Differential binding of chromosomal proteins HMG1 and HMG2 to superhelical DNA

The binding of chromosomal proteins HMG1 and HMG2 to various DNA structures was examined by a nitrocellulose filter binding assay using a 32P labelled supercoiled plasmid. Binding assays and competition experiments indicated that HMG2 has a higher affinity than HMG1 for supercoiled DNA. Studies at various ionic strengths and pH values reveal differences in the interaction of the two proteins with DNA. The results suggest that HMG1 and HMG2 are involved in distinguishable cellular functions.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

On the presence of the chromosomal proteins HMG I and HMG Y in rat organs

Using antiserum raised against HMG I, we have shown that HMG I and HMG Y are present in perchloric acid extracts of kidney, lung, heart, brain, liver and intestine in the rat, suggesting that the expression of these proteins may not be dependent upon proliferative activity. The results also show that the ratio between HMG I and HMG Y varies between different organs.     

A method for silver staining HMG chromosomal proteins in polyacrylamide electrophoretic gels

A method is presented for sensitive staining of the HMG14 and 17 proteins in polyacrylamide gels pre-stained with Coomassie Blue R250. The procedure involves binding negatively and positively charged polycyclic aromatic compounds to the proteins followed by staining with silver using the method of Wray et al. (1981).     

Occurrence of five different chromosomal HMG1 proteins in various maize tissues

The nuclear HMG1 proteins of higher plants are small non-histone proteins that have DNA-bending activity and are considered architectural factors in chromatin. The occurrence of the chromosomal HMG1 proteins, HMGa, HMGc1/2 and HMGd, in various maize tissues was analyzed, and in the course of these studies a novel HMG1 protein, now termed HMGe, was identified. Purification and characterization of HMGe (M_r 13 655) and cloning of the corresponding cDNA revealed that it displays only moderate similarity to other members of the plant HMG1 protein family. The five maize HMG1 proteins could be detected in kernels, leaves, roots and suspension culture cells, indicating that these proteins can be expressed simultaneously and occur relatively ubiquitously. However, the various HMG1 proteins are present in significantly different quantities with HMGa and HMGc1/2 being the most abundant HMG1 proteins in all tissues tested. Furthermore, the relative amounts of the various HMG1 proteins differ among the tissues examined. The HMG1 proteins were found to be relatively stable proteins in vivo, with HMGc1/2, HMGd and HMGe having a half-life of ca. 50 h in cultured cells, while the half-life of the HMGa protein is ca. 65 h. Collectively, these findings are compatible with the concept that the different plant HMG1 proteins might act as general architectural proteins in concert with site-specific factors in the assembly of certain nucleoprotein structures involved in various biological processes.     

Antibodies against chromosomal HMG proteins stain the cytoplasm of mammalian cells.

Antibodies specific to protein HMG-1 were purified by affinity chromatography on Sepharose columns to which HMG-1 was covalently bound. Immunofluorescence studies with these antibodies reveal that HMG-1 or components which immunologically cross-react with HMG-1 are present in the cytoplasm of Chinese hamster V-79, rat liver TR-12 and bovine trachea EBTr-NBL-4 cells. At selected antibody concentrations, the fluorescence present in the cytoplasm is more intense than that observed in the nucleus. The presence of HMG-1 protein in the cytoplasm of rat liver cells was verified by direct examination of the protein content of selected cytoplasmic fractions. A protein with electrophoretic mobility identical to HMG-1 was detected by electrophoresis on polyacrylamide gels containing either sodium dodecylsulfate or urea. Furthermore, the cytoplasmic extracts yielded a positive complement fixation with anti-HMG-1, while no reaction was obtained with control anti-H1 sera. We suggest that HMG protins, rather than functioning in the nucleus alone, are important structural elements of the entire cell.     

