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. 2022 Dec 12;11:e81086. doi: 10.7554/eLife.81086

Figure 3. Transcription-terminator-flanked cassettes prevent adjacent gene mis-regulation.

(A) Design of ‘insulated’ kanMX6-ins cassettes and proposed model. The DEG1 transcription terminator sequence was inserted 5′ of the TEF promoter and either the DEG1 or CYC1 terminator was inserted 3′ of the TEF terminator in pFA6a-kanMX6 (Longtine et al., 1998). “goi” in the cartoon is an abreviation for “gene of interest”. (B) A single representative trace showing growth of dbp1Δ::kanMX6 and dbp1Δ::kanMX6-ins strains compared to wild-type in YEP glycerol. Three independent replicates were performed and the doubling time for each replicate is shown in Figure 3—figure supplement 1A. (C) Western blots for Mrp51-3v5 levels in dbp1Δ::kanMX6 and dbp1Δ::kanMX6-ins cells compared to wild-type during mitotic exponential growth in rich media. For full blot scans see Figure 3—source data 1. (D) Quantification of western blots as in (C), with Mrp51 normalized to Tub1 (alpha tubulin). Data shown are two independent biological replicates. Statistical significance was determined by a one-way analysis of variance (ANOVA) adjusted for multiple comparisons using Dunnett’s multiple comparisons test where **padj < 0.005. (E) 5′ rapid amplification of cDNA ends (RACE) demonstrates the 5′ ends of transcripts produced in wild-type, dbp1Δ::kanMX6, and dbp1Δ::kanMX6-ins strains. Gel demonstrating 5′ end products sequenced for each sample and the exact 5′ mRNA end sequences listed in Figure 3—figure supplement 1B,C. (F) Schematics of improved selection cassettes cloned with terminators insulating the 5′ and 3′ cassette ends. (G) Top: schematic of the reversed orientation cassette inserted at the DBP1 locus. Bottom: a single representative growth curve of wild-type, dbp1∆::kanMX6, and dbp1∆::rev-kanMX6 cells grown in the non-fermentable carbon source glycerol. Four independent biological replicates were analyzed and doubling times for each replicate are shown in Figure 3—figure supplement 2A. (H) Design of two additional cassettes with minimal sequence overlap, to enable their paired use in the same strain, and insulated only on the 5′ cassette end. Growth data for these new cassettes in Figure 3—figure supplement 2B.

Figure 3—source data 1. Zipped folder containing uncropped tif image of western blot shown in Figure 3 with and without labels on the relevant samples and bands.

Figure 3.

Figure 3—figure supplement 1. DBP1 ORF replacement with kanMX6-ins cassettes yields wild-type MRP51 transcripts and growth rates.

Figure 3—figure supplement 1.

(A) Calculated doubling times of wild-type, dbp1∆::kanMX6, and dbp1∆::kanMX6-ins strains in the non-fermentable carbon source glycerol. Data from three independent replicates are shown and statistical significance was determined by one-way analysis of variance (ANOVA) corrected for multiple comparisons with Dunnett’s multiple comparison test where a *padj < 0.05. (B) PCR amplified 5′ ends of cDNA from wild-type, dbp1Δ::kanMX6, dbp1Δ::natMX6, dbp1Δ::kanMX6-ins1, and dbp1Δ::kanMX6ins-2. Colored dots represent the color each sample is depicted with in Figure 3E. Bottom image is darker exposure of the same gel to demonstrate lack of MRP51 LUTI in insulated cassette samples. See Figure 3—figure supplement 1—source data 1 for uncropped of gels. (C) The exact 5′ ends of transcripts sequenced to identify the start site of each transcript as well as the number of transcripts analyzed that mapped to a given position.
Figure 3—figure supplement 1—source data 1. Zipped folder containing uncropped tif image of agarose gel showing the amplified 5′ RACE products sequenced in Figure 3 and shown in Figure 3—figure supplement 1.
Figure 3—figure supplement 2. MRP51 mis-regulation is caused by cassette promoter-driven divergent transcription.

Figure 3—figure supplement 2.

(A) Calculated doubling times of wild-type, dbp1∆::kanMX6, and dbp1∆::rev-kanMX6 strains in the non-fermentable carbon source glycerol. Data from four independent replicates are shown and statistical significance was determined by use of a one-way analysis of variance (ANOVA) corrected for multiple comparisons with Dunnett’s multiple comparison test where a **padj < 0.005. (B) Growth curves of forward and reverse oriented 5′ insulated kanMX6 cassettes inserted at the DBP1 locus in the non-fermentable carbon source glycerol. For this experiment, a single biological replicate was analyzed. Cassettes used are shown in Figure 3H.