Histones of Drosophila Embryos

117

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaC1 has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCI. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaC1 concentrations in content of nonhistone chromosomal proteins. 40-60% of the nucleosomes were released by 0.3 M NaC1 with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaC1. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaC1. These fractions contained the DNA least available for micrococcal nuclease attack. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by involved in a special function.KEY WORDS nucleosome fractionation nucleosome packing 9 histone H1 interaction with nucleosomes 9 histone H1 stoichiometry 9 chromatin condensation mechanism Chromatin in the interphase nucleus in eukaryotic organisms is organized morphologically in more condensed and less condensed structures. RNA synthesis apparently occurs in the less condensed regions (30), and satellite DNA is thought to be present in condensed chromatin. Recent evidence 0.2 M NaC1 suggest that this population is that more than 85% of the nuclear DNA, including the actively transcribing genes as well as satellite DNA sequences, is complexed with histones in particulate structures called nucleosomes (3,6,23,24,39,42,46,52) suggests that the more-and less-condensed morphological features of the nucleus may reflect differences in the macromolecular composition and/or the packing of the nucleosomes. The differential condensation is preserved in nuclei isolated in the presence of divalent cations (51), and dilution or chelation of divalent cations causes the native nuclear organi-J. CELL BIOLOGY ~) The Rockefeller University Press

Section: Figurementioning

confidence: 80%

Section: Gel Electrophoresismentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

See 1 more Smart Citation

Fractionation of nucleosomes by salt elution from micrococcal nuclease-digested nuclei.

Sanders¹

1978

117

“…The SDS was removed by electrophoresis in the presence of 8 M urea and 1% Triton X-100 (Rohm & Haas Co., Philadelphia) to prevent the precipitation of the protein. 2 The samples were made acidic with acetic acid, and run on a 7.5% polyacrylamide, continuous acid-urea gel system at pH 2.5 (1). The proteins are positively charged at this pH and migrate toward the negative electrode.…”

Section: Sds and Acid-urea-polyacrylamide Gel Electrophoresismentioning

confidence: 99%

Is there a role for actin in virus budding?

Damsky¹,

Sheffield²,

Tuszynski³

et al. 1977

105

Electrophoretic data from both sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and acid-urea gels reveal a protein in purified murine mammary tumor virus (MuMTV) which co-migrates with purified chick skeletal muscle actin. ~zsI-labeling of intact and disrupted virus preparations shows that the actin-like protein is not artifactually adsorbed to the outside of virions during isolation. Quantitative SDS-PAGE and examination of negatively stained preparations show that the actin cannot be accounted for by a contaminating population of virus-free vesicles. The ultrastructure of mammary epithelial cells and of Rous sarcoma virus-transformed chick embryo fibroblasts shows that virus extrusion is associated with filament-containing cellular processes. In particular, MuMTV is released from the ends of long microvilli which contain a bundle of 6-8-nm microfilaments and share other structural features with intestinal microvilli. We suggest that virus nucleoids require an interaction with host cell contractile proteins for their extrusion from the cell.KEY WORDS mammary tumor virus actin microvilli virus budding virus assemblyIt has been assumed without much experimental basis that the interaction of a virus nucleoid with the cell surface membrane produces a localized alteration, sufficient for the virus to be extruded and pinched off without the need for a contractile apparatus (2-4, 15). We report here the presence of an actin-like protein associated with purified preparations of murine mammary tumor virus (MuMTV), a B-type RNA-containing, surfacebudding, enveloped virus produced by mammary tumor epithelium in vivo and in vitro. The ultrastructural and electrophoretic data presented in this report 1 show that the actin present in purified virus preparations is an internal protein of the virion, and not a contaminant of the isolation procedure, and suggest that it is derived from host microvillus microfilaments. Our results raise the possibility that interaction of the virus nucleoid with actin is required for the virus to be extruded from the cell. Biochemical evidence for the presence of actin in other enveloped viruses has been independently reported by Wang et al. (32). MATERIALS AND METHODSAn established line of mammary tumor cells in culture derived from a BALB/cfC3H mouse mammary tumor (13) was used for electron microscopy, and as a source of MuMTV. Cells were grown to a confluent monolayer in Hanks' balanced salt solution and Eagle's minimum essential medium (MEM) with 10% fetal calf serum, and then stimulated to produce MuMTV with 10 -a M dexamethasone (13, 20).

“…Histones were electrophoresed on Triton-urea-acetic acid slab gels in the first dimension and discontinuous SDS gels in the second as described previously (12,14), except that 0.37% Triton DF-16 and 8 M urea were used for the first dimension.…”

Section: Triton-urea-acetic Acid Gel Electrophoresismentioning

confidence: 99%

Identification of histone H2b as a heat-shock protein in Drosophila.

Sanders¹

1981

Total cell polypeptides synthesized, in cultured Drosophila cells under control (25°C) and heat-shock (37°C) conditions have been compared in two different two-dimensional polyacrylamide gel electrophoresis systems which, together, resolve polypeptides having a wide range of isoelectric points, including the most basic polypeptides of the cell . The electrophoresis of basic proteins showed that the most prominent basic polypeptide synthesized in heat shock comigrated with histone H2b. This heat-shock polypeptide was identified as histone H2b by two criteria: (a) it comigrated with authentic histone H2b in Triton-urea-acetic acid acrylamide gel electrophoresis after solubilization from nuclei with acid ; and (b) partial proteolysis peptide maps of the basic heat-shock protein and histone H2b were identical . The synthesis of histone H2b was induced threefold in heat shock, whereas synthesis of the other histones was reduced from two-to tenfold . The noncoordinate synthesis of histones in Drosophila in heat shock provides an interesting system in which to investigate transcriptional and translational controls of histone synthesis as well as assembly of histones into chromatin.The heat-shock response in Drosophila was first observed as a puffing response at nine specific loci on the polytene salivary chromosomes in larvae that were transferred from 25°C to 37°C (1). Subsequently, it has been found that the response involves the induction of synthesis of a number of RNAs, several of which code for a series of polypeptides synthesized during heat shock, the heat-shock proteins (2-6) . The mechanism of induction and the physiological meaning of the response are not understood, and up to this time none of the heat-shock polypeptides has been identified .I have characterized the heat-shock polypeptides in Drosophila by two-dimensional polyacrylamide gel electrophoresis (PAGE) of basic as well as neutral and acidic polypeptides from solubilized whole cells. These experiments have led to the identification of the smallest and most basic heat-shock protein as histone H2b . MATERIALS AND METHODS Cell Culture and Labeling ConditionsThe A4E6 line of Drosophila Kc cells (7) was obtained from Peter and Lucy Cherbas, of Harvard University, and was maintained at 25°C in tissue culture flasks in D22 medium (8) supplemented with 10% heat-inactivated fetal calf serum (FCS) at densities between l and 10 x 10' cells/ml. For heat-shock experiments the cells were centrifuged, washed once with D22 minus methionine, and resuspended at a density of l x 10' cells/ml in D22 minus methionine containing 10% dialyzed FCS. The culture was divided in half and 100 iuCi/ml ['Slmethionine (Amersham Corp., Arlington Heights, Ill .) was added to each culture 20 min after the beginning of the incubation period at 25°C (control) or 37°C (heat shock). At the end of the incubation the cells were centrifuged and the medium was removed .THE JOURNAL Of CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 579-583 © The Rockefeller University Press " 0021-9525/81/1...