Slit Scanning of <i>Saccharomyces cerevisiae</i> Cells: Quantification of Asymmetric Cell Division and Cell Cycle Progression in Asynchronous Culture

Block, David E.; Eitzman, P. D.; Wangensteen, Jeffrey D.; Srienc, Friedrich

doi:10.1021/bp00006a015

Cited by 34 publications

(20 citation statements)

References 32 publications

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“…However, the height of the fluorescence impulse of the latter cell is significantly decreased since the DNA is concentrated in two nuclear regions. A detailed description of this "slit scanning" flow cytometry measurement arrangement has been given previously (4). This population of cells exists in cultures treated with chloramphenicol as well as in untreated cultures.…”

Section: Resultsmentioning

confidence: 99%

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

et al. 1994

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The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identity the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of 0-galactosidase from these nrd-lacZ fusions in single-copy plasmids was Ribonucleotide reductase (RR) catalyzes the enzymatic reduction of ribonucleotides to deoxyribonucleotides and is the first enzyme in the pathway unique to DNA replication in Escherichia coli. The two nonidentical subunits of RR encoded by the nrdA and nrdB genes (20) are transcribed as a 3.2-kb polycistronic mRNA (23). The nrd operon has been sequenced, including approximately 3 kb of the upstream flanking sequence (8). The nrd promoter region was analyzed, and the operon's start of transcription was mapped at 110 bp upstream of the nrdA AUG start codon (50). Conditions that result in an increased mass/DNA ratio result in increased levels of RR (17). The synthesis of RR mRNA increases when DNA synthesis is specifically inhibited (17,18,23). Protein synthesis is necessary for both the initial induction and the continued increase of nrd mRNA synthesis during thymine starvation. Studies with cultures synchronized by either repeated phosphate starvation or sucrose gradient centrifugation demonstrated that the expression of the nrd operon in E. coli is cell cycle regulated (48). nrd mRNA synthesis increased at a time corresponding to the initiation of DNA replication. This type of regulation is similar to that observed in eukaryotic organisms (2,(13)(14)(15)(16)35 Flow cytometry is widely used in the analysis and sorting of viable heterogeneous cell populations. In the past decade, flow cytometry has been used for measuring cell cycle-related properties of a variety of mammalian cell cultures and has had major impact on cell cycle studies of mammalian cells. Recently, flow cytometry techniques have been successfully employed in cell cycle studies in the yeast Saccharomyces cerevisiae (11,12,43) and in DNA replication studies in the bacterium E. coli (6,38,41,(44)(45)(46)(47)52).In this study, we have used a series of nrd-lacZ fusion plasmid constructs to determine the cis-acting elements of the nrd regulatory region that are required for cell cycle-regulated nrd expression. Two different experimental approaches were used. In synchronized cultures, DNA replication and lac mRNA produced from the nrd-lac fusion located on singlecopy-number vectors as well as the nrd mRNA produced from the chromosomal gene were analyzed. Alteration in expression caused by mutations in the nrd promoter of the nrd-lac fusion on the vector could be compared with the chromosomal nrd promoter by comparing the lac mRNA and the nrd mRNA. In a second experimental approach, we used flow cytometry analysis with a combined immunofluorescent staining of n-galactosidase and a DNA-specific stain to quantitate 3-galacto-2415

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Section: Resultsmentioning

confidence: 99%

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

et al. 1994

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“…FACS and light microscopy analysis. Yeast cultures were grown in SC media and harvested in mid-logarithmic phase, and nuclei were stained with acriflavine by a modification of a method previously described (7). Cells were fixed in 70% EtOH for 20 min at 4°C, followed by a 20-min incubation at 22°C in 4 N HCl.…”

Section: Methodsmentioning

confidence: 99%

Association of Transcription Factor IIA with TATA Binding Protein Is Required for Transcriptional Activation of a Subset of Promoters and Cell Cycle Progression in Saccharomyces cerevisiae

