Paul J. Isackson

Isolation and separation of chicken erythrocyte high mobility group non-histone chromatin proteins by chromatography on phosphocellulose

The high mobility group (HMG) proteins are well characterized non-hi&one chromatin proteins that were first isolated from calf thymus in [ l]. The proteins can be extracted from calf thymus chromatin with 0.35 M NaCl, are soluble in 2% trichloroacetic acid, have relatively high mobflities in acid/ureapoly~~l~ide gel electrophoresis, and contain hlgh levels of both acid and basic amino acid residues [2]. Their distinctive physiochemical properties allow the HMG proteins to be readily identified (at least in higher o~a~sms) and have in large part been responsible for the proteins’ having been studied in many laboratories. In chicken erythrocytes, the HMG proteins are HMG-1, HMG-2, HMG-E, HMG-14, and HMG-17 f3,4]. We have shown that HMG-1, HMG-2, and HMG-E (sequence homologs [5], which we refer to as the high molecular weight HMG proteins) all have the distinctive property of binding selectively to single-stranded DNA at roughly physiological ionic strength [6]. Red cell HMG-14 and HMG-17 are associated preferentially with nucleosomes containing transcribed DNA sequences [7]. All of the chicken erythrocyte HMG proteins are thus likely to be the subjects of continued, intensive investigation. The principal large-scale procedures for purification of individual HMG proteins have been developed [8,9]. We report here the use of phosphocellulose chromatography as the first and most important step in an alternate purification method that has distinct advantages over existing procedures.

Comparison of the salt dissociations of high molecular weight HMG non-histone chromatin proteins from double-stranded DNA and from chromatin

The high mobility group (HMG) proteins are wellcharacterized mm-h&tone chromatin proteins first identified [I] in calf thymus. It has proved possible to identify proteins from phylogenetically diverse organisms that are clearly sequence homologs of the calf thymus HMG proteins and that, therefore, can reasonably be included in the HMG class. The HMG proteins fall into two distinct subsets. The first contains proteins with M, 26 000: the calf thymus prototypes, HMG-1 and HMG-2 [ 11; their direct analogs from other higher vertebrates [2,3]; HMG-E (or HMG-2a [4]>, p a rominent non-histone protein in chicken erythrocyte chromatic [2]; and HMG-T from trout testis [5]. We will refer to that group of proteins as the high molecular weight HMG proteins, since the second HMG subset contains prateins of substantially lower molecular weights: the sequence homologous eJf thymus prototypes, HMG 14, NY 11 000 [6], and HMG-17, off& IO 000 [7]; their direct analogs in other higher vertebrates [8,9]; and H-6 from trout testis [lo]. The high molecular weight HMG protein from chicken erythrocytes, calf thymus and cultured rat hepatoma cells possesses preferential a&tity for singie-stranded DNA [I 1,121. That property is poten< tially important in understanding the biological functions of the proteins, particularly in light of the fact that at 0.2 M NaQ (which presumably provides a roughly physiolog~ea~ ionic strength), the proteins appear to bind exclusively to single-stranded DNA [121.

Production of HMG-3 by limited trypsin digestion of purified high-mobility-group nonhistone chromatin proteins.

Three isolated nonhistone proteins (HMG-1, HMG-2 and HMG-E) have been purified from chicken erythrocyte chromatin without exposure to overt denaturing conditions, and subjected to limited proteolysis. When treated with trypsin, the three proteins exhibited similar patterns of degradation, as judged by SDS and acid/urea gel electrophoresis. In particular, the first product, P1 (a relatively stable intermediate in each digestion), was a protein analogous to HMG-3, a principal degradation product in preparations of calf thymus high-mobility-group proteins. At least in the case of HMG-E, the products formed by tryptic attack on P1 are the two individual DNA binding domains of HMG-E. P1 derived from HMG-E and one of the individual DNA binding domains of HMG-E were purified by chromatography on columns containing DNA-cellulose or phosphocellulose. The properties of these two portions of HMG-E are consistent with our recently postulated three-domain structure for HMG-1 and its homologs (Reeck, G.R., Isackson, P.J. and Teller, D.C. (1982) Nature 300, 76-78). Thus, P1 consists of two DNA-binding domains of approximately equal molecular weight covalently linked together. From chromatography on DNA-cellulose columns, it is clear that P1 binds to DNA more tightly than does HMG-E. The highly acidic C-terminal domain of HMG-E (which is removed by trypsin in generating P1) thus counteracts the DNA binding of the two other domains of HMG-E (at least in the proteins interaction with purified DNA).

Two-dimensional gel electrophoresis of nonhistone chromatin proteins with nonequilibrium pH gradient electrophoresis as the first dimension.

Abstract Proteins extracted from chicken erythrocyte chromatin with 0.35 m NaCl are analyzed by two-dimensional gel electrophoresis. The well-defined high mobility group (HMG) proteins are identified on the electrophoretogram. All but one of the HMG proteins as well as a number of other nonhistone proteins in the extract are sufficiently basic that they are observed only if the first dimension is carried out as nonequilibrium pH gradient electrophoresis. Protein fractions obtained by precipitation at several trichloroacetic acid concentrations are examined by two-dimensional gel electrophoresis and the term “HMG protein” discussed in light of the results.

Ionic interactions between proteins in nonequilibrium pH gradient electrophoresis: Histones affect the migration of high mobility group nonhistone chromatin proteins

In two-dimensional gel electrophoresis of the high mobility group (HMG) proteins, it has proved necessary to use nonequilibrium pH gradient electrophoresis (NEPHGE) in the first dimension rather than isoelectric focusing, because of the basic character of most of the HMG proteins [D. Tyrell, P. J. Isackson, and G. R. Reeck (1982) Anal. Biochem. 119, 433-439]. In this paper it is reported that in samples that contain histones, the mobilities of HMG proteins (particularly HMG-1, HMG-2, and HMG-E) are severely distorted in NEPHGE. This presumably results from formation of complexes between histones and HMG proteins through ionic interactions. Analysis of HMG proteins by NEPHGE/sodium dodecyl sulfate-gel electrophoresis is thus precluded in samples containing histones. Our results raise the possibility of similar artifacts occurring in NEPHGE (or isoelectric focusing) analysis of other proteins with regions of high charge density.

Removal of degradation products from calf thymus high mobility group non-histone chromatin proteins by chromatography on immobilized double-stranded DNA

The substantial protease activity in calf thymus chromatin inevitably produces some degradation of high mobility group (HMG) non-histone proteins in NaCl extracts of calf thymus chromatin. We have found that proteins considered to be degradation products can be conveniently and cleanly separated from intact high mobility group proteins 1 and 2 by chromatography on double-stranded DNA-cellulose in 0.2 M NaCl/1 mM Tris-HCl (pH 7.5). Under those conditions, only the presumptive degradation products are retained by the column.