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Results: 5

1.
Figure 2

Figure 2. From: The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II.

Position-dependent effects of CTR9 deletion on transcription and silencing. (A) The wild-type strains JHY49 and JHY25 each contain a single copy of URA3 integrated 11.5 Kb and 55 Kb, respectively, downstream of the natural left telomere of chromosome XV, as diagrammed above. Into these strains were introduced npt1Δ (HM193, HM190), bna1Δ (HM192, HM189) and ctr9Δ (HM191, HM188) deletions. Cells were diluted serially 10-fold and plated on SC, 5FOA or SC -ura as indicated. (B) The wild-type YCB647 strain carries a single copy of URA3 integrated into the left telomere of chromosome VII, as diagrammed above. Into this strain, set1Δ (HM206), set2Δ (HM207), ctr9Δ (HM180) or paf1Δ (HM205) deletions were introduced. Cells were diluted serially 5-fold and plated on SC, 5FOA, or SC -ura as indicated. (C) The wild-type YCB761 strain contains a single copy of ADE2 integrated into the right telomere of chromosome V as shown above. This and derivatives carrying npt1Δ (JS692) or ctr9Δ (HM182) deletions were plated on SC containing limiting adenine.

Heather A Marton, et al. BMC Mol Biol. 2008;9:4-4.
2.
Figure 3

Figure 3. From: The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II.

Impaired induction of GAL loci in cells deficient in Paf1 or Ctr9. (A) Induction of GAL1 transcripts. Wild-type (HM200), ctr9Δ (HM201) and paf1Δ (HM202) strains were grown at 30°C in YEP supplemented with 2% raffinose. Galactose was added to a final concentration of 2%. RNA was isolated at 0, 5, 15, 30, 45 and 60 min after addition of galactose and GAL1 (upper panels) and ACT1 (lower panels) transcripts were detected by northern blotting (above). The GAL1 signal intensities were normalized to those of ACT1 and plotted as a function of time (below). Filled squares, wild-type; filled triangles, ctr9Δ ; filled circles, paf1Δ. (B) Induction of GAL10 transcripts. Isolates of wild-type (HM200), ctr9Δ (HM201) and paf1Δ (HM202) were grown at 24°C in media indicated above. Galactose was added as described and RNA was isolated at 0, 15, 30, 45 and 60 min after addition. GAL10 (upper panels) and ACT1 (lower panels) transcripts were detected by northern blotting (above). GAL10 signal intensities were normalized as in (A) and plotted together with data obtained from an independent experiment using a second set of wild-type, ctr9Δ and paf1Δ isolates. Filled and open squares, wild-type; filled and open triangles, ctr9Δ; filled and open circles, paf1Δ.

Heather A Marton, et al. BMC Mol Biol. 2008;9:4-4.
3.
Figure 4

Figure 4. From: The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II.

Deletion of CTR9 or PAF1 impairs displacement of histones from the GAL1 locus upon induction. Wild-type (HM200), ctr9Δ (HM201) and paf1Δ (HM202) strains were grown at 24°C in YEP supplemented with 2% raffinose. Galactose was added to a final concentration of 2%. Nuclei were isolated at 0, 10 and 90 min after induction and digested with 0, 7, 15, 50 or 100 U of micrococcal nuclease. (A) Micrococcal nuclease digests of nuclei from wild-type (left), ctr9Δ (middle) and paf1Δ (right) strains were deproteinized and fractionated by agarose gel electrophoresis; DNA was detected by ethidium bromide staining. Inverse images of the stained gels are shown. Positions of markers and their sizes (in kb) are indicated at left. (B) Distribution of micrococcal nuclease digestion products at the GAL1 locus. DNA from gels in (A) was transferred to nylon and hybridized to a probe representing basepairs +422 to +925 of the GAL1 gene. Signal was detected by phosphorimaging. (C) Distributions of micrococcal nuclease products from the 50 U micrococcal nuclease reactions in (B) were quantified by densitometry for each strain at each time point. The signal intensities are plotted in arbitrary units.

