High-mobility Protein Research Articles

DPP4 Inhibition in Human Kidney Proximal Tubular Cells - Renoprotection in Diabetic Nephropathy?

Novel diabetic drugs that target the incretin system e.g dipeptidyl peptidase 4 (DPP4) inhibitors have pleiotropic effects other than inhibiting the cleavage of glucagon like peptide 1 (GLP-1). As DPP4 is located at the brush border of the kidney proximal tubular cells (PTC), we hypothesise that it is involved in the processing of luminal peptides influencing the kidney tubulointerstitium in diabetes. Using HK2 cells (human kidney proximal tubular cell line) in order to study the GLP-1 independent effects of DPP4 inhibition on the PTC cells were exposed to high glucose conditions, high mobility protein 1(HMGB1) and meprin beta. Relevant markers like DPP4, fibronectin, collagen IV, transforming growth factor beta (TGFβ), activator protein 1(AP-1) and nuclear factor-kappa B (NF-κB ) were measured. We show that linagliptin (DPP4 inhibitor) interferes with the activation of TGFβ and downstream reduction in fibronectin but not collagen IV expression. Linagliptin reduced HMGB1 but not high glucose induced NF-κB binding. Linagliptin also reduced high glucose induced AP-1 binding. Meprin beta peptides induce AP-1 binding and this was unchanged with linagliptin. Our data provides new insight into the GLP-1 independent role of DPP4 inhibition in kidney PTC exposed to stimuli relevant in diabetic renal complications. Identifying the functionally relevant renal substrates of DPP4 will help us understand and anticipate long term effects on the kidney in patients with diabetes.

Expressions and clinical significance of high mobility group box-1 mRNA and protein in laryngeal squamous cell carcinoma tissues and serum

To investigate the mRNA and protein expressions of high mobility group box-1 (HMGB1) in the tumor tissues and sera of patients with laryngeal squamous cell carcinoma (LSCC) and their clinical significance. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to detected the expressions of HMGB1 mRNA and protein in the tumors and adjacent normal epithelial tissues in 30 patients with LSCC. Serum HMGB1 protein levels in the patients with LSCC and in 10 healthy volunteers were detected with enzyme-linked immunosorbent adsorption experiment (ELISA). RT-PCR demonstrated that the mean relative mRNA expression levels of HMGB1 (HMGB1/GAPDH) in LSCC tissues and in adjacent normal epithelial tissues were 1.25 ± 0.12 and 0.32 ± 0.04, respectively (t = 40.27, P < 0.05). Western blot revealed that the mean relative protein expression levels of HMGB1 (HMGB1/β-actin) were 1.29 ± 0.10 and 0.34 ± 0.03 (t = 49.84, P < 0.05), respectively. Both mRNA and protein expression levels of HMGB1 were associated with T stage, clinical stage, lymph node metastasis status and smoking (all P < 0.05), but no significant correlation with age, alcohol consumption and primary tumor grade and location (all P > 0.05). Mean serum HMGB1 protein levels in patients with LSCC and healthy volunteers were (24.80 ± 14.08) ng/ml and (23.58 ± 14.69) ng/ml (t = 0.37, P > 0.05). Both mRNA and protein expressions of HMGB1 were obviously elevated in LSCC, which were associated closely with T stage, clinical stage and lymph node metastasis.

Expression of perforin–granzyme pathway genes in the bursa of infectious bursal disease virus-infected chickens

Infectious bursal disease (IBD) is an economically important immunosuppressive disease of chickens. The IBD virus (IBDV) actively replicates in B cells and causes severe bursal damage. Generally, T cells are refractory to infection with IBDV but are known to promote virus clearance. However, the mechanisms of T cell mediated viral clearance are not well understood. In this study, we evaluated the molecular mechanisms of cytotoxic T cell responses in the pathogenesis of IBD in chickens. Infection of chickens with IBDV was accompanied by the infiltration of CD4 + and CD8 + T cells in the bursa. There was an upregulation in the gene expression of important cytolytic molecules; perforin (PFN), granzyme-A (Gzm-A), DNA repair and apoptotic proteins; high mobility proteins group (HMG) and poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF) whereas expression of NK (natural killer) lysin was downregulated. Importantly, PFN producing CD4 + and CD8 + T cells were also detected in the bursa of IBDV-infected chickens by immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was also strongly upregulated, suggesting the activation of T cells. The findings of this study highlight the mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the clearance of virus-infected cells.

