Biomedical subjects

M Bustin

Publications and source records attributed to M Bustin.

At least 109 records · Page 6Linked to original sources

PubMed1985

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.

Binding Sites↗

PubMed1985

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene.

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.

Animals↗

PubMed1985

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.

Actins↗

PubMed1985

Evidence that high mobility group protein 17 is not phosphorylated in human colon carcinoma cells.

The high mobility group proteins 14 and 17 were reported previously to be phosphorylated in murine and human tumor cell lines. Recently, it was suggested that subgroups of HMG-14, HMG-14a and 14b, but not HMG-17, were phosphorylated in situ in HeLa cells. In order to definitively determine whether HMG-17 is indeed phosphorylated or whether the protein previously identified as [32P]HMG-17 was a subgroup of HMG-14, we have used the technique of electroblotting in conjunction with an immunochemical procedure utilizing anti-HMG-17 IgG. Our results indicate that HMG-17 was not phosphorylated in human colon carcinoma cell line HT-29 incubated for 18 h with 32Pi, but that HMG-14a and HMG-14b were phosphorylated. In contrast, HMG-14a, -14b and -17 were phosphorylated in vitro in isolated nuclei incubated with [gamma-32P]ATP.

Cell Line↗

PubMed1985

Antibodies to 5 M urea soluble chromosomal proteins from HeLa cells.

The tissue specificity of a chromosomal protein fraction, extractable from chromatin with 5 M urea at low ionic strength, has been examined in HeLa, A549 and HT 29 cells. Electrophoresis in polyacrylamide gels indicates that each cell type has a different content of 5 M urea soluble proteins which are distinguishable from the histones, from the tight DNA-binding proteins and from the high-mobility-group chromosomal proteins. Antibodies against 5 M urea soluble proteins extracted from HeLa cells were produced in mice. Although each of the mice tested prior to immunization contained a detectable amount of antibodies against both the 5 M urea soluble proteins and tight DNA-binding proteins, immunization elevated the level of the antibodies in the serum over 100-fold. The antibodies do not distinguish between the 5 M urea extracts obtained from different sources because most of the antibodies are directed against antigens shared by the cells studied. Immunofluorescence studies reveal that components which cross-react with 5 M urea soluble chromosomal proteins are also present in the cytoplasm. We conclude the following. (1) 5 M urea extracts from chromatin a group of proteins which differs among cells. (2) Mice contain detectable amounts of autoantibodies against these chromosomal proteins. (3) Immunization with the 5 M urea extractable fraction elicits antibodies against a restricted number of antigenic components which are shared among the cells studied. (4) 5 M urea extractable proteins are found both in the nucleus and cytoplasm; part of these may be cytoskeletal elements. Because the antisera do not react with histones, high-mobility-group proteins and tight DNA-binding proteins, they may be used for various functional studies on the 5 M urea extractable chromosomal protein fraction.

Animals↗

PubMed1985

The intracellular distribution and function of the high mobility group chromosomal proteins.

This brief review provides a framework for discussing current approaches being used to determine the cellular localization and function of the high mobility group chromosomal (HMG) proteins. The four main constituents of this group (HMG 1, 2, 14, 17) are present in all four eukaryotic kingdoms, have a relatively well conserved primary sequence and contain several functional domains which enable them to interact with DNA, histones and other components of the genome. The evolutionary conservation in the primary and tertiary structure as well as the observed correlations between cell phenotype and quantitative changes in protein levels and in post-synthesis modifications suggests that these proteins are components obligatory for proper cellular function. Proteins HMG 1, 2 are DNA-binding proteins which can distinguish between various types of single-stranded regions of the genome. Proteins HMG 14, 17 may be involved in maintaining specific chromatin regions in particular conformations. The data available presently suggests that these proteins are important structural elements of chromatin and chromosomes.

Amino Acid Sequence↗

PubMed1985

Site-specific carcinogen binding to DNA in polytene chromosomes.

