Comp. Biochem. Physiol. Vol. 94B, No. 3, pp. 511-519, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press plc STABILITY OF LIVER NUCLEAR PROTEINS IN INBRED STRAINS OF MICE JAN PALYGA Department of Genetics, Educational University of Kielce, 25-518 Kielce, Rewolucji Pa~dziernikowej 33, Poland (Recewed 21 March 1989) A~tract--1. A remarkable similarity in the gel patterns of liver nuclear proteins between four inbred strains of mice (A.CA, B10.A, CBA and DBA/2) was observed. 2. Only a very few quantitative differences were detected in the protein spot patterns of nucleoplasmic (spot of about 41 kDa) and chromatin (spot of about 37 kDa) non-histone proteins between those strains of mice. 3. Comparison of two-dimensional gel patterns of non-histone proteins from males and females revealed a few sex-linked spots. Nucleoplasmic protein with molecular weight of about 59 kDa and chromatin proteins with molecular weights of approximately 47 and 57 kDa were more abundant in liver nuclei of male mouse. INTRODUCTION MATERIALS AND METHODS The nuclear proteins play a significant role in structure and function of a cell nucleus because they encompass a great variety of structural and functional components such as histones, non-histone chromosomal proteins, nuclear matrix proteins, RNA-associated proteins and various enzymes that are needed for DNA packaging, DNA replication and transcription, RNA processing and regulation of gene expression. It is well known (Chiu and Hnilica, 1977; Bosshard et al., 1982) that non-histone nuclear proteins contain species-specific components. Furthermore, it has been shown (Reinecke and Sch6nmuth, 1973; Tallman et al., 1979; Lubofi et al., 1982; Amero et al., 1983; Fritschi et al., 1985; Patyga et al., 1989) that electrophoretic patterns of various classes of nuclear proteins differ at least quantitatively between distinct genetic groups (breeds, inbred lines, crosses, etc.) within a vertebrate species. Inbred strains of mice exhibited a genetic variation not only in activity of several enzymes (Bulfield et al., 1978) but also in electrophoretic patterns of hair keratins (Barnett and Bird-Stewart, 1985) and mitochondrial proteins (Jungblut and Klose, 1985). Fitch and Atchley (1985) have discovered a high diversity among inbred strains of mice after examina- tion of the genetic variation at 97 loci. It has been suggested (Blank et al., 1986) that genetic differences in inbred strains of mice might be accounted by introgression of chromosomes from multiple mouse species into the Mus domesticus stocks, from which inbred strains derived. This work aimed at comparing the polyacrylamide gel patterns of nucleoplasmic proteins, chromatin non-histone proteins and histones from liver of males and females of four inbred strains of mice to evaluate strain and/or sex differences in their composition. Females and males of inbred strains of mice (A.CA, BI0.A, CBA and DBA/2) at the age of 3 5 months were a gift of Prof. Alina Czarnomska from the Institute of Oncology in Warsaw. Animals were killed by a cervical dislocation and bled. Livers were perfused with a cold solution containing 0.13 M NaCI, 0.005 M KC1 and 0.008 M MgCI2 supplemented with 0.5 mM phenyl methane sulphonyl fluoride (PMSF) and frozen at -20°C. Livers from males and females of a given strain were processed independently. After thawing, two mouse livers of the same strain and sex were put together, minced with a pair of scissors and passed through a tissue press. Liver nuclei were isolated in the presence of Nonidet P-40 as outlined by Busch and Daskal (1977). Tissue pressate was homogenized in a medium containing 0.5% Nonidet P-40, 10 mM Tris-HC1 Loll 7.6), 10 mM NaCI and 1.5 mM MgCI: and centrifuged at low speed. The nuclear pellet was suspended in 0.25 M sucrose in the above buffer without Nonidet P-40 and centrifuged through a cushion of 0.88 M sucrose in the same medium. This procedure was repeated once more. Nuclear proteins were isolated by consecutive extraction of nuclei with (a) 0.075 M NaC1-0.025 M ethylene diamine tetracetic acid (EDTA), pH7.6, (b) 5M urea-0.05 M sodium phosphate, pH 7.6-0.02 M glycine and (c) 0.25 M HC1. All solutions used for isolation of nuclei and nuclear proteins were supplemented with 0.5 mM PMSF. Superna- tants containing non-histone nuclear proteins were precipitated by addition of appropriate volumes of 100% trichloroacetic acid to a final concentration of 10%. Precip- itates were washed with 70% ethanol, absolute ethanol and ethanol-ether (1:1, v/v), resuspended in 0.5 cm 3 5 mM 2- mercaptoethanol and lyophilized. Histones were precipi- tated overnight with 20% trichloroacetic acid and washed twice with acidified acetone and acetone. Histones were solubilized in 0.5 cm 3 5 mM acetic acid and lyophilized. Non-histone nuclear proteins and histories were separated in a 12.5% polyacrylamide slab gel in the presence of sodium dodecyl sulphate (SDS) according to the procedure of Laemmli (1970). Non-histone proteins were additionally separated in a two-dimensional gel similar to that of Orrick et al. (1973). Briefly, protein samples were dissolved in a 511