Isolation of a high-molecular-weight high-mobility-group-type non-histone protein from hen oviduct.

An organ-specific non-histone protein, with a mol.wt. of 95,000, was isolated from hen oviduct. This protein consists of approximately equal amounts of acidic and basic amino acids and has an isolectric point of 7.4. On the basis of its known characteristics, this protein is similar to the high-mobility-group proteins observed in other tissues.     

Crystallization and preliminary x-ray investigation of ubiquitin, a non-histone chromosomal protein.

Large crystals of bovine thymus ubiquitin, a non-histone chromosomal protein, were grown from polyethylene glycol solutions. The crystals are orthorhombic, space group P2₁2₁2₁, with $\text{a = 50·9}\text{a}\text{?}\text{, b = 42·9}\text{A}\text{?}\text{and}\text{c = 29·0}\text{A}\text{?}$. The asymmetric unit contains one ubiquitin molecule.     

The partial amino acid sequence of a non-histone chromosomal protein

The amino acid sequence of the first thirty nine residues of the nonhistone chromosomal protein HMG-17 has been determined. Results presented here give a molecular weight of 11,000 for the protein. Some interesting sequence homology with the trout specific histone, histone-T, is noted.     

Isolation and characterization of protein A24, a "histone-like" non-histone chromosomal protein.

In earlier studies, the nucleolar levels of protein A24 were found to be markedly decreased in the nucleolar hypertrophy induced by thioacetamide or during liver regeneration (Ballal, N.R., Goldknopf, I.L., Goldberg, D.A., and Busch, H. (1974) Life Sci. 14, 1835-1845; Ballal, N.R., Kang, Y.-J., Olson, M.O.-J., and Busch, H.J. Biol. Chem. 250, 5921-5925). To determine the role of protein A24, methods were developed for its isolation in highly purified form. Milligram quantities of highly purified protein A24 were isolated from the 0.4 N H2SO4-soluble proteins of calf thymus chromatin by exclusion chromatography on Sephadex G-100, followed by preparative polyacrylamide gel electrophoresis. Protein A24 was highly purified as shown by its migration as a single spot on two-dimensional polyacrylamide gel electrophoresis, its single NH2-terminal amino acid, methionine, and the production of approximately 50 peptides by tryptic digestion. Like histones 2A, 2B, 3, and 4. A24 was extractable from chromatin with 0.4 N H2SO4 or 3 M NaCl/7 M urea, but unlike most non-histone proteins or histone 1, protein A24 was not extracted with 0.35 M NaCl, 0.5 M HClO4, or 0.6 M NaCl. Protein A24 was present in only 1.9% of the total amount of histones 2A, 2B, 3 and 4; its molecular weight is 27,000.     

Conformational studies of two non-histone chromosomal proteins and their interactions with DNA.

The conformational properties of two non-histone chromosomal proteins (high-mobility-group proteins 1 and 2) have been studied by spectroscopic methods. The interaction of high-mobility-group protein 1 with DNA has also been studied. 1. Circular dichroism results indicate that in the presence of salt both proteins are 40-50% helical between pH 1 and 9. Above pH 9 denaturation takes place. In the absence of salt the proteins denature below pH 4. 2. Nuclear magnetic resonance spectra show the presence of ring-current shifted peaks and perturbed aromatic resonances, demonstrating that the helix formation is accompanied by specific tertiary folding. 3. Nuclear magnetic resonance spectra of compelxes between high mobility group protein 1 and DNA demonstrate that a low ionic strength a portion of the molecule rich in lysine and containing all the aromatic residues is bound to DNA, whilst a more acidic region of the chain remains free from the DNA.     

The isolation and partial sequence of peptides produced by cyanogen bromide cleavage of calf thymus non-histone chromosomal high-mobility-group protein 2. Sequence homology with non-histone chromosomal high-mobility-group protein 1.

Peptides produced by CNBr cleavage of non-histone chromosomal protein HMG 2 (CNBr peptides) were isolated and characterized, and their partial sequences were determined. The present sequence data account for over half of the sequence of the protein HMG (high-mobility-group) 2 molecule, and, together with previously published results, provide interesting information on the charge distribution within the molecule. Comparison of the CNBr-peptide-sequence data for protein HMG 2 with the previously published data on the CNBr peptides from protein HMG 1 reveals extensive sequence homology between the two proteins. Detailed evidence for the amino acid-sequence data has been deposited as Supplementary Publication SUP 50095 (6 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.     