HMGA proteins regulate the expression of FGF2 in uterine fibroids

In human fibroids genes encoding the high-mobility proteins containing the 'AT-hook' DNA-binding motif (HMGA) are frequently affected by non-random chromosomal rearrangements. Thus, the different proteins and their derivatives resulting from these genomic rearrangements can be assumed to be involved in the genesis of these tumors by activation of largely identical downstream pathways. Constructs encoding HMGA proteins and their relevant derivatives were overexpressed in human myometrial cells, and RNA isolated from these cells was hybridized to filter arrays. Four genes were either up- or down-regulated at least 2-fold after overexpression of either of the HMGA genes and their derivatives. FGF2 (fibroblast growth factor 2) was one of these genes, and we were then able to show by microarray analyses that tumors with rearrangements of the HMGA2 locus (n = 8) expressed significantly higher levels of FGF2 than those with an apparently normal karyotype (n = 47). Accordingly, by quantitative real-time PCR uterine leiomyomas with rearrangements of the HMGA2 locus were found to express significantly higher levels of FGF2 than those with an apparently normal karyotype with a linear relationship between the expression of FGF2 and the level of HMGA2 overexpression as well as the tumor size. The results of western blot analyses confirmed these findings. Moreover, stimulation of myometrial tissue by FGF1, a strong inducer of HMGA2, leads to an increase of HMGA2 as well as FGF2 expression. In conclusion, the results contribute to the understanding of the association between the overexpression of HMGA proteins, the regulation of FGF2 expression and the size of fibroids.

The Interaction of High Mobility Group Proteins with the p160 Coactivator SRC-1; a Role in Endocrine Resistance.

Abstract Breast cancer affects one woman in ten in the western world and despite the phenomenal advances in recent years, the mortality rate still remains at around 35%. Resistance to current endocrine therapies, including tamoxifen and aromatase inhibitors, is common with recurrence of disease in 30-40% of patients. The estrogen receptor (ER) coactivator protein SRC-1 plays an important role in this resistance. We examined SRC-1 protein expression in a large cohort of breast cancer patients (n=560) and found expression to be a strong predictor of disease recurrence in patients treated with adjuvant tamoxifen (hazard ratio: 5.032, p&amp;lt;0.0001). Identification of SRC-1 interacting proteins will provide key insights into the role of SRC-1 in the development of tumour recurrence. We employed a mass spectrometry-based screen to identify proteins which could differentially interact with SRC-1 in endocrine resistant compared with endocrine sensitive breast cancer cells. Selected SRC-1 interacting proteins relevant to endocrine resistance include the high mobility proteins HMGB1 and HMGB2, which have previously been reported to enhance binding of the estrogen receptor to its response elements in target gene promoters. Immunoprecipitation assays confirmed increased interactions between HMGB2 and SRC-1 following treatment of the endocrine resistant cell line LY2 with tamoxifen and estrogen. Steroid treatment also increased trafficking of the coactivator and the SRC-1 interacting protein to the nucleus of tumour epithelial cells. Treatment enhanced recruitment of SRC-1 to the promoter of the oncogene c-Myc and enhanced target gene expression. HMGB2 expression in the breast cancer patient TMA correlated with SRC-1 and poor disease-free survival. These findings would indicate an important role for HMGB2 in the development of endocrine resistance in breast cancer patients. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5138.

HMGA1a Protein Unfolds or Refolds Synthetic DNA–Chromophore Hybrid Polymers: A Chaperone‐Like Behavior

High group mobility protein, HMGA1a, was found to play a chaperone-like role in the folding or unfolding of hybrid polymers that contained well-defined synthetic chromophores and DNA sequences. The synthetic and biological hybrid polymers folded into hydrophobic chromophoric nanostructures in water, but existed as partially unfolded configurations in pH or salt buffers. The presence of HMGA1a induced unfolding of the hybrid DNA-chromophore polymer in pure water, whereas the protein promoted refolding of the same polymer in various pH or salt buffers. The origin of the chaperone-like properties probably comes from the ability of HMGA1a to reversibly bind both synthetic chromophores and single stranded DNA. The unfolding mechanisms and the binding stoichiometry of protein-hybrid polymers depended on the sequence of the synthetic polymers.