Treatment of Chironomus polytene chromosomes with the ultimate carcinogen benzo[a]pyrene diol epoxide I or in vivo administration of the parent hydrocarbon to larvae indicates that the carcinogen interacts with the genome in a nonrandom manner. Visualization of the carcinogen-DNA binding sites by immunofluorescence reveals that, in vivo, some sites are preferentially modified. The combined effects of DNA sequence, chromatin structure, and gene localization may lead to selective targeting of carcinogens to specific genomic regions. In polytene chromosomes the targeting effect is amplified, thereby making these chromosomes a uniquely suitable system for visualizing and studying site-specific interactions of carcinogens with the genome.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1984

Monoclonal antibodies against distinct determinants of histone H5 bind to chromatin.

A series of monoclonal antibodies specific for distinguishable epitopes in chromosomal protein histone H5 were obtained from mice immunized with either free H5 or H5 . RNA complexes. The antibodies elicited by H5 could be distinguished from those elicited by H5 . RNA by their binding to native or acid-denatured H5, by their interaction with the globular region of H5, and by their cross-reactivity with H1o. The specificity of the antibodies was assessed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting experiments. The antibodies could distinguish between H5 and the closely related histones H1 and H1o. The binding of some of the antibodies to the antigens was dependent on the type of assay used, suggesting nonrandom binding of the antigen to the solid supports used in ELISA and immunoblotting. Competitive ELISA experiments indicate that 8 of the 11 antibodies characterized bind to distinct epitopes. Three monoclonal antibodies bind to epitopes which are in close spatial proximity, causing mutual steric hindrance. The monoclonal antibodies bind to nuclei of fixed cells and to isolated chromatin, indicating that the epitopes are present both in the purified protein and in chromatin-complexed H5. These monoclonal antibodies can be used to study the organization of distinct regions of histones H5 and H1o in chromatin and chromosomes.

Animals↗

PubMed1984

Mapping the binding of monoclonal antibodies to histone H5.

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.

Animals↗

PubMed1984

Enrichment of acetylated histones in polynucleosomes containing high mobility group protein 17 revealed by immunoaffinity chromatography.

The possibility that chromatin domains containing acetylated histones are proximal to domains containing chromosomal high mobility group protein 17 (HMG-17) has been investigated. Oligonucleosomes containing [3H]acetate-labeled histones have been immunofractionated on anti-HMG-17 IgG-Sepharose columns. Ninety-one per cent of the 3H counts present in the oligonucleosomes specifically bound to the anti-HMG-17 column. Extraction of HMG-17 from chromatin by treatment with 0.4 M NaCl abolished the specific binding of acetylated chromatin to the Sepharose columns. Autoradiographic analysis of polyacrylamide gels of the bound fraction revealed that it contained all the major acetylated histone species. We conclude that acetylated histones are present on or near nucleosomes containing protein HMG-17.

Acetylation↗

PubMed1984

A survey of H1o-and H5-like protein structure and distribution in higher and lower eukaryotes.

A survey of H1o and H5-like proteins has been conducted through a range of higher and lower eukaryotic species. All mammals examined possessed H1o proteins, although in variable amounts, and the protein's structure was well conserved, though not invariant. The testis-specific histone Hlt (from rat) did not have an H1o-like structure and it appears that H1o does not occur in spermatocytes in any form. The results also show that Xenopus laevis contains H1o-like proteins, but lower, non-vertebrate eukaryotes (a crustacean, two fungi and a plant tissue) do not possess H1o or H5 proteins. The evidence suggests that H1o and H5 proteins may be considered as belonging to one family, distinct from H1 types. This H1o/H5 family may well be 'replacement histone' variants of H1. The results do not support suggestions of roles such as repression of DNA synthesis or of transcription for H1o/H5 proteins.

Animals↗

PubMed1984

Localization of HMG chromosomal proteins in the nucleus and cytoplasm by microinjection of functional antibody fragments into living fibroblasts.