Hierarchy of binding sites for chromosomal proteins HMG 1 and 2 in supercoiled deoxyribonucleic acid

The interaction of chromosomal proteins HMG 1 and 2 with various DNA structures has been examined with plasmid pPst-0.9, which contains DNA sequences that can form the Z-DNA conformation and palindromic sequences that can form cruciform structures. Direct binding and competition experiments with 32P-labeled plasmid indicated that proteins HMG 1 and 2 preferentially bind to supercoiled form I DNA as compared to double-stranded linear DNA. The preferential binding to form I is due to the presence of single-stranded regions in this DNA. The binding of HMG 1 and 2 to the form I plasmid results in inhibition of S1 nuclease digestion in a selective manner. The B-Z junction is preferentially protected as compared to the cruciform, which in turn is more protected than other minor S1-sensitive structures present in pPst-0.9. Our results indicate that the binding of HMG 1 and 2 proteins to DNA is not random in that HMG 1 and 2 can distinguish between various S1 nuclease sensitive sites in the plasmid. The existence of a hierarchy of DNA binding sites for these proteins suggests that they can selectively affect the structure of distinct regions in the genome.     

The isolation, characterization and partial sequences of the chicken erythrocyte non-histone chromosomal proteins HMG14 and HMG17. Comparison with the homologous calf thymus proteins.

Non-histone chromosomal proteins HMG14 and HMG17 were isolated from chicken erythrocyte nuclei. The proteins were characterized by amino acid analysis and by N-terminal sequence analyses. Comparison with the corresponding data for the calf thymus proteins shows that 11% of the residues in HMG14 protein and 5% of the residues in HMG17 protein differ between the two species. Proteins HMG14 and HMG17 therefore do not appear to exhibit the evolutionary stability shown by the nucleosome core histones. Detailed evidence for the amino acid sequence data has been deposited as Supplementary Publication SUP 50101 (4 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. 4. (1978) 169, 5.     

Conformation and domain structure of the non-histone chromosomal proteins HMG 1 and 2. Domain interactions

The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367-374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A + B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil----helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57--C-terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.     

Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells

The low-molecular-mass high-mobility-group (HMG) proteins from young rat thymus nuclei were fractionated by high-performance liquid chromatography. Two proteins analogous to calf HMG14 and HMG17 were found together with a third major component HMGI similar to that found in HeLa cells [Lund et al. (1983) FEBS Lett. 152, 163-167]. HMGI has as amino acid composition similar to but distinct from HMG14 and HMG17. The three proteins form a family of proteins with HMG14 having an amino acid composition intermediate between HMG17 and HMGI. HMGI is present in proliferating fibroblasts and embryos but is present in very low levels in rat liver, a non-dividing tissue, supporting the notion that HMGI is required for proliferating cells. Fibroblasts transformed with avian sarcoma virus have high levels of HMGI and an additional band HMGI' but the presence of HMGI and HMGI' is not dependent on a functional src gene product.     

Conformation and domain structure of the non-histone chromosomal proteins, HMG 1 and 2. Isolation of two folded fragments from HMG 1 and 2

Proteins HMG 1 and 2 have been digested with trypsin and two major products, stable to further digestion between 8 min and 2 h, have been purified (peptides A and B). Peptide B from HMG 1 has been identified as residues 12-75 and peptide A as residues 94/96-169 by amino acid analyses and Edman degradations. Peptide B spontaneously folds with the formation of 51% helix and exhibits the majority of the perturbed NMR resonances characteristic of folded intact HMG 1. Peptide B is stably folded in the presence of 150 mM NaCl between pH 3 and 10, like intact HMG 1. Peptide A forms 30% alpha-helix and also exhibits tertiary folding but is denatured by pH 10. The 11 N-terminal residues removed by trypsin contain both sites of post-synthetic acetylation (residues 2 and 11), a situation very similar to that found with core histones. It is proposed that HMG 1 and 2 consist of four structural domains, viz: (a) residues 1-11, (b) residues 12 to approximately 75, (c) residues 94-169 and (d) the very acidic region beyond residue 169. The instability of peptide A may mean that it is not a truly independent domain. No structural similarities to histone H1 are therefore observed in HMG 1 and 2.     

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.     

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.