|
| Status |
Public on Nov 30, 2007 |
| Title |
Yeast replication isw2 nhp10 MMS 45min a |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
S.cerevisiae, 45min post alpha factor, 0.015% MMS, unreplicated DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: YTT3306 (MATa ade2-1::pRS402 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5+ isw2::NatMX nhp10::HphMX)
Collected 45 minutes post alpha factor release.
Treated with MMS at 0.015%
Unreplicated DNA
|
| Biomaterial provider |
Jack Vincent / Tsukiyama Lab
|
| Growth protocol |
Density transfer experiments were performed essentially as described to isotopically label newly replicated DNA (see the Fangman-Brewer lab website at UW). Cells were grown a minimum of 7 generations in minimal medium containing 13C and 15N as the sole carbon and nitrogen sources (dense media). Cells were synchronized with α-factor for 105 minutes. Cells were then filtered and transferred to complete media (YC) containing 12C and 14N (light media) in the continued presence of α-factor for 75 minutes prior to release (termed conditioning phase). MMS was added to 0.015% 15 minutes into the conditioning phase resulting in a 60-minute exposure during G1 arrest. After the conditioning phase, cells were filtered, washed, and released in light media in the presence or absence of 0.015% MMS. This sample was collected at 45 min after release.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Standard Yeast genomic prep: Bead beating + phenol:chloroform extraction.
Unreplicated DNA was separated from replicated in a CsCl gradient (see the Fangman-Brewer lab website at UW.)
The CsCl gradient was drip-fractionated. Fractions were then applied to slot blots and probed for DNA content. Fractions from the bottom of the gradient (the most dense fractions) contain unreplicated DNA, while fractions from the top of the gradient (least dense fractions) contain newly replicated DNA. Fractions containing unreplicated DNA (or replicated DNA) were pooled and EtOH precipitated.
|
| Label |
Cy3
|
| Label protocol |
0.5ug of each sample was labeled with either Cy3 or Cy5-dUTP (GE Biosciences). 21ul DNA solution was heat denatured at 100°C for 5 min then quick chilled in an ice slurry. The following was added while samples were still on ice: 5ul 10x dNTP Mix (1.2mM each dATP, dCTP, dGTP; 0.6mM dTTP; 10mM Tris 8.0), 3ul Cy-dUTP, 1ul exo- Klenow (50000 U/ml, New England Biolabs). Samples were incubated at 37°C for 1hr. Reaction was stopped by adding 5ul 0.5M EDTA. 60ul of 1mg/ml tRNA was added to each reaction and unincorporated nucleotides were removed using a Biospin 30 column (BioRad). Oppositely labeled samples (i.e. Cy3-replicated and Cy5-unreplicated) from the same timepoint were combined for cohybridization. 20ul polydA::dT (1mg/ml; Sigma) was added to each probe mix, then DNA was precipitated after addition of 1/10 volume 3M NaOAc and 2 volumes 100% ethanol.
|
| |
|
| Channel 2 |
| Source name |
S.cerevisiae, 45min post alpha factor, 0.015% MMS, replicated DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain: YTT3306 (MATa ade2-1::pRS402 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 RAD5+ isw2::NatMX nhp10::HphMX)
Collected 45 minutes post alpha factor release.
Treated with MMS at 0.015%
replicated DNA
|
| Biomaterial provider |
Jack Vincent / Tsukiyama Lab
|
| Growth protocol |
same as channel 1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
same as channel 1
|
| Label |
Cy5
|
| Label protocol |
same as channel 1
|
| |
|
| |
| Hybridization protocol |
• hybridization buffer:
3X SSC + 10%SDS + 1 mg/mL poly(dA)
• blocking agent:
no prehybridization
• slide blocking:
no prehybridization
• probe blocking:
1mg/mL polydA during hybridization
• wash procedure:
wash1: 1X SSC, 0.03% SDS
wash2: 1X SSC dip slides 15 times
wash3: 0.2X SSC: shake slides 75rpm for 20minutes
wash4: 0.05X SSC: shake slides 75rpm for 10 minutes
spin-dry slides in centrifuge 50g for 5 minutes.
• quantity of labelled target used:
all material generated from 0.5 ug of EcoRI digested genomic DNA
• time, concentration, volume, temperature:
16h, 26 microliters at 63°C
• Hybridization instrument:
Manual, TelChem hybridization chambers in waterbath
|
| Scan protocol |
Scanning hardware
GenePix 4000B scanner (Molecular Devices)
Image analysis
GenePix Pro v6.0 (Molecular Devices)
|
| Description |
Unreplicated vs Replicated DNA during MMS treatment
isw2 nhp10 double mutant strain
45 min time-point
|
| Data processing |
Percent replication for each spot was determined as described (Alvino, GM et al, Mol Cell Bio, 2007). The calculation of percent replication at each time point is dependent on two measurements; the percentage of total genomic replication in the population at each time point and the number of cycling cells over the entire time course.
• Total genomic % replication:
The total percent replication for the genome was determined after fractionation of each CsCl gradient. Fractions were blotted to a nylon membrane and then hybridized to a radiolabeled genomic probe (as described above). Percent replication was determined by quantifying the signal from each fraction (by phosphorimager), plotting the value for each fraction, and then comparing the area under the curve corresponding to replicated and unreplicated fractions (see http://fangman-brewer.genetics.washington.edu/density_transfer.html). The total % replication for this sample was as follows:
45min 14%
• Ratio of cycling cells:
The ratio of cycling cells to non-cycling cells was estimated from the maximum percentage of budded cells obtained during the time course. The maximum percent budded was 86% for isw2 nhp10 experiments.
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| |
|
| Submission date |
Sep 20, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Jack A. Vincent |
| E-mail(s) |
[email protected]
|
| Phone |
206-667-2986
|
| Organization name |
Fred Hutchinson CRC
|
| Department |
Basic Sciences
|
| Lab |
Tsukiyama
|
| Street address |
1100 Fairview Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL1914 |
| Series (1) |
| GSE9122 |
Yeast replication in the presence of MMS: WT and isw2 nhp10 strains |
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