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M Bustin

Publications and source records attributed to M Bustin.

At least 19 recordsLinked to original sources
PubMed1992

The transcriptionally-active MMTV promoter is depleted of histone H1.

We have used an ultraviolet light cross-linking and immunoadsorption assay to demonstrate that histones H1 and H2B are bound to the repressed MMTV promoter. Hormone activation results in reduced H1 content with little or no change in H2B. High resolution analysis of the glucocorticoid-inducible DNaseI hypersensitive region demonstrates an NF-1 footprint as well as specific sites of enhanced cleavage on nucleosome B and in the nucleosome B/nucleosome A linker. These results are consistent with a model in which binding of the glucocorticoid receptor to glucocorticoid regulatory elements on the surface of nucleosome B induces a chromatin transition that is necessary for transcription factor (NF-1 and TFIID) recruitment to the MMTV promoter. We hypothesize that association of histone H1 with important cis-elements on the promoter masks these sites, and glucocorticoid-induced displacement of H1 is necessary to expose factor binding sites at the 3' edge of nucleosome B, in the nucleosome B/nucleosome A linker and at the 5' edge of nucleosome A.

Base Sequence↗
PubMed1979

Serological analysis of species specificity in the high mobility group chromosomal proteins.

The non-histone chromosomal protein of the high mobility group (HMG-1) present in mouse liver was purified to homogeneity. Antibodies against this protein as well as pure HMG-1 derived from calf thymus and HMG-E purified from duck erythrocytes were elicited in rabbits. The interaction between the antibodies and the immunogens was measured by passive hemoagglutination and by quantitative microcomplement fixation. Quantitative microcomplement fixation assays revealed that the immunological distance between HMG-1 from calf thymus and HMG-1 from mouse liver and duck erythrocytes was 15. This corresponds to 3% sequence differences. It was estimated that amino acid substitution occurred at about seven positions in the polypeptide chain. Thus, HMG-1 proteins display remarkable evolutionary conservation in their primary sequence, similar to that displayed by histones H4 and H3, suggesting that their biological function is dependent on stringent structural requirements. HMG-E protein is significantly different from both HMG-1 and HMG-2 derived from calf thymus. As such, it is a protein unique to avian erythrocytes.

Amino Acid Sequence↗
PubMed1979

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.

Animals↗
PubMed1979

Injected histone antibodies interfere with transcription of lampbrush chromosome loops in oocytes of Pleurodeles.

Antibodies to calf thymus histone H2B were purified by chromatography on DEAE-cellulose and injected into oocyte nuclei of Pleurodeles waltlii. As shown by indirect immunofluorescence these antibodies cross-reacted strongly with corresponding histones associated with lampbrush chromosomes. Shortly after injection the lateral loops of the chromosomes retracted into the chromomeres and by 3 h postinjection the 'lampbrush' appearance was completely lost and the chromosomes appeared in light-microscopic preparations as rod-like structures consisting of longitudinally coalesced chromomeres. In control oocytes injected with non-immune immunoglobulins or antibodies against a ubiquitous transcript-associated protein no morphological alterations of the lampbrush chromosomes could be observed. Electron microscopic spreads of chromosomes prepared at various times after injection of anti-H2B revealed a progressive loss of transcriptional complexes from the loop axes. Finally, higher-order chromatin configurations, like supranucleosomal globules ('superbeads') or cable-like chromatin strands 50-60 nm thick predominated, indicating complete transcriptional inactivation of all chromosomal regions. The results indicate that H2B antibodies react specifically with histones associated with the transcribed DNA of lateral loops in their native state. The resulting antigen-antibody complexes seem to inhibit progression of the RNA polymerases along the template, thus causing the premature release of transcripts, a process analogous to the stripping effect of actinomycin D. The demonstration of histones associated with heavily transcribed regions, which are not compacted into nucleosomes but largely extended, supports the current concept that unfolding of nucleosomes to allow transcription of the DNA does not involve dissociation of histones. In contrast, amplified ribosomal RNA genes are unaffected by injected H2B antibodies. This does not necessarily indicate absence of histones from nucleolar chromatin, since we do not know whether it is accessible in vivo to antibodies or whether the histone antigenic determinants are masked by the presence of other proteins. The technique of injecting specific antibodies should be widely applicable when analysing the in vivo distribution of chromosomal components at the electron-microscopic level and when studying complex metabolic processes, like the cleavage and modification of RNA, by selective inhibition of defined enzymic steps.

