Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines

Knop, Michael; Siegers, Katja; Pereira, Gislene; Zachariae, Wolfgang; Winsor, Barbara; Nasmyth, Kim; Schiebel, Elmar

doi:10.1002/(sici)1097-0061(199907)15:10b<963::aid-yea399>3.0.co;2-w

Cited by 998 publications

(716 citation statements)

References 26 publications

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“…A complete list of oligonucleotides and yeast strains used in this study is presented in Tables 1 and 2. Plasmid K643 was generated by inserting the DNA sequence for a 3xHA epitope amplified by PCR from pYM1 (Knop et al 1999) into BbsI-and BssHII-restricted pFA6a-MN-KanMX6 (Schmid et al 2004). K643 served as a template in PCRs using the primers listed in Table 2 to amplify MN-3HAϻKanMX6 cassettes.…”

Section: Plasmids and Yeast Strainsmentioning

confidence: 99%

“…The amplified fragments were framed by 35-50 bp of DNA sequence homologous with the 3Ј end excluding the stop codon (in-frame with the MNase coding sequence at the 3Ј end), and 35-50 bp of sequence homologous with the DNA downstream from the stop codon of the respective target gene. The DNA in the PCR reactions was precipitated with ethanol and used for transformation of yeast cells, as described (Knop et al 1999). After selection, proper integration was confirmed by PCR and/or Southern blot analysis.…”

Section: Plasmids and Yeast Strainsmentioning

confidence: 99%

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Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Merz¹,

Hondele²,

Goetze³

et al. 2008

Genes Dev.

168

213

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Synthesis of ribosomal RNAs (rRNAs) is the major transcriptional event in proliferating cells. In eukaryotes, ribosomal DNA (rDNA) is transcribed by RNA polymerase I from a multicopy locus coexisting in at least two different chromatin states. This heterogeneity of rDNA chromatin has been an obstacle to defining its molecular composition. We developed an approach to analyze differential protein association with each of the two rDNA chromatin states in vivo in the yeast Saccharomyces cerevisiae. We demonstrate that actively transcribed rRNA genes are largely devoid of histone molecules, but instead associate with the high-mobility group protein Hmo1.[Keywords: Chromatin; transcription; ribosomal DNA] Supplemental material is available at http://www.genesdev.org. Received December 11, 2007; revised version accepted February 29, 2008. In the yeast Saccharomyces cerevisiae (hereafter called yeast), >60% of total cellular transcripts are produced from the ribosomal DNA (rDNA) locus by a specific multiprotein enzyme, RNA Polymerase I (Pol I), and an assorted set of additional transcription factors (Reeder 1999;Moss and Stefanovsky 2002;Russell and Zomerdijk 2006). The basal Pol I transcription machinery has been largely defined and consists of two multiprotein complexes, the upstream activating factor (UAF) , and the core factor (CF) (Keys et al. 1994), as well as the TATA-box-binding protein (TBP) (Steffan et al. 1996).The template of RNA Pol I transcription, the yeast ribosomal RNA (rRNA) genes are located on the right arm of chromosome XII in a tandem array of 150-200 copies (Fig. 1), representing almost 10% of the yeast genome. Each of the repeats contains the Pol I-transcribed 35S rRNA gene (precusor of the 5.8S, 18S, and 25S rRNAs) and the RNA Polymerase III (Pol III)-transcribed 5S rRNA gene (Fig. 1). Three different regulatory DNA elements have been identified within the 35S rRNA gene sequence, two of which are located at the 5Ј end of the 35S rDNA, spanning only ∼170 base pairs (bp) (Musters et al. 1989;Kulkens et al. 1991): the upstream element (UE), which is the binding site for the UAF; and the core promoter (CP), where CF can be recruited (Fig. 1). The third element, called the enhancer (ENH), is located at the 3Ј end of the 35S transcription unit (Fig. 1) and was originally identified as exhibiting a stimulatory effect on Pol I transcription in experiments using episomal reporter systems Warner 1984, 1986). However, this sequence has been shown subsequently to be dispensable for RNA Pol I transcription in the chromosomal context (Wai et al. 2001).In each cell, Pol I-transcribed rDNA coexists in two different chromatin states (Conconi et al. 1989). In these studies, cells were treated with the DNA cross-linking drug psoralen, and it was shown that actively transcribed and transcriptionally inactive rRNA genes differed substantially in their degree of psoralen incorporation. The different migration behavior of the corresponding restriction fragments in agarose gel electrophoresis led to the terms s-ban...

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Section: Plasmids and Yeast Strainsmentioning

confidence: 99%

Section: Plasmids and Yeast Strainsmentioning

confidence: 99%

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Merz¹,

Hondele²,

Goetze³

et al. 2008

Genes Dev.