Fetal Hemoglobin in Sickle Cell Anemia: Associations with Single Nucleotide Polymorphisms in Quantitative Trait Loci on Chromsomes 8q12 and Xp22.

Fetal hemoglobin (HbF) inhibits the polymerization of sickle hemoglobin modulating some of the subphenotypes of sickle cell anemia. HbF concentrations vary considerably among patients and this variation is regulated as a multigenic trait; some regulatory regions previously identified are linked to the β-globin gene-like cluster and are also within quantitative trait loci (QTL) on chromosomes 6q, 8q and Xp. We genotyped panels of haplotype tagging (ht)SNPs in the β-globin gene-like cluster and covering the 8q and Xp QTL in two independent patient samples and applied two independent analytical methods to study their association with HbF levels. The first sample included 327 patients (135 htSNPs) and the second sample had 987 patients (102 htSNPs). Genotyping was done using a custom 384 multiplex design and the Illumina platform. Single SNP association was investigated by multiple linear regression analysis with simultaneous adjustment for age, sex and the co-existence of α thalassemia; we performed a permutation procedure to correct for multiple testing. The nonlinear regression Random Forest method was used for a joint analysis of covariates and all SNPs. In the smaller patient sample, 7 SNPs in TOX (thymus high mobility protein; 8q12.1) gene showed significant association with HbF (p-value <0.05), but none passed the multiple test correction. Another 11 SNPs in TOX also showed significant association in the larger patient sample and SNP rs7817609 passed the multiple test correction. This SNP in TOX was significantly associated with HbF under codominant and dominant models (empirical p-values 0.0154 and 0.0067) with the C allele associated with a high level of HbF expression; the level of HbF in subjects with CC and CG genotypes was on average 25 percent higher than that in subjects with GG genotype. Three SNPs, including the Xmn I, −158 C T SNP 5′ to HBG2 within the β-globin gene-like cluster, also showed association with HbF. This SNP was the 2nd most important SNP in predicting HbF expression using Random Forest analysis indicating that it might interact non-linearly with other regulatory factors. Joint analysis of all SNPs and covariates revealed that the most important variables for predicting HbF matched most of the SNPs identified by single SNP association studies in both data sets. Random Forest analysis also identified SNPs in EGFL6, GPM6B and FIGF that have a strong effect on predicting HbF level and that lie within the Xp22.2–p22.3 QTL. This suggests that genes within the Xp QTL might be involved in interaction with other genes to regulate the expression of HbF. TOX belongs to one of the high mobility group (HMG) box protein families, which contains proteins with a single HMG box that bind with high sequence specificity to variants of the DNA sequence (A/T)(A/T)CAAAG, inducing a sharp bend in DNA, altering local chromatin structure and modulating the formation of multi-protein regulatory complexes. Within 50 kb 5′ and 40 kb 3′ to the HBG2 there were 50 matches to this binding sequence. TOX might bind to the β-globin gene cluster forming multi-protein regulatory complexes with other proteins to regulate the expression of HbF. We have identified new candidate genes, or linkage disequilibrium with candidate genes, that might play a role in HbF regulation, but further genetic and biological studies are required for validation.

Detection of Transgenic and Endogenous Plant DNA Fragments in the Blood, Tissues, and Digesta of Broilers

The aim was to determine the fate of transgenic and endogenous plant DNA fragments in the blood, tissues, and digesta of broilers. Male broiler chicks (n = 24) were allocated at 1 day old to each of four treatment diets designated T1-T4. T1 and T2 contained the near isogenic nongenetically modified (GM) maize grain, whereas T3 and T4 contained GM maize grain [cry1a(b) gene]; T1 and T3 also contained the near isogenic non-GM soybean meal, whereas T2 and T4 contained GM soybean meal (cp4epsps gene). Four days prior to slaughter at 39-42 days old, 50% of the broilers on T2-T4 had the source(s) of GM ingredients replaced by their non-GM counterparts. Detection of specific DNA sequences in feed, tissue, and digesta samples was completed by polymerase chain reaction analysis. Seven primer pairs were used to amplify fragments ( approximately 200 bp) from single copy genes (maize high mobility protein, soya lectin, and transgenes in the GM feeds) and multicopy genes (poultry mitochondrial cytochrome b, maize, and soya rubisco). There was no effect of treatment on the measured growth performance parameters. Except for a single detection of lectin (nontransgenic single copy gene; unsubstantiated) in the extracted DNA from one bursa tissue sample, there was no positive detection of any endogenous or transgenic single copy genes in either blood or tissue DNA samples. However, the multicopy rubisco gene was detected in a proportion of samples from all tissue types (23% of total across all tissues studied) and in low numbers in blood. Feed-derived DNA was found to survive complete degradation up to the large intestine. Transgenic DNA was detected in gizzard digesta but not in intestinal digesta 96 h after the last feeding of treatment diets containing a source of GM maize and/or soybean meal.