Animals↗
PubMed1978

Immunological relatedness of high mobility group chromosomal proteins from calf thymus.

The non-histone proteins HMG-1, HMG-2, HMG-3, HMB-8, HMG-14, and HMG-17 (Goodwin, G. H., SANDERS, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14) were purified from calf thymus. The apparent molecular weights on polyacrylamide gels run in the presence of sodium dodecyl sulfate of the high mobility group (HMB) proteins were determined. Those for HBG-1 and HMG-2 agreed with the molecular weights determined by sedimentation; that for HMG-17 was anomalously high. Antibodies against HMG-1 were elicited in rabbits. The interaction between HMG-1 and anti-HBG-1 was measured by quantitative precipitation and by the microcomplement fixation technique. Quantitative microcomplement fixation assays revealed that the indices of dissimilarity between HMG-1 and HMG-2, HMG-3, HMG-8, HMG-14, and HMG-17 were 2.0, 1.0, 3.8, 10.0, and 6.1, respectively. These correspond to 6%, 0%, 12%, 20%, and 16% sequence difference between HMG-1 and the other five HMG proteins, although the immunological distance between HMG-1 and HMG-14 may be too large to allow a good correlation between the sequence and the immunological reaction. Antibodies to HMB-1 bind to chromatin purified from calf thymus. Therefore, we suggest that the in situ organization of HMG proteins in chromatin and chromosomes may be studied by serological techniques.

Animals↗
PubMed1978

Histone localization in polytene chromosomes by immunofluorescence.

Polytene chromosomes of Chironomus thummi were treated with antisera elicited by purified calf thymus histone fractions, and the location of each histone type was visualized by the indirect immunofluorescence technique. Each of the antisera produced specific and distinct patterns of fluorescence, suggesting that it is possible to use the indirect immunofluorescence technique to study the in situ organization of each histone in the various regions of the chromosomes. H1 and H2A antisera produced diffuse fluorescence patterns in acetic acid-fixed chromosomes which become more defined in formaldehyde-fixed preparations. Antisera to H2B, H3 and H4, when reacted with either formaldehyde- or acetic acid-fixed chromosomes, produce distinct banding patterns closely resembling the banding of acetoorcein-stained or phase-contrast-differentiated chromosomal preparations. These antisera produce corresponding patterns of fluorescence for each chromosome, suggesting that the overall organization of the histones is similar in the various bands. Because the dense band regions stain more brightly with antihistone sera than the less compacted interband areas, we believe that the number of antigenic sites of chromosome-bound histones is related to the amount of DNA present, and that the accessibility of histone determinants does not differ between the bands and interbands.

Animals↗
PubMed1978

Binding of E. coli RNA polymerase to chromatin subunits.

Chromatin subunits were prepared from purified rat liver nuclei and the template properties of the nucleosome preparation studied. It was found that: 1) The fundamental template restriction of chromatin (as compared to deproteinized DNA) is retained in the isolated nucleosomes, 2) On the average one molecule of RNA polymerase is bound to one molecule of DNA purified from nucleosomes, 3) The number of RNA polymerase binding sites on chromatin subunits is 6 to 20 times lower than that of the DNA extracted from these subunits, 4) Transcription can proceed through nucleosomes resulting in RNA chains approximately 150 nucleotides long.

Animals↗
PubMed1977

Molecular homogeneity of the histone content of HeLa chromatin subunits.

Interaction of affinity chromatographically purified antihistone H3 and antihistone H4 with isolated HeLa core particles, followed by separation of unreacted and reacted particles by sedimentation, demonstrates that every core particle contains these histones. Taken together with our previous data indicating the presence of H2B in every nucleosome (Simpson, R. T., and Bustin, M. (1976), Biochemistry 15, 4305), these data lead to the conclusion that each core particle contains two each of the four smaller histones. In contrast to the lack of interference in binding of more than one molecule of antibody to a single species of histone to the core particle, steric hindrance exists when attempts are made to bind both anti-H3 and anti-H4 to core particles.