Heather A Marton, et al. BMC Mol Biol. 2008;9:4-4.
4.
Figure 5

Figure 5. From: The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II.

Depletion of Ctr9 or Paf1 delays removal of histone H3 and alters the distribution of RNA pol II upon induction of GAL1 transcription. (A) Schematic representation of the GAL1 locus. The TATA region, translation start site and ORF are indicated. Black bars below indicate positions of PCR probes used in ChIP assays. (B – G) Association of histone H3 (B – D) or RNA pol II (E – G) with 5' (B, E), middle (C, F) and 3' (D, G) regions of the GAL1 locus as a function of time after induction. Wild-type (HM200), ctr9Δ (HM201) and paf1Δ (HM202) strains were grown at 24°C in YEP supplemented with 2% raffinose. Galactose was added to final concentration of 2%. Samples were taken at 0, 5, 10, 20, 40, 60 and 90 min after induction. Association of histone H3 (B – D) and Rpb3 (E – G) with 5' (B, E), middle (C, F) and 3' (D, G) regions of GAL1 were assayed by ChIP and real-time PCR for wild-type (gray), ctr9Δ (peach) and paf1Δ (turquoise) strains. All samples were also assayed with primers specific for an intergenic region (B – D) or FBA1 (E – G) as standards for normalization of the GAL1-specific signals. The normalized association of H3 or Rpb3 with each region of GAL1 at 0 min was set to 1; all other values represent the fold difference relative to the 0 min sample. Each value is the average of two experiments; error bars correspond to the standard errors of the mean. Statistical significance of pairwise comparisons between wild-type and ctr9Δ or paf1Δ strains was determined using a two-tailed t-test. ***, P < 0.0005; **, P < 0.004; *, P < 0.04.

Heather A Marton, et al. BMC Mol Biol. 2008;9:4-4.
5.
Figure 1

Figure 1. From: The Paf1 complex promotes displacement of histones upon rapid induction of transcription by RNA polymerase II.

Gene occupancy by Ctr9 and Paf1. (A) Expression of endogenous, c-myc-tagged Ctr9 and Paf1 proteins. Protein from three Ctr9-myc-expressing isolates (HM198), three Paf1-myc-expressing isolates (HM199) or the untagged HM177 parental strain (U) were fractionated by SDS-PAGE. Protein was detected by immunoblotting with the 9E10 antibody. (B) Locations of gene-specific PCR probes. Each ORF is represented by a box. Light gray, genes whose expression decreased upon removal of Ctr9; dark gray, genes whose expression increased; white, genes whose expression remained unchanged. The positions and relative sizes of PCR products are indicated by dark bars above. (C) Assays of gene occupancy by ChIP. Association of Ctr9 and Paf1 with the regions indicated in (B) was monitored in cells maintained in YPD medium at 30°C. Upper panel, amplification of anti-myc immunoprecipitates; lower panel, amplification of input chromatin. The middle and upper bands in each lane represent amplified segments of the ORFs indicated (middle/upper) at bottom. The lowest band in each lane is an amplified fragment from the promoter region of ARN1, which was used as an internal reference [16]. (D) Correlation of transcriptional initiation frequency with gene occupancy by Ctr9 and Paf1. The relative signal intensities for each of six gene fragments associated with Ctr9-myc and Paf1-myc are plotted as a function of their transcriptional initiation frequencies as estimated in reference [25]. Diamonds, Paf1-myc; squares, Ctr9-myc; circles, untagged control. (E) Effect of acute Ctr9 depletion on intracellular NAD+ levels. Wild-type (HM167) and ctr9Δ (HM158) strains containing the Ctr9 expression plasmid pgCTR9M2 were initially grown in SC -leu + 2% galactose. Cells were then transferred to SC -leu + 2% glucose and maintained at 30°C. Relative intracellular levels of NAD+ were measured at 0, 4, 8 and 12 hours after switch to glucose. NAD+ levels are expressed on the Y axis as absorbance at 340 nm. Values represent mean and standard deviation (n = 3).

Heather A Marton, et al. BMC Mol Biol. 2008;9:4-4.

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