Ernest W. Johns

An improved large scale fractionation of high mobility group non-histone chromatin proteins

1. Methodology is presented for the large scale preparation and fractionation of high mobility group proteins from calf thymus chromatin. The total high mobility group protein from approx. 1 kg calf thymus tissue can be separated into five fractions by CM-Sephadex C25 ion-exchange chromatography. High mobility group proteins 1 and 2 comprise two fo the fractions. From a third fraction two more chromatin proteins, protein 3 and 17, can be isolated by trichloroacetic acid precipitation and CM-cellulose chromatography at pH 5.5. 2. The four proteins thus purified are lysine-rich proteins. Proteins 1 and 2 are additionally characterised by their high contents of acidic amino acids, as described previously (Goodwin, G. H. and Johns, E. W. (1973) Eur. J. Biochem. 40, 215-219). Proteins 3 and 17, having lower contents of acidic amino acids, are basic proteins similar to the histones. All four proteins exhibit single N-terminal amino acids; glycine is the N-terminal group of proteins 1, 2 and 3; protein 17 has a proline N-terminal amino acid. The proteins are not highly phosphorylated nor are they associated with appreciable quantities of nucleic acid.

The presence of high mobility group non-histone chromatin proteins in isolated nucleosomes

Recent models of the structure of chromatin suggest that it is composed of repeating subunits, each subunit or nucleosome consisting of about 200 base pairs of DNA associated with the five histones [ 1,2]. It is thought that 140 base pairs of DNA are associated with an octamer of two each of the four histones H2A, H2B, H3 and H4 [3], whilst Hl is probably bound to the section of DNA linking the subunits [4]. Little is know as regards how the non-histone chromosomal proteins fit into this model and it is not known which, if any, of the non-histone proteins are bound to nucleosomes. Our laboratory is currently studying a particular group of non-histone chromosomal proteins, called the High Mobility Group (HMG) proteins [5], and it was therefore of interest to find out whether these proteins are associated with nucleosomes. The HMG proteins are a group of proteins loosely bound to the chromatin, being dissociated from it with 0.35 M NaCl [6]. They have some properties in common with histones, e.g., having approximately 25% basic amino acids and are extractable with acids [7]. However, they are present in much smaller quantities than the histones, the total HMG protein being about 3% by weight of the DNA in thymus. Three of the HMG proteins, HMGl, HMG2 and HMG17, have been isolated in a pure form [6] and the complete amino acid sequence of protein HMG17 has recently been determined [8]. In this paper we demonstrate that the HMG proteins are present in nucleosomes which have been isolated by gel-filtration chromatography following nuclease digestion of rabbit thymus nuclei.

Structural homology between a mammalian H1° subfraction and avian erythrocyte‐specific histone H5

In 1969 Panyim and Chalkley described a lysinerich histone, now known as Hl”, whose presence seemed to correlate with a lack of DNA synthesis [ 11. These and other data which have emerged since add weight to the idea that Hl’ is a repressor of DNA synthesis [2]. We have been studying Hl” and have found it to be characteristically different from all the other Hl subfractions from the same source and that it can also be separated into subfractions itself [3]. Here we report that homologies exist between the amino acid sequences of one of the subfractions of bovine Hl o and the avian erythrocyte-specific histone H5.

The isolation of the high mobility group non-histone chromosomal protein HMG 14

Chromatin contains a group of non-histone proteins called the High Mobility Group (HMG) proteins [l] . There are four main HMG proteins, HMG 1,2, 14 and 17, in thymus which we have shown to be present in isolated nucleosomes [2]. Three of these proteins, HMG 1,2 and 17, and an HMG protein from trout testis, HMG T, have been isolated in a pure form [3,4,5] . The amino acid sequence of HMG 17 has recently been determined [6]. The fourth thymus HMG protein, HMG 14, is present in chromatin in much smaller quantities than the other three and has been more difficult to isolate in a pure form. In this paper we report the large scale isolation of this protein from pig thymus.

