New method for fractionating histones for physical and chemical studies

Kr, Sommer; Chalkley, Roger

doi:10.1021/bi00702a029

Cited by 50 publications

(32 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Electrophoresis in long 2.5 M urea-15% polyacrylamide gels was performed by the method of Panyim and Chalkley (21). Gels were stained in fast green and were scanned in a Gilford spectrophotometer (22 (27). These (25) or acetylated subspecies of F2A1 (20).…”

Section: Methodsmentioning

confidence: 99%

Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

Gorovsky¹,

Keevert²

1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Macronuclei of the ciliated protozoan Tetrahymena pyriformis contain at least five classes of histones, including two with properties like those of histones F3 and F1 of higher eukaryotes. Micronuclei isolated under identical conditions contain little or no detectable F3 or Fl. Digestion of both macronuclei and micronuclei with staphylococcal nuclease results in DNA fragments of discrete sizes. The electrophoretic mobilities of the larger fragments suggest that they are oligomers of the smallest ones. These resu s indicate that the periodic subunit structure observed in the chromatin of higher organisms also occurs in protozoans, and that this structure does not depend on the presence of either histone F1 or F3, even in an organism which has the genetic information for synthesizing these proteins.Recent studies indicate that the chromatin of higher organisms has an underlying subunit structure which can be visualized directly by electron microscopic techniques (1-5). The existence of this-repeating structure can also be inferred by digesting isolated nuclei or chromatin with relatively nonspecific (endogenous or exogenous) nucleases. When used under conditions which completely degrade free DNA, these nucleases digest only about 50% of the DNA in chromatin to acid-soluble fragments (6). The majority of the undigested DNA is found in a nucleoprotein particle (7), and can be isolated as fragments containing approximately 50-200 base pairs (7-11). If nuclei or chromatin are only partially digested under conditions in which 10-30% of their DNA content becomes acid-soluble, the remaining DNA is found in an oligomeric series of nucleoprotein particles (10, 12). As first reported by Hewish and Burgoyne (13), the DNA molecules of such partial digests have discrete sizes. They also form an oligomeric series, the size of the larger fragments being integral multiples of the smallest fragment, which contains 100-200 base pairs (10,12,13).Based on results of the type described above (10) (14). One possible test of this postulated role for F3 and F2A1 in chromatin would be to examine the structure of chromatin lacking one (or both) of these histones.The macro-and micronuclei of the ciliated protozoan Tetrahymena pyriformis offer an opportunity to assess the role played by histone F3 in the structure of a naturally occurring chromatin. In ciliates, macro-and micronuclei are formed from daughter products of a mitotic division during the sexual process of conjugation and, in Tetrahymena, 80-90% of the DNA sequences in the two nuclei are the same (18). In this report, we present evidence that macronuclei isolated from Tetrahymena pyriformis contain histone F3, but that micronuclei do not. Nonetheless, when micronuclei are digested with staphylococcal nuclease, a series of DNA molecules of discrete sizes is obtained which is similar to that produced when macronuclei or calf thymus nuclei are digested under identical conditions. MATERIALS AND METHODS

show abstract

Section: Methodsmentioning

confidence: 99%

Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

Gorovsky¹,

Keevert²

1975

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…When invertebrate (6,7) and protozoan H 1 histones (8-1 1) were compared with each other and with the corresponding vertebrate fractions, many differences were also found. In general, available data on plant histones indicate that the HI fraction has a slower migration on acrylamide gels (12)(13)(14), a more complex electrophoretic pattern (two or more subfractions), and higher molecular weight (up to 3000) than its counterparts in animal cells.…”

Section: Introductionmentioning

confidence: 99%

Comparative study of rye and thymus histones: amino acid analysis and tryptic fingerprinting

Nadeau¹,

Pallotta²,

Lafontaine³

1977

Can. J. Biochem.