Antibodies↗
PubMed1977

Lectins as probes of chromatin structure. Binding of concanavalin A to purified rat liver chromatin.

Concanavalin A (Con A) binds specifically to rat liver chromatin. The extent of binding is directly proportional to both chromatin and concanavalin A concentration. It is reversible and inhibited by specific sugars for which concanavalin A has a binding site. Scatchard analysis reveals the presence of one type of Con A-binding site, with an apparent dissociation constant of 3 X 10(-7) M. A maximum of 10 pmol of Con A binds to 10 microgram of chromatin, indicating an average of one binding site/1400 base pairs of DNA. To identify the polypeptide chains which contain Con A-binding sites, chromosomal proteins were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. Con A receptors were localized by incubating the gel in 125I-Con A and subsequent autoradiography. Three major polypeptide bands which bind Con A were identified among the nonhistone chromosomal proteins. The apparent molecular weights of these glycoproteins are 135,000, 125,000, and 69,000. We suggest that lectins may serve as probes for the study of the organization of specific components in chromatin.

Animals↗
PubMed1977

Immunological cross-reaction between calf and Drosophila histones.

Antibodies elicited by purified calf thymus histone fractions are used to quantitate the immunological cross-reactions between calf and Drosophila histones. The index of dissimilarity between calf and Drosophila histones are: H4, 1.0; H2B, 1.0; H3, 1.6; H2A, 2.0; H1, 16. These values respectively correspond to 0, 0, 4, 6, and 27% difference in amino acid composition. The differences in amino acid composition assessed by immunological techniques are in agreement with values obtained by amino acid analysis (Alfageme, C. R., Zweidler, A., Mahowald, A., and Cohen, L. H. (1974) J Biol. Chem. 249, 3729-3736 and Oliver, D., and Chalkley, R. (1972) Exp. Cell. Res. 73, 295-302). The strong immunological cross-reaction between calf and Drosophila histones H4, H3, H2A, and H2B leads us to suggest that antibodies elicited against purified histone fractions derived from calf thymus can serve as "universal" reagents for various studies of chromatin-bound histones. The use of standard preparations of antisera will facilitate comparisons of results obtained in different laboratories with various experimental systems.

Amino Acids↗
PubMed1976

Histone compostion of chromatin subunits studied by immunosedimentation.

Chromatin subunits were prepared from HeLa cells by in situ digestion of nuclear DNA with micrococcal nuclease followed by sucrose gradient sedimentation. These 11S chromosomal particles (nucleosomes) contain a DNA fragment 140--180 base pairs long and an equal mass of histones, H2A, H2B, H3, and h4. nucleosomes were incubated with purified antibodies to histones H2A and H2B and to hemoglobin A, and the resulting complexes were analyzed by ultracentrifugation. Of these, only anti-H2B bound specifically to nucleosomes. When sufficient antibody was present, all (greater than 98%) the nucleosomes sedimented with increased velocities, indicating that all chromosomal particles contain H2B, as suggested by previous electron microscopic studies (Bustin, M., Goldblatt, D., and Sperling, R. (1976), Cell 7, 297). The amount of antibody reacting with H2B in the nucleosome was quantitated by densitometric scanning of gel electrophoresis patterns of the proteins in various nucleosome-anti-H2B complexes separated by sedimentation on isokinetic sucrose gradients. Under conditions where all particles had increased sedimentation velocities, from 1 to 3 IgG molecules are bound to each nucleosome, the ratio increasing from top to bottom of the sedimenting peak. When nucleosomes are thus dispersed on the basis of reaction with anti-H2B, the ratios of H2A to H4 and of (H2B + H3) to H4 are identical (+/- 8%) for all fractions, suggesting that each nucleosome has an identical histone complement, two each of histones H2A, H2B, H3, and H4. Confidence limits for exclusion of other possible octamers are presented. The variation in ratio of bound antibody to nucleosome probably reflects a normal distribution during the titration, although differential exposure of H2B antigenic determinants in several populations of nucleosomes cannot be excluded as an explanation. The method use should be generally applicable to further studies of the composition and function of nucleosomes.

Animals↗