Isolation of Nuclei From Yeast

Bhargava, Madhu M.; Halvorson, Harlyn O.

doi:10.1083/jcb.49.2.423

Cited by 99 publications

(37 citation statements)

References 20 publications

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“…Fluorescence microscopy revealed that at the higher detergent concentration the nuclei appeared more disrupted, and electron microscopy showed that the nuclear membranes were removed from most nuclei. The overall recovery of DNA from A. nidulans was less than reported for S. cerevisiae (Bhargava & Halvorson, 1971;Doi & Doi, 1974;Wintersberger et al, 1973) or for the N. crassa slime mutant (Hsiang & Cole, 1973). However, our recovery was better than that of Hsiang & Cole (1973)~ who obtained a recovery of less than 10 % from wildtype N. crassa using the disruption method of Reich & Tsuda (1961).…”

Section: Nuclei In Situmentioning

confidence: 80%

“…The nuclear membrane was not complete in all nuclei and this may have been a direct result of our procedure. Membrane connexions between the nucleus and endoplasmic reticulum have been described in other fungi (Bracker, 1967) and it is also possible that breakage of such a connexion during homogenization resulted in the rupture of the nuclear membrane. Nuclei which lacked part of their envelope were, nevertheless, not disrupted.…”

Section: Nuclei In Situmentioning

confidence: 99%

“…Although protoplast formation using Aspergillus nidulans has been reported (Peberdy & Gibson, 1971) we have been unable to establish conditions under which it is possible to process sufficient quantities of mycelia to obtain nuclei for biochemical analysis. Physical disruption of the cell wall has been used to isolate nuclei from Saccharomyces cerevisiae (Bhargava & Halvorson, 1971)~ Achyla bisexualis (Jaworski & Horgen, 1973)~ and Neurospora crassa (Reich & Tsuda, 1961). Freezing of mycelium in liquid nitrogen before homogenization has been used to isolate N, crassa mitochondria (Minssen & Munkres, 1973) and polyribosomes (Sargent, 1973).…”

Section: Introductionmentioning

confidence: 99%

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The Isolation of Nuclei from the Filamentous Fungus Aspergillus nidulans

Gealt

Sheir-Neiss

Morris

1976

Journal of General Microbiology

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A method has been developed for isolating nuclei from the filamentous fungus Aspergillus nidulans. In this procedure, the mycelia from 14 to 16 h spore-derived cultures of A. nidulans M I , a stable diploid strain, were frozen with liquid nitrogen and homogenized in a Waring blender. After homogenization, the mycelia were warmed to 4 "C and the nuclei were purified from the homogenate by differential centrifugation followed by sedimentation through 2' I M-sucrose. The final nuclear yield was 15 to 20 ?<, based on DNA estimations. The purified nuclear pellet was free of whole cells. The morphology of the isolated nuclei resembled that of the in situ nuclei; they contained a nucleolus, chromatin, and had a surrounding double membrane. The purified nuclei were characterized by a DNA:RNA: protein ratio of I : 2.8 : 8.7.

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Section: Nuclei In Situmentioning

confidence: 80%

Section: Nuclei In Situmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Isolation of Nuclei from the Filamentous Fungus Aspergillus nidulans

Gealt

Sheir-Neiss

Morris

1976

Journal of General Microbiology

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“…Assessment of normality involves applying the concept of balanced growth. [1][2][3][4][5][6][7][8][9][10] As defined by Campbell,10 balanced growth is the doubling of every bio chemical component of the cells within the time of one cycle without change in the rate of growth. In considering this, it must be remembered that most synchronous cultures are batch cultures and therefore the medium changes in composition during the experiment, thus tending to make growth unbalanced.…”

Section: Methods Of Studying the Cell Cyclementioning

confidence: 99%

The Cell Cycle in Yeast-a Review

Duffus

1971

Journal of the Institute of Brewing

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“…Yeast RNA polymerase I, II, and III were purified from 60 g of frozen cells. Frozen cells were lysed in an Eaton press at 9000 lb/inch2 (62 MPa) (17) 10,000 X g. Nucleic acids in the clarified extract were removed by precipitation with 0.5% protamine sulfate followed by centrifugation. The protamine sulfate supernatant was diluted with two volumes of buffer A and adsorbed onto a DEAE-Sephadex A-25 column.…”

Section: Abstracimentioning

confidence: 99%

Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

Plevani¹,

Chang²

1977

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

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cation show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. DNA polymerases (DNA nucleotidyltransferases) from prokaryotic and eukaryotic cells do not initiate chains de novo (1, 2). In the replication of single-stranded coliphage DNA templates in vitro, a rifampicin-resistant RNA polymerase (RNA nucleotidyltransferase) is believed to be required for the conversion to double-stranded replicative form for kX174 and G4 DNAs (3). The conversion of phage M13 DNA depends on a rifampicin-sensitive RNA polymerase (3). The enzymatic replication of phage T4 and T7 DNA templates is stimulated X by the presence of rNTPs, although RNA polymerase activity is not required (4, 5). Ribonucleotide initiation of DNA synthesis had been implicated in the replication of polyoma DNA in isolated nuclei (6), slime molds (7), and mammalian cells (8).In the replication of synthetic DNA templates, DNA polymerase-a from calf thymus uses oligoribonucleotide initiators while DNA polymerase-,B does not use oligoribonucleotides efficiently (9). On single-stranded HeLa DNA, RNA polymerase from Escherichia coli initiates DNA synthesis catalyzed by HeLa cell DNA polymerase-a, but not by DNA polymerases-f and --y (10). Eukaryotic enzymes capable of initiating DNA synthesis have not been demonstrated. Two DNA polymerases are present in yeast (11,12). The properties of yeast DNA polymerase I are similar to those of mammalian DNA polymerase-a. DNA polymerase II is similar to prokaryotic DNA polymerases in that it has an associated 3 '-exonuclease (11-14). Experiments using synthetic DNA templates showed that DNA polymerase I readily accepts an oligoribonucleotide initiator while DNA polymerase II does not (12). The availability of yeast RNA polymerases permits investigation of enzymatic initiation of DNA synthesis in a homologous system. The results reported here, using a singlestranded circular DNA template, show that efficient enzymatic initiation occurs only with DNA polymerase I. All Tris-HCI, pH 7.9/0.5 mM dithiothreitol/0.1 mM EDTA/10% (wt/vol) glycerol]. The lysate was treated for 30 sec at 100 W in a Branson Sonifier to reduce the viscosity, and was then clarified by centrifugation for 20 min at 10,000 X g. Nucleic acids in the clarified extract were removed by precipitation with 0.5% protamine sulfate followed by centrifugation. The protamine sulfate supernatant was diluted with two volumes of buffer A and adsorbed onto a DEAE-Sephadex A-25 column. RNA polymerases were eluted with a linear gradient of (NH42S04 from 0.05-0.425 M. Active fractions were pooled for RNA polymerases I, II, and III, and the protein in each pool was precipitated by dialysis against 90% saturated (NH4)2SO4 in buffer A. The precipitates were collected by centrifugation, redissolved in 50 mM Tris-HCI, pH 7.9/0.1 mM EDTA/0.5 mM dithiothreitol/50% (wt/vol) glycerol, and stored at -20°.Enzyme Assays. DNA polymerase activities were assayed as described (12). One DNA polymerase ...

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Isolation of Nuclei From Yeast

Cited by 99 publications

References 20 publications

The Isolation of Nuclei from the Filamentous Fungus Aspergillus nidulans

The Isolation of Nuclei from the Filamentous Fungus Aspergillus nidulans

The Cell Cycle in Yeast-a Review

Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

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