Ozer

Lezina

Ewing

et al. 1998

Molecular and Cellular Biology

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The general transcription factor IIA (TFIIA) interacts with the TATA binding protein (TBP) and promoter DNA to mediate transcription activation in vitro. To determine if this interaction is generally required for activation of all class II genes in vivo, we have constructed substitution mutations in yeast TFIIA which compromise its ability to bind TBP. Substitution mutations in the small subunit of TFIIA (Toa2) at residue Y69 or W76 significantly impaired the ability of TFIIA to stimulate TBP-promoter binding in vitro. Gene replacement of wild-type TOA2 with a W76E or Y69A/W76A mutant was lethal in Saccharomyces cerevisiae, while the Y69F/W76F mutant exhibited extremely slow growth at 30°C. Both the Y69A and W76A mutants were conditionally lethal at higher temperatures. Light microscopy indicated that viable toa2 mutant strains accumulate as equal-size dumbbells and multibudded clumps. Transcription of the cell cycle-regulatory genes CLB1, CLB2, CLN1, and CTS1 was significantly reduced in the toa2 mutant strains, while the noncycling genes PMA1 and ENO2 were only modestly affected, suggesting that these toa2 mutant alleles disrupt cell cycle progression. The differential effect of these toa2 mutants on gene transcription was examined for a number of other genes. toa2 mutant strains supported high levels of CUP1, PHO5, TRP3, and GAL1 gene activation, but the constitutive expression of DED1 was significantly reduced. Activator-induced start site expression for HIS3, GAL80, URA1, and URA3 promoters was defective in toa2 mutant strains, suggesting that the TFIIA-TBP complex is important for promoters which require an activator-dependent start site selection from constitutive to regulated expression. We present evidence to indicate that transcription defects in toa2 mutants can be both activator and promoter dependent. These results suggest that the association of TFIIA with TBP regulates activator-induced start site selection and cell cycle progression in S. cerevisiae.The RNA polymerase II general transcription factors are an evolutionarily conserved set of proteins required for the regulation and recognition of specific promoter start sites (reviewed in references 56 and 60). In higher eukaryotes, the general transcription factor IID (TFIID) binds to core promoter elements and can nucleate the assembly of an active preinitiation complex in vitro (reviewed in reference 10). TFIID consists of the TATA binding protein (TBP) and TBPassociated factors (TAF II s), which modify the promoter recognition and transcriptional activities of TBP (reviewed in reference 75). In addition to the TAF II s, multiple other factors can associate with TBP and regulate transcription initiation by modulating the binding of TBP to the core promoter (5,19,27,35,48,52). The general transcription factor IIA (TFIIA) is a positive modulator of TBP binding to TATA box elements and is essential for regulated transcription in vitro. However, the precise function and general requirement for a TFIIA-TBP association in vivo have not been completely ...

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“…Other Methods. Flow cytometry was performed by the method described (22). Gap-repair mapping analysis of the cdc45-1 mutant was as described (3).…”

Section: Methodsmentioning

confidence: 99%

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

Hopwood

Dalton

1996

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

107

101

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We report the isolation and characterization of CDC45, which encodes a polypeptide of650 amino acids that is essential for the initiation of chromosomal DNA replication in the budding yeast, Saccharomyces cerevisiae. CDC45 genetically interacts with at least two members of the MCM (minichromosome maintenance) family of replication genes, CDC46 and CDC47, which are proposed to perform a role in restricting initiation of DNA replication to once per cell cycle. family of polypeptides are essential for the initiation of chromosomal DNA replication, minichromosome maintenance, and, in S. cerevisiae, are only present in the nucleus from the end of mitosis until the G1-S transition (4-6). However, MCM homologues in Schizosaccharomyces pombe (7,8), Drosophila melanogaster (9), X laevis (10) and mammals (11)(12)(13)(14) are located in the nucleus throughout the cell cycle where they associate with chromatin during GI and dissociate during S-phase. A common feature of all MCM proteins, however, is that they bind to, and are displaced from, chromatin in a manner consistent with them licensing DNA (15-18).It is clear from characterization of RLF activity in Xenopus that components besides MCM polypeptides are required for licensing activity (19). Cdc45p is a likely component of MCM complexes in yeast based on observations showing that CDC45 is essential for chromosomal replication and that two MCMs genetically interact with CDC45 (20). In this report we describe the isolation and characterization of the CDC45 gene and show that Cdc45p is assembled into a complex with one member of the MCM family, Cdc46p/Mcm5p. This suggests Cdc45p as an additional component of RLF complexes in budding yeast. MATERIALS AND METHODSYeast Strains, Media, Isolation of CDC45 and DNA Manipulations. Yeast media used in this study are as described in Guthrie and Fink (27). All yeast strains used in this study were isogenic with W303-la (MATa, ura3-52, trpl-1, ade2-1, lys2-801, leu2-3, his3-11, 15, canl-100 [psi']) and were either generated by direct gene replacement or by backcrossing the original mutant to W303-la at least three times. The CDC45 gene was isolated by rescue of the cdc45-1 cold-sensitive mutation at 12°C in the strain DBY2027 (from D. Botstein, Stanford University) essentially as described (6). Approximately 25,000 transformants were screened. Plasmids were recovered from primary yeast transformants showing plasmid-dependent cold-sensitive rescue by transformation into bacteria, and then tested for complementing activity by retransformation of plasmid DNA into the original cold-sensitive strain. Rescuing plasmids were characterized by restriction mapping. All sequencing was performed on both strands with customized primers using Applied Biosystems Taq Dye Deoxy Terminator cycle sequencing kits according to the manufacturers instructions.A strain expressing Cdc45p(1-650)-GFP was constructed by inserting the gene fusion at the CDC45 locus by a one-step gene replacement (6). A single HA epitope tag was inserted directly after c...

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Slit Scanning of Saccharomyces cerevisiae Cells: Quantification of Asymmetric Cell Division and Cell Cycle Progression in Asynchronous Culture

Cited by 34 publications

References 32 publications

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

Association of Transcription Factor IIA with TATA Binding Protein Is Required for Transcriptional Activation of a Subset of Promoters and Cell Cycle Progression in Saccharomyces cerevisiae

Cdc45p assembles into a complex with Cdc46p/Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication.

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