Surfactant-based methods for prevention of protein adsorption in capillary electrophoresis.

Surfactants such as didodecyldimethyl ammonium bromide (DDAB) and 1,2-dilauroyl-sn-phosphatidylcholine (DLPC) form bilayers at the walls of bare silica capillaries. Once formed, these bilayers are stable in the absence of surfactant in the buffer. DDAB provides a cationic bilayer coating which yields a strong reversed EOF and is effective for separation of cationic proteins. DLPC provides a zwitterionic bilayer coating which is effective for both cationic and anionic proteins. The electroosmotic flow (EOF) is strongly suppressed in DLPC-coated capillaries, thus low mobility proteins are slow to elute, and so the coating is favored for separation of high mobility proteins.

Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows.

The objective was to determine the presence or absence of transgenic and endogenous plant DNA in ruminal fluid, duodenal digesta, milk, blood, and feces, and if found, to determine fragment size. Six multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas received a total mixed ration. There were two treatments (T). In T1, the concentrate contained genetically modified (GM) soybean meal (cp4epsps gene) and GM corn grain (cry1a[b] gene), whereas T2 contained the near isogenic non-GM counterparts. Polymerase chain reaction analysis was used to determine the presence or absence of DNA sequences. Primers were selected to amplify small fragments from single-copy genes (soy lectin and corn high-mobility protein and cp4epsps and cry1a[b] genes from the GM crops) and multicopy genes (bovine mitochondrial cytochrome b and rubisco). Single-copy genes were only detected in the solid phase of rumen and duodenal digesta. In contrast, fragments of the rubisco gene were detected in the majority of samples analyzed in both the liquid and solid phases of ruminal and duodenal digesta, milk, and feces, but rarely in blood. The size of the rubisco gene fragments detected decreased from 1176 bp in ruminal and duodenal digesta to 351 bp in fecal samples.

High mobility group protein HMGA1 expression in breast cancer reveals a positive correlation with tumour grade.

Members of the HMGA protein (high mobility group protein A) family act as master switches of the chromatin structure by bending DNA and thus modulating the formation of transcription factor complexes of a number of target genes. Accordingly, HMGA proteins have been shown to be associated with the development and/or progression of a variety of benign and malignant tumours. Nevertheless, the HMGA1 expression studies published so far have not included primary breast cancer samples. In this study we have investigated the HMGA1 expression patterns in a series of 170 breast cancer samples by immunohistochemistry. We have found a strong variation in HMGA1 expression between the tumours. Based on an immunoreactive score (IRS) 14.1% of the tumour samples were scored to IRS 8-12 (strong positivity for HMGA1), 24.7% were scored to IRS 4-6 (moderate positivity), 25.3% were scored to IRS 1-3 (weak positivity), and 35.9% showed no positivity at all. Immunoreaction could be detected in all histological types of breast cancers analysed with the exception of invasive papillary and cribriform carcinoma. Statistical analysis revealed a strong correlation between tumour grade and HMGA1 expression (rs=0.3516, p<0.0001). Thus, the HMGA1 expression level can be considered a potential prognostic marker for breast cancer.

