FETAL AND NEWBORN CALF THYMUS AS A SOURCE OF CHROMATIN PROTEINS - PURIFICATION OF HMG-1 AND HMG-2

The high mobility group (HMG) non-histone chromosomal proteins were first isolated from calf thymus1 but were later found in numerous organs of many vertebrates.2 The proteins can be extracted from calf thymus with 0.35 M NaCl and they are quite soluble in 2% trichloroacetic acid.1 We have shown that members of the HMG-1 family (i.e., HMG-1, HMG-2, and HMG-E) exhibit a preferential affinity for single-stranded DNA at roughly physiological ionic strength.3-5 Members of this family have other intriguing properties (see references 6 and 7 for recent reviews), including the ability to assemble nucleosomes in vitro8. The architecture of the proteins strongly suggests that they are designed to interact simultaneously with histones and with DNA through physically distinct domains.6,9 A special emphasis in our laboratory has been developing procedures for the large-scale isolation of HMG-1 and its homologs without exposure to overt denaturing conditions.6 We have described, for instance, chromatography of 0.35 M extracts of chicken erythrocyte chromatin on phosphocellulose and demonstrated the value of that chromatography in the purification of chicken erythrocyte HMG-1, HMG-2, and HMG-E.10 The importance of avoiding denaturing conditions in isolating members of the HMG-1 family has been emphasized by the demonstration by Cockerill et al.11 that acid treatment procedures apparently irreversible effects on the folding of HMC-1. Calf thymus is an attractive source of nuclei and nuclear components from several standpoints. These include the ease of obtaining thymus glands, the high content (per unit mass) of nuclei in thymus, and the large body of information about calf thymus nuclear components, including amino acid sequence information about HMG-1 and HMG-2.12 Calf thymus has, however, had an over-riding disadvantage. It contains an avid protease that, at neutral pH, rapidly degrades proteins in nuclei, chromatin, or NaCl extracts of them. (See, for example references 13 and 14. We report here that fetal and newborn calf thymus extracts are apparently devoid of such protease activity. We also demonstrate in this paper that Bio-Rex 70 (a cation-exchanger) and the Mono Q support (an anion-exchanger) are both useful supports for chromatographic separation of calf thymus HMG-1 and HMG-2.