The primary structures of non-histone chromosomal proteins HMG 1 and 2

It is now generally accepted that in all eukaryotes the DNA and the histone proteins are complexed together in a repeating unit called the nucleosome (review [ 11). The nucleosome consists of -200 basepairs of DNA complexed with an octamer of histones H2A, H2B, H3 and H4, and interacts with 140-145 basepairs of DNA to form the core particle of the nucleosome. The fifth histone, Hl , is associated with a variable length of spacer or linker DNA which separates the repeating units. The amino acid sequences of all 5 histones have now been known for a number of years. However, until recently very little sequence information has been available for any of the nonhistone chromosomal proteins involved in the nucleosome structure. We have been studying a particular group of non-histone chromosomal proteins called the HMG proteins (review [2]). The presence of HMG proteins in a variety of organisms and tissues, including avian erythrocytes [3,4], trout testis and trout liver [5,6], wheat and yeast [7] and insects [g] implies a widespread occurrence in eukaryotic nuclei. There are 4 main HMG proteins in thymus, HMG 1,2,14 and 17. All 4 of these proteins have been isolated in a pure form from both pig and calf thymus [9-l I], and have all been shown to be present in isolated nucleosomes [ 121. The primary structures of both HMG 14 and 17 have been determined [ 13 ,141. Because of the quantities of the HMG proteins present in the nucleus

Chapter 15 The Isolation and Purification of the High Mobility Group (HMG) Nonhistone Chromosomal Proteins

Publisher Summary This chapter describes the isolation and purification of the high mobility group (HMG) nonhistone chromosomal proteins. Chromatin contains a group of nonhistone chromosomal proteins that are less firmly bound than the histones and can be extracted from the chromatin. This nonhistone protein fraction is highly heterogenous; it is made up of structural proteins, nuclear enzymes, and small amounts of regulatory proteins. The HMG proteins bind to DNA and histones, and are the chromatin structural proteins, though being present in smaller quantities than the histones they have a more specific function. One of the HMG proteins, HMG2, exhibits multiple forms that could be because of sequence microheterogeneity or postsynthetic modifications such as methylation or acetylation. The chapter describes the large-scale isolation of proteins HMG1, 2, and 17 and also the isolation of the HMG2 subfractions.

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.

The primary structure of the nucleosome-associated chromosomal protein HMG 14.

Chromatin contains a group of non-histone proteins called the high mobility group (HMG) proteins [ 11. There are four main HMG proteins in thymus, HMG 1,2,14 and 17, which have all been shown to be present in isolated nucleosomes [2]. All four of these proteins have been isolated in a pure form from both pig and calf thymus [3---S], and an HMG protein from trout testis, HMG-T, has also been isolated [6]. Because of the quantities of the HMG proteins present in the nucleus (lo’-lo6 molecules of each protein) we feel that the HMG proteins are structural proteins, possibly involved in the higher ordered structure of the chromatin, and not involved in specific gene control. As part of our characterisation of the HMG proteins we are determining the amino acid sequences of the four calf thymus proteins. We have published partial sequences for HMG 1 and 2 [6,7] and the complete amino acid sequence of calf thymus HMG 17 [8]. We now report the complete amino acid sequence of calf thymus HMG 14.

Chapter 11 The Isolation and Purification of Histones

Publisher Summary This chapter describes the methods for fractionating, isolating, and characterizing different histone fraction. The histone fractions F1 and F2B are lysine-rich histones, F3 is an arginine-rich histone, and the fraction F2A is a mixture of two histones components—that is, F2A1 and F2A2. F2Al is another arginine-rich histone and F2A2 is an intermediate type having a molar ratio of lysine to arginine of about one. The chapter discusses the purification of these histone fractions. Although very useful for the purification of fractions prepared on a large scale by other methods, gel exclusion chromatography is not selective enough to separate all five histone fractions during one run. The preparation of the nucleated erythrocyte specific histone F2C is discussed. The techniques for characterization of the products obtained are: total amino acid analysis, N-terminal amino acid analysis, and polyacrylamide gel electrophoresis. The histone fractions prepared and dried are white powders easily soluble in water. They should be stored in a vacuum desiccator over silica gel at room temperature.

Studies on the degradation of high mobility group non-histone chromosomal proteins

During the isolation of high mobility group non-histone proteins from calf thymus chromatin by methods described previously (e.g. Goodwin, G.H., Nicolas, R.H. and Johns, E.W. (1975) Biochim. Biophys, Acta. 405, 280--291) protein degradation occurs resulting in a number of proteins appearing in the chromatin extracts which are not present in high mobility group protein preparations in which proteolysis has been completely inhibited. These extra proteins, formerly numbered high mobility group proteins 3, 5, 6 and 8, are thus probably degradation products of other nuclear proteins, produced during the isolation procedure. From the amino acid analyses, tryptic peptides and N-terminal sequences, it is concluded that high mobility group protein 3 is probably a degradation product of high mobility group protein 1. The amino acid analysis of high mobility group protein 8 is very similar to that of the N-terminal half of histone H1 suggesting that high mobility group protein 8 is a degradation product of this histone.