Death Detection

Apoptotic cells in organisms are removed through their recognition and ingestion by phagocytes. Apoptotic cells are recognized in part by the presence of phosphatidylserine on the cell surface, which flips from the inner leaflet of the plasma membrane during apoptosis. Two reports this week point to additional control mechanisms--regulation of a "detachment" signal from a receptor that alters the duration of interactions with macrophages and regulation of a secreted signal that stimulates the inflammatory response when released from necrotic cells, but is retained in the nucleus of apoptotic cells. Brown et al . detected the protein CD31 as a mediator of the interaction of human neutrophils with macrophages. CD31 is a cell-surface adhesion molecule of the immunoglobulin superfamily and is expressed in leukocytes and macrophages. Homophilic interactions of CD31 on leukocytes with CD31 molecules on endothelial cells contribute to the response to infection. Brown et al . found that, under flow conditions, both viable and apoptotic leukocytes bound at low temperature to a surface coated with CD31, but at higher temperatures, only viable leukocytes were released. The binding appeared to be mediated by CD31 on the leukocytes, as Jurkat lines selected for low expression of CD31 did not adhere to CD31. Signaling by the CD31 molecule seemed to be required for release, as cells expressing modified CD31 that cannot signal acted like apoptotic cells and were not released. The authors propose that viable cells interact only transiently with macrophages because of CD31-mediated release, whereas CD31 function is somehow altered in apoptotic cells, which prolongs interaction with and, ultimately, ingestion by macrophages. Cells that die by necrosis in an organism trigger a protective inflammatory response, whereas apoptotic cells do not and are removed by macrophages. Scaffidi et al . show that the HMGB1 protein (high mobility group 1 protein) may be an important signal that allows cells to distinguish between these events. The authors found that HMGB1 tagged with green fluorescent protein bound to chromatin, but it was released when the cell membrane was permeabilized, as occurs in necrosis. However, in permeabilized apoptotic cells, HMGB1 remained tightly bound to chromatin. Nuclei from HMGB1 −/− cells bound recombinant HMGB1 only if the cells were induced to undergo apoptosis before the nuclei were isolated. Thus, a change in chromatin in apoptotic cells enhances binding of HMGB1. Trichostatin A, a general inhibitor of deacetylases, prevented retention of HMGB1 in nuclei of apoptotic cells. Released HMBG1 binds to a receptor and enhances the inflammatory response. Thus, release of HMBG1 appears to be a signal of necrosis that is disabled during the process of apoptosis. S. Brown, I. Heinisch, E. Ross, K. Shaw, C. D. Buckley, J. Savill, Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature 418 , 200-203 (2002). [Online Journal] P. Scaffidi, T. Mistell, M. E. Bianchi, Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418 , 191-195 (2002). [Online Journal]

The Binding Interaction of HMG-1 with the TATA-binding Protein/TATA Complex

High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP.

Characterisation of genes isolated from a potato swellingstolon cDNA library

In screening to isolate a full-length copy of a previously isolated cDNA clone, a further three cDNAs were also isolated from a library prepared from sub-apical swelling-stolon tissue of potato (Solanum tuberosum L.). Sequence analysis showed these clones to be similar to extensin-like protein genes, acyl carrier protein thioesterase genes and high mobility group protein genes, respectively. A further cDNA, isolated by subtractive hybridisation, was similar to a tomato cDNA previously isolated on the basis of its down-regulation following nematode infection. While all the newly isolated genes were expressed in swelling stolons, for most, maximal expression was seen to be in stem tissue. Possible roles for these genes in the development of potato plants are discussed, as is the significance of gene expression in stems and stolons to the process of tuberisation.

Deletion of HMG17 in Uterine Leiomyomas with Ring Chromosome 1

Uterine leiomyomas are characterized by several subgroups with characteristic chromosomal aberrations, mainly 12q14–15, 6p21, or interstitial deletions of chromosomes 3 and 7. For the first two subgroups, aberrations of the HMGIC and HMGIY genes have been described and are held responsible for tumor initiation. For other subgroups no molecular findings have been described as of yet. We focus here on a smaller subgroup of uterine leiomyomas with a ring chromosome 1 either as the only karyotypic deviation or occurring along with other abnormalities. In the p-arm of chromosome 1 HMG17, another member of the high-mobility group of proteins has been localized to the short arm of chromosome 1 (1p35) with two PAC clones on metaphase spreads of a uterine leiomyoma ring(1). Hybridization signals for these probes were not detected within the ring chromosome consistent with loss or deletion of HMG17. These findings suggest that HMG17 does not play a mechanistic role in leiomyoma similar to that observed with other high-mobility proteins.

Specific interaction between H1 histone and high mobility protein HMG1.

