC1- and 5 M urea-soluble proteins were, however, not affected by fasting. INTRODUCTION MATERIALS AND METHODS Non-histone nuclear proteins contain a great variety of components differing in their mol. wts, isoelectric points and biological properties (Klyszejko-Stefanowicz and Hnilica, 1983). These proteins are organized into several nuclear sub- structures with distinct functions in the nucleus. The non-histone proteins encompass structural com- ponents of chromosomes (Adolph, 1984) and nuclear matrix (Pogo and Procyk, 1985), various nuclear enzymes (van Holde, 1989) and factors controlling genetic regulation (Stein et aL, 1983). A body of evidence suggests that at least quantitat- ive differences in some non-histone proteins can be detected between various breeds, strains and inbred lines within an animal species (Lubofi et aL, 1982; Bosshard and Stranzinger, 1983; Fritschi et al., 1985; Palyga, 1989; Jungblut et aL, 1989; Palyga et aL, 1990; Ramagli et al., 1990). It seems that animals with different genotypes which appeared as a result of selection for some inheritable traits and properties might exhibit some alterations in the structure and/or function of their chromatins. The observed shifts in the rate of RNA synthesis in the inbred lines of rats (Amero et al., 1983) might support this assumption. It was shown that avian nuclear proteins could alter under environmental stress (Schlesinger et al., 1989) including starvation (Patyga, 1985, 1987a, b). Since birds can be transiently deprived of food both in their natural environment and, accidentally, in the large breeding farms, this work was aimed at compar- ing the polyacrylamide gel patterns of nucleoplasmic and chromatin non-histone proteins from the liver of Japanese quail divergently selected during several generations for relative susceptibility or resistance to short-term starvation. Japanese quails (Coturnix coturnix) variety Pharaoh, from a flock bred at the Academy of Agriculture in Lublin (Poland) were used. Birds were divergently selected for six generations for body weight reduction following a 4-day deprivation of food (water ad libitum) at 12 weeks of age. By mating the birds with the largest or smallest body weight decreases, two well-separated lines of animals were obtained: one line relatively more sensitive and the other relatively more resistant to starvation. Control birds were bred at random (Witkowski, 1986). Studies were carried out using females 6 months old from the seventh generation. The quails were divided into two groups of 18 birds each. One group was fed ad libitum and the other was starved for 4 days. All animals had free access to water. After completing the experiment, the following subgroups of animals were obtained: S, quails of the line relatively susceptible to body weight reduction after star- vation; R, quails of the line more resistant to body weight decreases after starvation; C, control quails; SS, quails of the susceptible line which had been fasted for 4 days; RS, quails of the resistant line following 4-day food deprivation; CS, control birds after fasting. The birds were killed by decapitation and after opening the abdomen cavity, the livers were immediately perfused with a cold solution of 0.15 M NaC1 and 0.015 M sodium citrate. Liver nuclei were isolated according to the procedure of Bloom and Anderson (1978). Nucleoplasmic proteins were prepared by a double extraction of nuclear pellets with 0.075 M NaCI, 0.025 M ethylene diamine tetra-acetic acid, pH 6.5. The nuclear residue was washed twice with a solution of 0.35 M NaC1 and 0.0015 M sodium citrate, pH 7.0. The resulting chromatin was extracted twice with 5M urea in 0.15 M Tris-HC1 buffer, pH 8.1, containing 0.02 M glycine (Kane et aL, 1982). All the solutions used for liver perfusion, nuclei isolation and protein extraction were supplemented with 0.5 mM phenyl methane sulphonyl fluoride, 1 mM N-ethylrnaleinimide and 0. I mM CdSO4 as protease inhibitors. The supernatants containing the appro- priate protein fractions were saved and combined. After 627