We have used microinjection and cell fractionation to localize the chromosomal high mobility group proteins (HMG) in human fibroblasts. Electrophoretic analysis of nuclear and cytoplasmic fractions from the fibroblasts indicates that the concentration of HMG-1,2 in the cytoplasm is 2.9 times larger than in the nucleus indicating that the majority of the cellular HMG-1,2 is present in the cytoplasm. In contrast, HMG-17 remains predominant in the nuclear fraction. We conclude that the cellular distribution of HMG-1,2 is significantly different from that of HMG-17. To avoid possible artifacts due to cell fractionation, fluoresceinated HMG-1 and HMG antibodies were microinjected into living fibroblasts. The cellular distribution of the injected proteins was monitored using fluorescent microscopy. Fluoresceinated HMG-1 microinjected into the cytoplasm moves very rapidly into the nucleus and concentrates in the nucleolus of living human fibroblasts. However, some control non-nuclear proteins also migrated into the nucleus raising the possibility that exogenous injected proteins do not always distribute in the same pattern as the endogenous proteins. The localization of microinjected F(ab)2 fragments derived from anti-HMG-1 was compared to that of microinjected F(ab)2 derived from anti-histones. Whereas the anti-histone F(ab)2 when injected into the cytoplasm migrated into the nucleus, the anti-HMG-1 F(ab)2 remained in the cytoplasm. Microinjection of anti-HMG-17 and anti-histone inhibited transcription in living cells, anti-HMG-1,2 did not. We conclude that HMG-1,2 proteins are present in both the nucleus and cytoplasm of living fibroblasts.

Antibodies↗

PubMed1984

Functional histone antibody fragments traverse the nuclear envelope.

Factors important in the translocation process of proteins across the nuclear membrane were studied by microinjecting either fluoresceinated nonimmune IgG and F(ab)2 or the corresponding molecules, prepared from antisera to histones, into the nucleus and cytoplasm of human fibroblasts. Intact IgG from both preparations remained at the site of injection regardless of whether it was injected into the nucleus or the cytoplasm. In contrast, nonimmune F(ab)2 distributed uniformly throughout the cell. The F(ab)2 derived from affinity-pure antihistone moves into the nucleus after cytoplasmic injection and remains in the nucleus after nuclear microinjection. The migration of the antihistone F(ab)2 into the nucleus results in inhibition of uridine incorporation in the nuclei of the microinjected cells. We conclude that non-nuclear proteins, devoid of specific signal sequences, traverse the nuclear membrane and accumulate in the nucleus provided their radius of gyration is less than 55A and the nucleus contains binding sites for these molecules. These findings support the model of "quasibifunctional binding sites" as a driving force for nuclear accumulation of proteins. The results also indicate that active F(ab)2 fragments, microinjected into somatic cells, can bind to their antigenic sites suggesting that microinjection of active antibody fragments can be used to study the location and function of nuclear components in living cells.

Biological Transport↗

PubMed1983

Specific binding of Alu sequences by HeLa nuclear extracts.

Plasmid Blur 8 which contains the 300bp human Alu consensus sequence and plasmid pBR322 were digested with restriction enzymes and the fragments obtained end labelled with 32P-gamma-ATP. The end labelled fragments were incubated with HeLa nuclear extracts and the incubation mixtures passed through a nitrocellulose filter. The 300bp alu consensus sequence was preferentially retained on the filter. The HeLa nuclear extract did not preferentially bind any fragments generated from pBR322 and histones which bind nonspecifically all DNA fragments did not preferentially bind the alu sequence. We conclude that the HeLa nuclear extract contains components which specifically bind the human alu sequence.

Base Sequence↗

PubMed1983

The binding of a carcinogen to the nucleosomal and non-nucleosomal regions of the simian virus 40 chromosome in vivo.