High mobility group proteins HMG1 and -2 and histone H1 are structural components of chromatin. Previously, we reported that HMG1 interacts with H1 histone in a way that modulates the ability of H1 to condense DNA in vitro, suggesting that these proteins may act together in vivo to regulate locally the condensation state of chromatin, possibly affecting replication and/or transcription. Here we show that reduced (native) HMG1 binds to H1 cooperatively at pH 6.0 as a tetramer with a dissociation constant of 3.4 x 10(-8) M, and at pH 7.5 as a monomer with a dissociation constant less than 10(-9) M. Denaturation through oxidation of sulfhydryl groups has a strong effect on the interaction of HMG1 with H1 histone, suggesting that the reduced state of HMG1 is critical to its function. Oxidized HMG1 failed to bind H1 at pH 7.5, and its binding at pH 6 was biphasic; the first three (or two) molecules of H1 were bound with a dissociation constant of 2 x 10(-8) M with negative cooperativity, and the last one (or two) H1's were bound cooperatively with KD = 1.8 x 10(-7) M. Regulation of the pH or the concentration of some other ion may be used in vivo to alter the interactions between HMG1 and -2, H1 histone, and DNA.

Cis-Diamminedichloroplatinum (II) modified chromatin and nucleosomal core particle probed with DNase I

Chromatin and nucleosomal core particles were modified with cis-diamminedichloroplatinum (II) and the nucleoprotein complexes then digested with DNase I. Limited digestion of the modified chromatin containing cis-Pt(NH 3) 2Cl 2 mediated cross-links involving the non-histone chromosomal proteins (Scovell et al. (1987) Biochem. Biophys. Res. Commun. 142, 826–835) does not release the low mobility proteins and excises only about 20% of the high mobility proteins 1, 2, and E. This supports previous findings that the low mobility proteins are involved primarily in protein-protein cross-links. In addition, the covalent cross-links between DNA and the high mobility proteins 1, 2, and E are relatively inaccessible to DNase I, in marked contrast to their accessibility to micrococcal nuclease. Furthermore, gels of the denatured DNA fragments obtained from digestion of both modified chromatin and nucleosomal core particle reveal virtually no difference in the 10 n base repeat pattern, indicating no detectable change in the DNA-protein interactions upon DNA modification. This suggests that the predominant modification produced on core particle DNA, whether contained within higher order chromatin structure or in the core particle itself, is one which does not significantly alter the helical twist of the DNA within these nucleoprotein assemblies.

Differential stimulation of histone and high mobility group chromatin protein synthesis in the rat uterus by estrogen.

Uterine tissue isolated from immature rats at different times after estradiol injection was incubated with medium containing [3H]lysine. The acid-extractable protein from the uterine tissue was subjected to electrophoresis on sodium dodecyl sulfate and acid-urea-Triton X-100 polyacrylamide gels, and the rate of chromatin protein synthesis determined by densitometric analysis of the fluorographs of the gels. Synthesis of chromatin proteins (histones and high mobility group chromatin proteins) was stimulated by 3 h after estrogen treatment and reached a peak at 9 h, several hours before DNA synthesis was stimulated. Synthesis of chromatin proteins occurred at the same time as total cellular protein synthesis. Estrogen stimulated the synthesis of histone variants at different rates, but the accumulation of histone proteins remained coordinated such that equivalent amounts of histone proteins were being produced at any one time.

Protein HMG1 is different from a DNA helix unwinding protein in calf thymus.

A number of criteria were used--chromatography on columns with single-stranded and double-stranded DNA, electrophoresis, peptide analysis, immunological tests and thermal denaturation of DNA--to show that protein (high mobility group) HMG1 and an unwinding protein from calf thymus are two distinct, unrelated proteins. While both proteins are thought to be related to DNA replication this might involve different mechanisms of action.

The interaction of high mobility proteins HMG14 and 17 with nucleosomes

The interaction of the high mobility group proteins, HMG14 and HMG17, with nucleosome core particles has been studied. The results show that two molecules of HMG14/17 can be bound tightly but reversibly to each core particle and that their affinity for core particles is greater than their affinity for histone-free DNA of core size. Thermal denaturation and nuclease digestion studies suggest that major sites of interaction are located near the ends of the nucleosome core DNA. When nucleosome preparations from chicken erythrocyte nuclei stripped of HMG proteins are partially titrated with HMG14/17, the nucleosome-HMG complex fraction is enriched in beta-globin gene sequences.