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M Bustin

Publications and source records attributed to M Bustin.

35 records · Page 2Linked to original sources
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↗
PubMed1976

Chromatin structure visualization by immunoelectron microscopy.

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 A, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 A. This size is compatible with a model where one layer of gamma globulin molecules 110 A long encircles a sphere of chromatin 100 A in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting the most of the chromatin beads contain this type of histone.

Antigen-Antibody Reactions↗
PubMed1976

Immunological cross-reactivity of antibodies to a synthetic undecapeptide analogous to the amino terminal segment of carcinoembryonic antigen, with the intact protein and with human sera.

A peptide corresponding to the 11 amino acid residues of the NH2-terminal portion in the sequence of carcinoembryonic antigen(CNTHETIC CEA(1-11) peptide was attached by means of a water-soluble carbodiimide reagent to multichain poly(DL-alanine( as well as to bovine serum albumin. Both macromolecular conjugates provoked in rabbit anti-CEA(1-11) peptide antibodies. The specificity of this immunological system and the crossreactivity between the peptide and intact CEA were investigated by two methods--passive hemagglutination and modified bacteriophage inactivation. Hemmagglutination experiments showed that not only anti-CEA(1-11) sera, but also anti-CEA sera, agglutinated CEA(1-11)-coated sheep erythrocytes, and both these reactions were inhibited with CEA(1-11) peptide. In experiments with the chemically modified bacteriophage technique CEA(1-11)-coated phase was efficiently inactivated with antisera against the CEA(1-11) conjugates, and the inactivation reaction could be totally inhibited with the free peptide. The semipure CEA, but not the pure protein, could also inhibit the phage inactivation, even though less efficiently. On the basis of the above results, sera of some cancer patients were tested for their capacity to inhibit the inactivation of CEA(1-11)-coated phage by means of anti-CEA(1-11) antiserum. The results indicate that sera from a large proportion of patients with adenocarcinomas of the digestive tract, pancreas, and breast are capable of inhibiting the above inactivation, whereas most normal sera do not inhibit.

Adult↗
PubMed1976

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.

Animals↗
PubMed1976

Histone and DNA detection in swollen spermatozoa and somatic cells, by immunofluorescence.

A method of swelling spermatozoa and other cells, which leads to the exposure of nuclear antigens is described. By applying the indirect IFT on these swollen cells with sera containing antibodies to nuclear antigens, and by comparing the results to those obtained in other tests (measuring anti-nuclear antibodies), the following conclusions could be drawn: (a) By swelling human spermatoza, nuclear antigens of the sperm are exposed, and can be used for the detection of antibodies directed against them. (b) Heterlogous antibodies to histones F2al, FIa2 and F3 which can not be detected in the indirect IFT on rat liver cells, become detectable after swelling of these cells. (c) Mature human spermatozoa contain, in addition to double-stranded DNA and protamine, small amounts of histone F2b and F2a2. (d) In mature human spermatozoa histone F1 is absent.

Antibodies, Antinuclear↗
PubMed1975

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.

Animals↗
PubMed1975

Immunological comparison of basic encephalitogen and histone F2A1.

The extent of immunological cross-reaction between basic encephalitogen and histone F2A1 on both the humoral antibody level and on the cellular level has been established. The extent of humoral cross-reaction was tested by direct complement fixation employing both anti-histone F2A1 and antisera to basic encephalitogen, by inhibition of complement fixation, by radioimmunoassay and by passive cutaneous anaphylaxis. The data obtained failed to reveal immunological cross-reaction at the cellular level was tested by the lymphocyte stimulation technique in rabbits and guinea pigs, by inhibition of lymphocyte stimulation and by delayed hypersensitivity skin reactions. A slight but significant cross-reaction between the two proteins on the cellular level was detected by inhibition of lymphocyte stimulation and by the delayed hypersensitivity test. It is concluded that the immunological studies provide limited evidence that the two proteins share antigenic determinants.

Animals↗
PubMed1975

Exposure of histone antigenic determinants in chromatin.

The exposure of antigenic determinants of histones present in "native" chromatin was studied by: (1) testing their ability to elicit anti-histone antibodies and (2) measuring their ability to interact with anti-histone sera. To this end, antisera specific to purified histone fractions and to purified rat liver chromatin were elicited in rabbits. The anti-chromatin sera did not react with pure histone fractions and pure histone fractions F2b, F3, F2a1, and F2a2 failed to inhibit the complement fixation resulting from the binding of anti-chromatin to chromatin. These results suggest that in native chromatin, determinants in these histones are not immunogenic. Histone F1, however, inhibited the reaction between chromatin and anti-chromatin. Antisera elicited by histone fractions reacted weakly with "native" chromatin. The maximal complement fixations (obtained with 5-10 mug of chromatin DNA) were as follows: 60% with anti-F2b, 20% with anti-F1 and anti-F3, and less than 5% with either anti-F2a1 or anti-F2a2. Studies of the interaction between anti-histone antibodies and chromatin in which chromatin was used as an immunoadsorbent indicated that antibodies against different histones were adsorbed to a different degree by the same amount of chromatin. Differences in the immunoadsorbing capacity between sonicated and nonsonicated chromatin were found. Quantitative adsorbtion studies revealed that in the "native" chromatin structure, antigenic determinants of F1 and F2b were more available to interact with homologous antibody than those of F3 and F2a1 and that determinants in F2a2 were the least available. It could be calculated that the "equivalent antigenicity" of the histones in chromatin was 9.6% for F1, 3.2% for F2b, and 0.90% for F3 and F2a1. Upon sonication these values did not change for F1 but increased two-, three-, and fourfold for F2b, F3, and F2a1, respectively. Digestion of chromatin with trypsin totally abolished the ability of chromatin to adsorb anti-histone antibodies.

Animals↗