View full text Add to dashboard Cite

Amino acid composition and tryptic fingerprints of rye (Secal cereale) H1, H2B (PH1), and H2A(PHII) histones indicate the presence of major differences between these and the corresponding calf or rabbit fractions. In addition to variations for other amino acids, fraction H1 from rye contains twice as much arginine as the corresponding animal fraction; the plant H2B (PHI) and H2A (PHII) histones show lysine to arginine ratios greater than those of their animal counterparts. The tryptic maps of the same proteins appear to differ between plants and animals by the number and the general pattern of the peptides, as well as by the quantity and distribution of the arginine-containing peptides. Such results suggest the presence of differences in the primary structure of the calf and rye lysine-rich and moderately lysine-rich histones. Furthermore, the possibility is ruled out that each of these plant histones consists of an animal-like protein with an additional segment of 20--30 amino acid residues. On the other hand, the rye and calf arginine-rich fractions H3 and H4 show similar amino acid compositions and tryptic peptides maps.

show abstract

“…Purification of Histones. D. melanogaster embryo histones were extracted from nuclei (10), chromatographed on Bio-Gel P-100, and assayed by absorbance at 230 nm (19). Histones in the A23o peaks were precipitated with trichloroacetic acid (8).…”

mentioning

confidence: 99%

Drosophila nucleosomes contain an unusual histone-like protein.

Palmer¹,

Snyder²,

Blumenfeld³

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Histones are major structural components of the basic repeating subunit of chromatin, the nucleosome (1). Core histone sequences are remarkably conserved during evolution. Nonetheless, a number of histone variants are known (2-4). Synthesis of core histone variants is stage-specific during sea urchin development (3, 4), tissue-specific in mammals (5), and correlated with erythroid differentiation in Friend leukemia cells (6, 7). These observations suggest that nucleosome heterogeneity is related to the functional specialization of specific elements of chromatin. In this context, it may be important that mononucleosomes isolated from Drosophila chromatin contain large amounts of a histone-like protein that is not a standard core histone. This protein was first identified by Alfageme et al. (8), and designated D2, or "Drosophila 2."In this communication, we demonstrate that D2 is nucleosomal and histone-like, yet is readily distinguished from each of the four "standard" core histones. It is also conserved during the evolution of Drosophila. The curious features of D2 raise questions about histone evolution and the organization of histone genes in Drosophila.MATERIALS AND METHODS Purification of Nuclei. D. melanogaster embryo nuclei and adult head nuclei were prepared with three modifications of published procedures (9, 10): (i) buffer A of Hewish and Burgoyne (11), containing 1 mM EDTA, 0.2 mM ethylene glycol bis(f3-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA), and 0.2 mM phenylmethylsulfonyl fluoride was used for the nuclear isolation; (ii) the crude nuclear pellet was washed in buffer containing 0.1% Triton X-100; (iii) the detergent-washed nuclei were then resuspended and recentrifuged three times in buffer lacking Triton X-100.Purification of Mononucleosomes. Nuclei were suspended in buffer A of Hewish and Burgoyne (11), made 4 mM in CaCl2, and digested at 250C to 25-29% acid solubility with staphylococcal nuclease (EC 3.1.31.1). Digested nuclei were lysed according to Noll and Kornberg (12). Digests were fractionated by sedimentation through linear (5-20%) sucrose gradients containing 0.5 M NaCl (13) or by electrophoresis in 5% acrylamide gels (14).Analysis of Nucleosomal DNA. DNA was extracted from pooled, sucrose-gradient-purified nucleosomes (15), omitting RNase treatment. DNA was released from electrophoretically purified nucleosomes with 1% NaDodSO4 and proteinase K at 100 ,ug/ml. DNA was electrophoresed on 5% acrylamide slab gels (16) and visualized by staining with ethidium bromide.Analysis of Nucleosomal Proteins. Gradient-purified nucleosomes were precipitated with 10 mM MgCl2 (17) and extracted with protamine/urea/acetic acid (18). The released chromosomal proteins were applied directly to acetic acid/ urea/Triton DF-16 gels (see below). Electrophoretically purified nucleosomes were electrophoresed into dialysis bags and extracted with 0.25 M HCO (8). The extracted proteins were analyzed by two-dimensional electrophoresis (see below). Purification of Histones. D. melanogaster embryo histones w...

show abstract

New method for fractionating histones for physical and chemical studies

Abstract: We have developed a method for the fractionation of histones using a single column (120 X 2.5 cm) packed with

Cited by 50 publications

References 17 publications

Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

Subunit structure of a naturally occurring chromatin lacking histones F1 and F3.

Comparative study of rye and thymus histones: amino acid analysis and tryptic fingerprinting

Drosophila nucleosomes contain an unusual histone-like protein.

Contact Info

Product

Resources

About