168

213

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“…Plasmid pYM9, kindly provided by E. Schiebel (The Beatson Institute for Cancer Research, Glasgow, UK; Knop et al, 1999) was used to amplify an integrative PrA-KanMX6 fusion cassette using primers pOLE1-S2 and pOLE1-S3. Purified PCR-fragments were transformed into wild-type and ts mutant strains.…”

Section: Generation Of Gfp-and Protein A-tagged Desaturase Allelesmentioning

confidence: 99%

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Tatzer¹,

Zellnig

Kohlwein

et al. 2002

MBoC

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The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C., and Yaffe, M.P. ). J. Cell Biol. 115, 1249 -1257. We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.

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“…The cdc13-1 and stn1-13 strains were obtained from M. Charbonneau (Ecole Normale Superìeure de Lyon, Lyon, France) (Grandin et al, 2001). Epitope tagging of EXO1 and MRE11 was performed by a PCR-based strategy (Knop et al, 1999;Hirano and Sugimoto, 2006). The CDC13-myc strain was constructed using the integration plasmid as described previously (Taggart et al, 2002).…”

mentioning

confidence: 99%

Cdc13 Telomere Capping Decreases Mec1 Association but Does Not Affect Tel1 Association with DNA Ends

Hirano

Sugimoto

2007

MBoC

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Chromosome ends, known as telomeres, have to be distinguished from DNA breaks that activate DNA damage checkpoint. Two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR), control not only checkpoint activation but also telomere length. In budding yeast, Mec1 and Tel1 correspond to ATR and ATM, respectively. Here, we show that Cdc13-dependent telomere capping attenuates Mec1 association with DNA ends. The telomeric TG repeat sequence inhibits DNA degradation and decreases Mec1 accumulation at the DNA end. The TG-mediated degradation block requires binding of multiple Cdc13 proteins. The Mre11-Rad50-Xrs2 complex and Exo1 contribute to DNA degradation at DNA ends. Although the TG sequence impedes Exo1 association with DNA ends, it allows Mre11 association. Moreover, the TG sequence does not affect Tel1 association with the DNA end. Our results suggest that the Cdc13 telomere cap coordinates Mec1 and Tel1 accumulation rather than simply covering the DNA ends at telomeres. INTRODUCTIONDNA double-strand breaks (DSBs) are induced by exogenous DNA-damaging agents and carcinogens or by endogenous by-products, including reactive oxygen species (Friedberg et al., 2006). The repair of DSBs is crucial for maintaining genome stability. All organisms respond to DSBs by promptly launching the DNA damage response. This response involves the recruitment of DNA repair factors and the activation of signal transduction pathways, often termed DNA damage checkpoint pathways (Zhou and Elledge, 2000). Activation of the checkpoint pathway induces cell cycle arrest and expression of genes required for DNA repair.The checkpoint signals are initiated through two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR) (Zhou and Elledge, 2000;Abraham, 2001). ATM and ATR are highly conserved among eukaryotes. ATR is closely related to Mec1 in the budding yeast Saccharomyces cerevisiae and Rad3 in the fission yeast Schizosaccharomyces pombe. ATM homologues are termed Tel1 in both budding and fission yeasts. Current evidence indicates that the Mre11-Rad50 -Nbs1 (Xrs2 in budding yeast) complex is the primary sensor that recruits ATR/ Mec1 and ATM/Tel1 to DSBs (Nakada et al., 2003a(Nakada et al., , 2004Falck et al., 2005;You et al., 2005). In budding yeast, there is compelling evidence that the Mre11-Rad50 -Xrs2 (MRX) complex collaborates with exonuclease 1 (Exo1) in the generation of single-stranded DNA (ssDNA) at DSB ends (Krogh and Symington, 2004). ATM/Tel1 interacts with the C terminus of Nbs1/Xrs2 to localize to DNA ends (Nakada et al., 2003a;Falck et al., 2005;You et al., 2005). Meanwhile, the ssDNA that is generated at the DSB is covered with replication protein A (RPA) (Wold, 1997;Krogh and Symington, 2004). RPA-covered DNA recruits ATR/Mec1 to a region near the DSB end (Zou and Elledge, 2003;Nakada et al., 2005). ATM and ATR activate the downstream kinases CHK1 and CHK2 with assistance of checkpoint mediators, including 53BP1, BRCA1, and MDC1 (Zhou and Elledge, 2000;Bakke...

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Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines

Cited by 998 publications

References 26 publications

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

Lipid-dependent Subcellular Relocalization of the Acyl Chain Desaturase in Yeast

Cdc13 Telomere Capping Decreases Mec1 Association but Does Not Affect Tel1 Association with DNA Ends

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