Microbial Determinations by Flow Cytometry

Hutter, K.‐J.; Eipel, H. E.

doi:10.1099/00221287-113-2-369

Cited by 202 publications

(107 citation statements)

References 3 publications

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“…Immunoltuorescent staining of microtubules was carried out as described by IGlmartin and Adams (1984) as modified by Jacobs et ai. (1988) Eipel (1979) using the DNA stain propidium iodide (Sigma Chemical Co., St. Louis, MO). Stained cells were analyzed on a Becton Dickinson FACSCAN ® flow cytometer using the CELLFIT and LYSYS software packages to obtain and analyze data.…”

Section: Cytological Techniquesmentioning

confidence: 99%

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

Winey

Hoyt

Chan

et al. 1993

The Journal of Cell Biology

146

139

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Abstract. The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional lethal mutation ndcl-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in (31 after a-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.SEMBLY of the mitotic spindle in a eucaryotic cell is dependent on the formation of two centrosomelike organelles from which spindle microtubules emanate. The apparent duplication of centrosomal components to generate spindle poles is well described cytologically (reviewed by McIntosh, 1983;Brinkley, 1985;Sluder, 1989), but few molecular details of the underlying mechanism are known. The relevant organeLle in the yeast Saccharomyces cerevisiae is the spindle pole body (SPB)/ which is situated within the nuclear envelope (reviewed by Winey and Byers, 1992). As the sole microtubule organizing center in S. cerevisiae, the SPB forms microtubular arrays in both the cytoplasm and the nucleus. Electron microscopy of wild-type and mutant yeast strains has permitted description of the SPB duplication pathway and spindle formation (Byers, 1981a;Winey et al., 1991). A crucial early step in this pathway is the formation of the satellite on the outer surface of the half-bridge structure adjacent to the extant SPB. At START in G1, the satellite-bearing SPB is transformed

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Section: Cytological Techniquesmentioning

confidence: 99%

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

Winey

Hoyt

Chan

et al. 1993

The Journal of Cell Biology

146

139

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“…Flow Cytometry-Cells were prepared for flow cytomtery essentially as described by Hutter and Eipel (1979). Approximately 5 ϫ 10 6 cells were collected and fixed in 1 ml of 70% ethanol for 10 min.…”

Section: Materials-chemicalsmentioning

confidence: 99%

Casein Kinase II Is Required for Cell Cycle Progression during G1 and G2/M in Saccharomyces cerevisiae

Hanna

Rethinaswamy

Glover

1995

Journal of Biological Chemistry

208

198

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The catalytic subunit of Saccharomyces cerevisiae casein kinase II (Sc CKII) is encoded by the CKA1 and CKA2 genes, which together are essential for viability. Five independent temperature-sensitive alleles of the CKA2 gene were isolated and used to analyze the function of CKII during the cell cycle. Following a shift to the nonpermissive temperature, cka2 ts strains arrested within a single cell cycle and exhibited a dual arrest phenotype consisting of 50% unbudded and 50% largebudded cells. The unbudded half of the arrested population contained a single nucleus and a single focus of microtubule staining, consistent with arrest in G 1 . Most of the large-budded fraction contained segregated chromatin and an extended spindle, indicative of arrest in anaphase, though a fraction contained an undivided nucleus with a short thick intranuclear spindle, indicative of arrest in G 2 and/or metaphase. Flow cytometry of pheromone-synchronized cells confirmed that CKII is required in G 1 , at a point which must lie at or beyond Start but prior to DNA synthesis. Similar analysis of hydroxyurea-synchronized cells indicated that CKII is not required for completion of previously initiated DNA replication but confirmed that the enzyme is again required for cell cycle progression in G 2 and/or mitosis. These results establish a role for CKII in regulation and/or execution of the eukaryotic cell cycle.Casein kinase II (CKII) 1 is a serine/threonine protein kinase which is ubiquitous among eukaryotic organisms (for review, see Issinger, 1993;Pinna, 1990;Tuazon and Traugh, 1991). The enzyme is composed of a catalytic ␣ and regulatory ␤ subunit that combine to form a native ␣ 2 ␤ 2 holoenzyme which is constitutively active in vitro. How (and indeed whether) the enzyme is regulated in vivo is unknown, though regulation via allosteric effectors (e.g. polyamines), covalent modification, cellular redistribution, and substrate-directed effects have all been proposed. CKII recognizes a Ser of Thr residue followed by a series of acidic residues and phosphorylates a broad and intriguing spectrum of both nuclear and cytoplasmic substrates.Although the physiological role of CKII is not known, several lines of evidence suggest a role for the enzyme in cell prolifer-

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“…Visualization of bud scars was achieved by staining with 0.1% Calcofluor 100 M (Cyanamid, Bound Brook, NJ) (Pringle et al, 1991). Differential interference contrast microscopy was performed with a Nikon Microphot FX microscope by using ethanol-fixed cells (Hutter and Eipel, 1979) mounted in water.…”

Section: Microscopymentioning

confidence: 99%

“…Yeast cells were prepared for flow cytometry by fixation in ethanol and staining with propidium iodide (Sigma) (Hutter and Eipel, 1979 …”

Section: Microscopymentioning

confidence: 99%

Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

McGrew

Goetsch

Byers

et al. 1992

MBoC

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Mutations in the ESP1 gene of Saccharomyces cerevisiae disrupt normal cell-cycle control and cause many cells in a mutant population to accumulate extra spindle pole bodies. To determine the stage at which the esp1 gene product becomes essential for normal cell-cycle progression, synchronous cultures of ESP1 mutant cells were exposed to the nonpermissive temperature for various periods of time. The mutant cells retained viability until the onset of mitosis, when their viability dropped markedly. Examination of these cells by fluorescence and electron microscopy showed the first detectable defect to be a structural failure in the spindle. Additionally, flow cytometric analysis of DNA content demonstrated that massive chromosome missegregation accompanied this failure of spindle function. Cytokinesis occurred despite the aberrant nuclear division, which often resulted in segregation of both spindle poles to the same cell. At later times, the missegregated spindle pole bodies entered a new cycle of duplication, thereby leading to the accumulation of extra spindle pole bodies within a single nucleus. The DNA sequence predicts a protein product similar to those of two other genes that are also required for nuclear division: the cut1 gene of Schizosaccharomyces pombe and the bimB gene of Aspergillus nidulans.

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Microbial Determinations by Flow Cytometry

Cited by 202 publications

References 3 publications

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

Casein Kinase II Is Required for Cell Cycle Progression during G1 and G2/M in Saccharomyces cerevisiae

Requirement for ESP1 in the nuclear division of Saccharomyces cerevisiae.

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