The effect of chromatin structure on the binding of a chemical carcinogen to the genomic DNA was studied. The binding in vivo of the ultimate carcinogen, benzo-pyrene 7,8,-diol,-9,10-epoxide, to various regions of the SV40 chromosome was revealed by an immunological method. Particular attention was given to restriction fragments which include the origin of replication which is "non-nucleosomal" in a significant fraction of the chromosomes. The distribution of (+/-) trans-7,8-dihydrobenzo[alpha]pyrene-7,8-diol-9,10-epoxide (BPDE) adducts was studied in 1) SV40 DNA modified in vitro to a level of 20 adducts/molecule, 2) DNA from SV40 chromosomes modified in vivo to a level of less than 1 adduct, and 3) DNA from only those chromosomes with an open origin of replication. In other experiments, the binding of BPDE to the origin region was compared to the binding to nucleosome core particle DNA from the viral chromosome. The origin region bound 1.7-fold more BPDE than core DNA, while linker DNA is 3-fold more modified than core DNA. However, the origin region was only about 20% more modified than any other region of the chromosome. We conclude that while the conformation of the DNA in chromatin has a slight effect on its accessibility to the carcinogen, the SV40 chromosome does not contain a particular "hot spot" which is preferentially modified by BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1983

Selective exposure of antigenic determinants in chromosomal proteins upon gene activation in polytene chromosomes.

The immunological accessibility of the nucleosomal core protein H3 and non-histone protein, HMG-1, was studied in transcriptionally active regions of Chironomus thummi polytene chromosomes. Chromosomal loci were decondensed by incubating isolated salivary glands in various salt solutions or hemolymph. Indirect immunofluorescence studies on these chromosomes using anti-sera to histone H3 revealed that the puffed regions were depleted of fluorescence. The lack of fluorescence could be correlated with the degree of puffing and the level of transcriptional activity. The puffed regions fluoresce after anti-H3 addition if the chromosomes are not cross-linked with formaldehyde, and if prior to the addition of antibodies the chromosomes are treated with 45% acetic acid. We conclude that, whereas histone H3 is present in the puffed regions, its antigenic determinants are sterically hindered by components which are extractable by 45% acetic acid. On the other hand, the antigenic determinants of protein HMG-1 are always available to antibody binding in puffed regions, as well as other chromosomal areas.

Animals↗

PubMed1983

Effect of X-radiation on DNA and histone synthesis in ataxia telangiectasia and normal lymphoblastoid cells.

The possibility that the radiosensitivity of lymphoblastoid cell lines from patients with ataxia telangiectasia (A-T) is due to an aberrant content of histones has been examined. The histone pattern of lymphoblastoid cell lines derived from A-T patients was found to be indistinguishable from that obtained from normal individuals. X-ray irradiation led to a greater decrease in cell growth rate in the A-T cells than in the normal cells but was accompanied by a greater decrease of DNA synthesis rate in the normal cells. This difference in radiosensitivity was not reflected in differences in the content or rates of synthesis of histones or of major non-histone proteins in these cells. Reduction in the rate of DNA synthesis was not associated with the appearance of the lysine-rich histone variant H1. We conclude that the hypersensitivity to ionizing radiation in A-T cells is not due to fundamental differences in the composition or synthesis of the major chromosomal proteins.

Ataxia Telangiectasia↗

PubMed1983

Immunological evidence for the in vivo occurrence of a crosslinked complex of poly(ADP-ribosylated) histone H1.

The poly(ADP-ribosylation) of histones, which occurs within a limited and functionally specific domain of chromatin, is a novel post-translational modification. However, in the past it has been difficult to study this process in living cells because the substrate of the reaction (NAD) does not permeate the plasma membrane. In the current study, antibodies specific for histone H1 and poly(ADP-ribose) were used to study the occurrence of poly(ADP-ribose)+ species of H1 in vivo. Perchloric acid-extracted proteins from synchronously growing HeLa cells were fractionated by electrophoresis and transferred to nitrocellulose, and the transferred moieties were allowed to react with the specific antibodies and then with 125I-labeled protein A. The results conclusively demonstrate the natural occurrence of poly(ADP-ribose)-crosslinked complexes of histone H1 (i.e., H1 dimer), at the S/G2 phase transition of the cell cycle.

Cell Cycle↗