Abstract
In vivo DNA-protein interactions are usually studied at the molecular level using DNA-degrading agents of low molecular weight. In order to be useful, macromolecular probes of chromatin structure, such as enzymes must first cross the cell membrane. In this paper we describe the introduction and evaluation of macromolecules with enzymatic activity into yeast spheroplasts treated with the polyene antibiotic nystatin. We report the low resolution analysis of chromatin structure in the promoter region of the Saccharomyces cerevisiae gene encoding DNA topoisomerase I by this technique using micrococcal nuclease and restriction enzymes.
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Refereneces
Almer A, Herz W (1986) Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PH05/PH03 locus in yeast. EMBO J 5:2681–2687
Axelrod JD, Majors J (1989) An improved method for photofootprinting yeast genes in vivo using Taq polymerase Nucleic. Acids Res 17:171–183
Bolard J (1986) How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim Biophys Acta 864:257–304
Burgoyne LA, Hewish DR (1978) The cell nucleus, Vol 4. Academic Press, pp 48–74
Carlson M, Botstein D (1982) Two differentially regulated mRNAs with different 5′ ends encode secreted intracellular forms of yeast invertase. Cell 28:145–153
Cerenghini S, Yaniv M (1984) Assembly of transfected DNA into chromatin: structural changes in the origin-promoter-enhancer region upon replication. EMBO J 3:1243–1253
Gross DS, Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57:159–197
Kitagawa T, Andoh T (1978) Stimulation by amphotericin B of uridine transport, RNA synthesis and DNA synthesis in density-inhibited fibroblasts. Exp Cell Res 115:37–46
Kornberg A (1980) DNA replication. Freeman and Company, San Francisco, pp 377–379
Kuwano M, Akiyama S, Endo H, Koga M (1972) Potentiation of fusidic acid and lentinian effect upon normal and transformed fibroblastic cells by amphoterician B. Biochem Biophys Res Commun 49:1241–1248
Marquet R, Colson P, Matton AM, Housson C (1988) Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations. J Biol Struct Dynam 5:839–857
Medoff G, Kobayashi GF, Kwan CN, Schlessinger D, Vonkov P (1972) Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proc Natl Acad Sci USA 69:196–199
Mueller PL, Wold B (1989) In vivo footprint of a muscle specific enhancer by ligation mediated PCR. Science 246:780–786
Philippsen P, Stotz A, Scherf C (1991) DNA of Saccharomyces cerevisiae. Methods Enzymol 194:169–181
Plevani P, Badaracco G, Mamiroli N, Cassani G (1975) In vivo and in vitro effects of rifampicin and streptolydigin on transcription of Kluyveromyces lactis in the presence of nystatin. Nucleic Acids Res 2:239–255
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbbr, New York
Sherman F, Fink GR, Lawrence C (1983) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Thrash C, Bankier AT, Barrel BG, Sternglanz R (1985) Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene. Proc Natl Acad Sci USA 82:4374–4378
Wu C (1980) The 5′ end of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. Nature 286:854–860
Wu C, Bingham PM, Livak KJ, Holmgren R, Elgin SCR (1979) The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence. Cell 16:797–806
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Communicated by C.P. Hollenberg
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Venditti, S., Camilloni, G. In vivo analysis of chromatin following nystatin-mediated import of active enzymes into Saccharomyces cerevisiae . Molec. Gen. Genet. 242, 100–104 (1994). https://doi.org/10.1007/BF00277353
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DOI: https://doi.org/10.1007/BF00277353