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An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

Kofoed M, Milbury KL, Chiang JH, Sinha S, Ben-Aroya S, Giaever G, Nislow C, Hieter P, Stirling PC - G3 (Bethesda) (2015)

Bottom Line: We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles.We also use high-throughput approaches to describe the relative ts behavior of the alleles.By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.

View Article: PubMed Central - PubMed

Affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada.

No MeSH data available.


Related in: MedlinePlus

Detection of flanking barcodes in ts alleles by microarray. (A) The raw score of the barcodes detected in a pooled scrapate sorted by intensity for the 3′ downtag (top) and the 5′ uptag (bottom). (B) Comparison of downtag and uptag scores for each allele. 581 alleles had both barcodes represented on the microarray. For our purposes an arbitrary score cut-off of 100 was applied to group the data. Colored boxes indicate the groups chosen and the number of alleles in each group is shown in the upper left. Although most alleles retain a functional downtag, less than half have a functional uptag. (C) Competitive growth analysis of the ts allele collection outgrown at the indicated temperature. The microarray intensity score of each allele under control or experimental conditions is expressed as a ratio. The gray triangle highlights scores lower at 25°, whereas the white area shows higher scores. The numbers of alleles in each group are noted on the upper right. Note the shift of alleles into the white area at 37°. The average of three replicates is used in (C) and all axes are logarithmic scales.
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fig3: Detection of flanking barcodes in ts alleles by microarray. (A) The raw score of the barcodes detected in a pooled scrapate sorted by intensity for the 3′ downtag (top) and the 5′ uptag (bottom). (B) Comparison of downtag and uptag scores for each allele. 581 alleles had both barcodes represented on the microarray. For our purposes an arbitrary score cut-off of 100 was applied to group the data. Colored boxes indicate the groups chosen and the number of alleles in each group is shown in the upper left. Although most alleles retain a functional downtag, less than half have a functional uptag. (C) Competitive growth analysis of the ts allele collection outgrown at the indicated temperature. The microarray intensity score of each allele under control or experimental conditions is expressed as a ratio. The gray triangle highlights scores lower at 25°, whereas the white area shows higher scores. The numbers of alleles in each group are noted on the upper right. Note the shift of alleles into the white area at 37°. The average of three replicates is used in (C) and all axes are logarithmic scales.

Mentions: One potentially useful byproduct of the diploid-shuffle method is to link each ts allele to the cognate barcode assigned to the heterozygous deletion mutant corresponding to the gene of interest (Figure 1). However, because the promoter region of each ts allele is duplicated upstream of the 5′ barcode in the genome, the alleles may be integrated in such a way that the 5′ barcode is deleted or subsequently lost due to direct repeat recombination between the duplicated promoter (Figure 1A). The 3′ barcode from the deletion strain being targeted should remain intact due to selection of the URA3 marker that sits between the direct repeats on the 3′ end of both the allele and the original deletion strain. To measure the integrity of the barcodes and determine their use in pooled growth experiments, we performed barcode microarrays (Giaever et al. 2002) of the pooled ts collection either directly after scraping from the array plate or after 20 generations of growth at various temperatures. Analysis of the ts collection scrapate confirmed that the 3′ barcodes (downtags) were more likely to be intact (Figure 3A and Table S4). More than 500 of the 581 alleles with downtags on the array met our arbitrary cutoff of normalized signal intensity 100, whereas less than half of the 5′ barcodes (uptags) met this threshold (Figure 3A). Both tags were present for 256 of the alleles, whereas 260 had only downtags and only 30 scored strongly for uptags without having a detectable downtag (Figure 3B). Comparing the behavior of downtags after 20 generations of outgrowth across temperature showed a temperature-dependant loss of tag intensity. The ratios of tag scores at 25° and 30° cluster around 1, whereas the ratios of 37° to 25° are shifted to less than 1 (Figure 3C). Together these data show that most alleles in the collection can be interrogated using their 3′ barcode and open the door to pooled growth analyses of the ts collection under various environmental or stress conditions.


