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| Status |
Public on Sep 18, 2008 |
| Title |
Global mRNA expression analysis in myo1 delta strains of the budding yeast Saccharomyces cerevisiae |
| Organism |
Saccharomyces cerevisiae |
| Experiment type |
Expression profiling by array
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| Summary |
The Saccharomyces cerevisiae MYO1 gene encodes the myosin type II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Deletion of the MYO1 gene prevents actomyosin-driven cytokinesis thereby activating an alternative mechanism that involves the synthesis of a remedial septum. Myo1p deficiency in yeast (myo1) also causes the formation of attached cells, abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, and increased chitin synthesis. To determine how the differential expression of genes is related to these diverse phenotypes, we analyzed the global mRNA expression profile of myo1 strains. Global mRNA expression profiles of myo1 strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes were identified with 263 up-regulated and 284 down regulated genes in the myo1 strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories.
Genes involved in cell wall assembly (GAS1, PSA1, CIS3, FIT1, WSC2), MAP kinase activity (SLT2), Rho1p Guanine Exchange Factor (ROM1), and regulation of cell proliferation (RAS1) were also differentially expressed in myo1 strains.
Conclusions:
We have presented a global mRNA expression analysis of yeast myo1 strains and hypothesized about possible correlations with morphological and biochemical phenotypes observed in these strains. We report 547 differentially regulated genes in the myo1 mutant strains. Genes involved in the control of cell proliferation, protein synthesis and maturation, DNA replication, and cell division processes were largely down regulated, suggesting a mechanism for delayed cell cycle progression and growth that involves coordinated regulation of these processes in myo1 strains. Other genes involved in the cellular response to cell wall stress and cell wall organization were largely up-regulated suggesting that cell wall biogenesis is important for the completion of cytokinesis and cell wall morphogenetic processes that may also be affected by myosin II deficiency. Gene set enrichment analysis indicates that stress response and protein biosynthesis gene categories are inversely correlated in this mutant.
Keywords: Comparative genomic hybridization
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| Overall design |
All the experiments were performed using Saccharomyces cerevisiae wild type and myo1 strains. Six biological replicates were performed for this experiment, three arrays comparing myo1 vs. wild type from (MGD353-46D vs.YJR6) and three from (YJR12 vs.YJR13). YJR6 is a myo1::HIS5 strain generated by homologous recombination in the parental haploid wild type strain, MGD-353-46D using a PCR based method. YJR12 (wild type) and YJR13 (myo1∆) strains were obtained as haploid segregants from a cross between YJR6 and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) or histidine dropout media (CSM-HIS-) with continuous shaking at 200 rpm. Total RNA was extracted from 4 X 10^7 cells derived from triplicate biological replicate cultures of strains MGD343-56D, YJR6, YJR12, and YJR13 using the RNeasy Mini Kit for isolation of total RNA following manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following manufacturer’s instructions. 1.0 ug of total RNA from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). The amplified cRNA was labeled with 10mM Cyanine 5-CTP (Cy5) or Cyanine 3-CTP (Cy3) (Perkin Elmer Life Sciences). Labeled cRNA’s were purified with Qiagen RNeasy mini spin columns and dye incorporation was monitored on an Agilent Bioanalyzer. Hybridization of Cy5 and Cy3 labeled cRNA’s were performed using Yeast Oligo Microarray slides and hybridization kit from Agilent Technologies in 1012AG hybridization chambers (Sheldon Manufacturing) at 60ºC for 17 hours. Slides were washed at high stringency and scanned with a VersArray Chip Reader system (BioRad, Hercules, CA) at a resolution of 5um with detector sensitivity values between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software (Biodiscovery) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files. The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.01 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma program.
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| Contributor(s) |
Rodríguez-Quiñones JF, Irizarry RA, Gómez-Garzón D, Rivera-Molina FE, Rodríguez-Medina JR |
| Citation(s) |
18215314, 19426543 |
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| Submission date |
Sep 28, 2006 |
| Last update date |
Dec 06, 2012 |
| Contact name |
José R Rodríguez-Medina |
| E-mail(s) |
jorrodriguez@rcm.upr.edu
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| Organization name |
University of Puerto Rico Medical Sciences Campus
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| Department |
Biochemistry
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| Lab |
A-633
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| Street address |
PO BOX 365067
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| City |
San Juan |
| State/province |
PR |
| ZIP/Postal code |
00936-5067 |
| Country |
USA |
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| Platforms (1) |
| GPL884 |
Agilent-011447 Yeast Oligo Microarray G4140A (Feature Number version) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA97381 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE5931_RAW.tar |
15.9 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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