Hepatocyte chromosomal non-histone proteins in developing rats.

Rat hepatocytes taken a different stages of the perinatal period were partially purified. On sodium dodecylsulphate acrylamide electrophoresis chromosomal non-histone proteins showed important variations in complexity during development. Chromosomal phosvitin kinase strongly increased during the last days of fetal life; it strongly decreased just after birth and increased again for a short time, while the cytosol phosvitin kinase increased more significantly after birth. Chromosomal non-histone proteins prepared at varoius stages were incubated with [gamma-32P]ATP and resolved on polyacrylamide gel. The incorporation was very low in sample taken at the 15th say of the fetal life. A dramatic increase was observed at the 17th day. This incorporation strongly decreased in the samples taken thereafter and it was negligible in proteins from adult rats. The variations in protein kinase and in 32P incorporation into non-histone proteins were correlated with the pattern of appearance of enzymes in this period of life, with cell growth and with the hormone-induced maturation.     

Nuclear protein phosphokinase activities and phosphorylation of chromosomal proteins in embryonic and adult muscles of the chick

Summary Nuclei isolated from chick embryonic (11-days old) and adult muscles contain 25% and 15%, respectively, of the total cellular protein phosphokinase activity. The specific activity, on a DNA basis, of the adult enzyme is approximaely 4 times lower than that of the nuclei from 11-day-embryos. Nuclear extracts of either stage of development give rise on DEAE cellulose chromatography to two main protein kinase activity peaks, which elute at 20mm and 300mm phosphate buffer, pH 7 (protein kinases A and C, respectively). Nuclei of adult muscles contain 10 fold and 3 fold less protein kinases A and C, respectively, than embryonic nuclei. The properties (substrate specificity, effect on activity of NaCl and phosphate, cyclic AMP activability, molecular weight) of protein kinases A and C are different, but those of embryonic and adult protein kinase A or C are indistinguishable, indicating that the transition from nuclei with an active mitosis (embryonic) to a resting state (adult) is accompanied by a quantitative, but not a qualitative, change in the protein kinase pattern.Phosphorylation with [-³²P] ATP of intact nuclei arising from 11 day and 20 day embryos results in the labelling of histone and nonhistone chromosomal proteins, their specific radioactivity being 2 and 3 times lower, respectively, in the older nuclei than in those from 11 day embryos.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.     

Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei

The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complex-lamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. 5-day-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear 'activity'.     

Isolation of the mature subunit of delta-aminolaevulinate synthase from embryonic chick liver.

We presented evidence indicating that the established procedure for purifying delta-aminolaevulinate (ALA) synthase from embryonic-chick liver yielded an enzyme with a partially degraded subunit of molecular weight 51000 [Ades & Harpe (1981) J. Biol. Chem. 256, 9329-9333]. We now report the purification from livers of porphyric embryos of a preparation of ALA synthase which consisted primarily of a 63000-Da polypeptide and a component migrating as a smear of polypeptides with a minimum molecular weight of 52 000. Neither component could be recovered from liver mitochondria of normal embryos, where the amounts of ALA synthase were relatively low. The 52 000-Da component had been established to be the partially degraded subunit of the enzyme. Peptide-mapping analyses indicated that the 63 000- and the 52 000-Da components possessed significant structural homologies, and it was concluded that the 63 000-Da polypeptide represented the mature subunit of ALA synthase.     

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.     

Detection of monoclonal antibody to high-mobility-group protein 17 from chick oviduct.

Total chromosomal HMG (high-mobility-group) proteins have been isolated from oestrogen-stimulated chick oviduct. The antibodies against these proteins were induced in mice and subsequently their spleen cells were fused with myeloma cells to form hybridomas. A highly purified HMG protein, 17, was used to select for the hybridomas that produce antibody against HMG protein 17. The hybridomas were cultured and injected into mice to produce ascites. The antibody against HMG protein 17 in the IgG (immunoglobulin G) fraction of the ascites fluid was obtained by Protein A-Sepharose column chromatography. We have devised a solid-phase radioimmunoassay and enzyme-linked serological assay for the detection and characterization of this antibody directed against HMG protein 17. This anti-(HMG protein 17) IgG interacted only with HMG protein 17, but not with other chromosomal proteins, e.g. histone H1, "95K protein' (a chick oviduct-specific chromosomal protein) and HMG proteins 1, 2 and 14. The monospecific nature of this anti-(HMG protein 17) IgG fraction is confirmed.     

Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.