An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

Kofoed M, Milbury KL, Chiang JH, Sinha S, Ben-Aroya S, Giaever G, Nislow C, Hieter P, Stirling PC - G3 (Bethesda) (2015)

Detection of flanking barcodes in ts alleles by microarray. (A) The raw score of the barcodes detected in a pooled scrapate sorted by intensity for the 3′ downtag (top) and the 5′ uptag (bottom). (B) Comparison of downtag and uptag scores for each allele. 581 alleles had both barcodes represented on the microarray. For our purposes an arbitrary score cut-off of 100 was applied to group the data. Colored boxes indicate the groups chosen and the number of alleles in each group is shown in the upper left. Although most alleles retain a functional downtag, less than half have a functional uptag. (C) Competitive growth analysis of the ts allele collection outgrown at the indicated temperature. The microarray intensity score of each allele under control or experimental conditions is expressed as a ratio. The gray triangle highlights scores lower at 25°, whereas the white area shows higher scores. The numbers of alleles in each group are noted on the upper right. Note the shift of alleles into the white area at 37°. The average of three replicates is used in (C) and all axes are logarithmic scales.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555224&req=5

fig3: Detection of flanking barcodes in ts alleles by microarray. (A) The raw score of the barcodes detected in a pooled scrapate sorted by intensity for the 3′ downtag (top) and the 5′ uptag (bottom). (B) Comparison of downtag and uptag scores for each allele. 581 alleles had both barcodes represented on the microarray. For our purposes an arbitrary score cut-off of 100 was applied to group the data. Colored boxes indicate the groups chosen and the number of alleles in each group is shown in the upper left. Although most alleles retain a functional downtag, less than half have a functional uptag. (C) Competitive growth analysis of the ts allele collection outgrown at the indicated temperature. The microarray intensity score of each allele under control or experimental conditions is expressed as a ratio. The gray triangle highlights scores lower at 25°, whereas the white area shows higher scores. The numbers of alleles in each group are noted on the upper right. Note the shift of alleles into the white area at 37°. The average of three replicates is used in (C) and all axes are logarithmic scales.
Mentions: One potentially useful byproduct of the diploid-shuffle method is to link each ts allele to the cognate barcode assigned to the heterozygous deletion mutant corresponding to the gene of interest (Figure 1). However, because the promoter region of each ts allele is duplicated upstream of the 5′ barcode in the genome, the alleles may be integrated in such a way that the 5′ barcode is deleted or subsequently lost due to direct repeat recombination between the duplicated promoter (Figure 1A). The 3′ barcode from the deletion strain being targeted should remain intact due to selection of the URA3 marker that sits between the direct repeats on the 3′ end of both the allele and the original deletion strain. To measure the integrity of the barcodes and determine their use in pooled growth experiments, we performed barcode microarrays (Giaever et al. 2002) of the pooled ts collection either directly after scraping from the array plate or after 20 generations of growth at various temperatures. Analysis of the ts collection scrapate confirmed that the 3′ barcodes (downtags) were more likely to be intact (Figure 3A and Table S4). More than 500 of the 581 alleles with downtags on the array met our arbitrary cutoff of normalized signal intensity 100, whereas less than half of the 5′ barcodes (uptags) met this threshold (Figure 3A). Both tags were present for 256 of the alleles, whereas 260 had only downtags and only 30 scored strongly for uptags without having a detectable downtag (Figure 3B). Comparing the behavior of downtags after 20 generations of outgrowth across temperature showed a temperature-dependant loss of tag intensity. The ratios of tag scores at 25° and 30° cluster around 1, whereas the ratios of 37° to 25° are shifted to less than 1 (Figure 3C). Together these data show that most alleles in the collection can be interrogated using their 3′ barcode and open the door to pooled growth analyses of the ts collection under various environmental or stress conditions.

Bottom Line: We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles.We also use high-throughput approaches to describe the relative ts behavior of the alleles.By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes.

View Article: PubMed Central - PubMed

Affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada.

No MeSH data available.


Related in: MedlinePlus