Properties of a membrane-attached form of the folded chromosome of Escherichia coli

Worcel, Abraham; Burgi, Elizabeth

doi:10.1016/0022-2836(74)90576-2

Cited by 212 publications

(100 citation statements)

References 17 publications

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“…1 B and C. FI is characterized by an S value of about 4600, and FIT by an S value of about 3000. The S values were determined using the E. coil membrane-attached folded chromosome as a marker (13,14). The S value of the E. coil chromosome (about 4600 S) was determined using T4 bacteriophage (1000 S) as a marker (15).…”

Section: Methodsmentioning

confidence: 99%

Isolation of folded chromosomes from the yeast Saccharomyces cerevisiae.

Piñon¹,

Salts²

1977

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

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Two fast-sedimenting chromatin complexes with sedimentation velocities of approximately 4600 and 3000 S can be isolated from logarithmically growing diploid Saccharomyces cerevisiae cells. The DNA in both structures appears to be folded into at least 60 domains and characterized by a negative superhelical density. Sensitivity to proteases and insensitivity to RNases suggest ta proteins an not RNA are important in maintaining the organization of the chromosomes in both structures. The 4600S and 3000S complexes represent folded genomes isolated from diploid cells in the G2 and GI stages of the cell cycle, respectively.Recent studies (1-3) have provided a rather consistent and unified picture of the subunit structure of chromatin as a flexible chain of spherical particles (nucleosomes), but information on how the nucleosomes are organized into higher order structures is still fragmentary. Although higher eukaryotic cell types provide suitable material for cytological or ultrastructural studies, it seems clear that at present the very large genomes of such cells will make it difficult to carry out well-defined physical and biochemical studies on the long-range features of chromosome organization. The microbial eukaryote, Saccharomyces cerevisiae, with a genome size about 3.3 times that of Escherichia coli (4), may provide a more amenable system for these types of studies. The work reported here indicates the feasibility of using the yeast system in studying the structural and functional aspects of chromosome organization in a eukaryotic cell.We have found that the nuclear genome from logarithmically growing diploid cells can be isolated as two compact, fast-sedimenting chromosome complexes containing RNA, protein, and a trace of membrane components and characterized by sedimentation velocities of approximately 4600 S and 3000 S. The DNA in both structures is characterized by a negative superhelical density, as judged by sedimentation studies with ethidium bromide (EtBr Preparation of Chromosome Complexes. Logarithmic (1 to 1.5 X 107 cells per ml) cultures of strain 131 (10-30 ml) were harvested by centrifugation (2 min at 1150 X g). After the cell pellet was washed twice in cold glass-distilled water, it was resuspended in 5 ml of 75 mM thioglycollate/0. 1 M Tris-HCl at pH 8.8, made up just before use, and incubated for 15 min at 00. The cells were then washed twice in cold glass-distilled water, resuspended in 2.5 ml of 1 M sorbitol and 2% (vol/vol) glusulase (Endo Laboratories), and incubated for 10-15 min at 370. Spheroplast formation was monitored by phase microscopy. In all cases reported here, over 95% of the cells had been converted to spheroplasts by 10-15 min. The spheroplasts were washed by centrifugation (4 min at 1150 X g) three times with 5 ml of cold 1 M sorbitol. After each centrifugation the spheroplast pellet was resuspended very gently with a pasteur pipette. The last centrifugation was done in a small (10 X 75 mm) test tube. Spheroplasts were lysed by a modification of the method used by H...

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Section: Methodsmentioning

confidence: 99%

Isolation of folded chromosomes from the yeast Saccharomyces cerevisiae.

Piñon¹,

Salts²

1977

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

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“…Worcel and Burgi (1974) (Marunouchi and Messer, 1973). This conflict was recently resolved by Korch et al (1976).…”

Section: Ultraviolet Radiation Damage and Repairmentioning

confidence: 95%

“…Lysis at 0-4oC results in 3200S nucleoids, whereas lysis at 20°C yields nucleoids with a mean sedimentation coefficient of 1600S. The more rapidly-sedimenting nucleoids were shown to be associated wi th a specific fraction of the cell membrane due to the presence of specifïc membrane proteins, lipids, and phospholipids (Pettijohn et al ~ 1973;Worcel and Burgi, 1974). Direct examination of such nucleoids in the electron microscope also revealed membrane fragments as.sociated with the DNA (Pettijohn et al~ 1973;Delius and Worcel, 1974).…”

Section: Ultraviolet Radiation Damage and Repairmentioning

confidence: 99%

Rate zonal density gradient ultracentrifugation analysis of repair of radiation damage to the folded chromosome of Escherichia coli

Ulmer¹

1978

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“…phr-l rec{l argt3 Sancar et al . (1979) Twigg & Sherratt(1980) Sutcl iffe (1978) Srosrus (1984) Hershf ield et al (1974) (Miller, 1972) G. Hershey's Salts (Worcel and Burgi , 1974) …”

Section: ¿¿ Hedia and Cø¡mpnly Used Solutionsmentioning

confidence: 99%