1. Volume 18, Issue 4, Pages 491-498 (May 2005)
Negative Control Contributes to an Extensive Program of Meiotic Splicing in Fission Yeast 
Nicole Averbeck, Sham Sunder, Nicole Sample, Jo Ann Wise, Janet Leatherwood 
Molecular Cell 
Volume 18, Issue 4, Pages (May 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Meiosis-Specific Splicing of 12 pre-mRNAs
(A) pat1–114 strain F90 was shifted to 34°C at time 0 to induce meiosis. RT-PCR products from total RNA were resolved on 2% agarose gels and detected by ethidium bromide staining. Faint intermediate-sized bands for spo4, meu13, and SPAPB8E5.10 may be partially spliced intermediates or arise via use of cryptic splice sites, which is quite common in fission yeast (Alvarez and Wise, 2001).
(B) Gene annotation for meiotically spliced RNAs. The penultimate column indicates the classification of each gene according to the timing of transcript induction. In the last column, the first number indicates the position of the intron(s) assayed within the transcript, and the second number indicates the total number of introns.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 crs1 Is Induced and Spliced during Meiosis
(A) Northern blot of crs1 and rad25 transcripts after shifting the pat1–114 strain to 34°C.
(B) The same total RNA preparations were amplified by RT-PCR using primers specific for crs1, rem1, and mes1. Meiotic divisions MI and MII are indicated. The regions amplified are diagrammed (exons, open boxes; introns, lines). As a control, genomic DNA was amplified with the same primers.
(C) Meiosis was induced in diploid JLP560 by nitrogen starvation (time 0) and splicing assayed as in (B).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Effect of Expressing nmt1-crs1 in Vegetative Cells
(A) Micrograph showing cell cycle defects. A wild-type (wt) strain (F14) was transformed with either of two plasmids that express transcripts from the nmt1 promoter: pJL205 (GST control) or pJL556 (GST-crs1 insert). Cells were grown for 18 hr in medium lacking thiamine to derepress the promoter and stained with DAPI. Arrows indicate aberrant divisions and hypercondensed DNA.
(B) RT-PCR assays of splicing. Wt cells (F14) were transformed with either pJL554 carrying the crs1 coding region inserted into the nmt1 expression cassette or the empty vector pREP4x. After derepression as in (A), splicing was assayed by RT-PCR on total RNA from two independent transformants carrying pJL554 (T1, T2) or the empty vector pREP4x. Primer pair 1/2 amplifies cDNA produced from both the nmt1-crs1 plasmid pJL554 and chromosomal crs1, whereas 3/4 amplifies only cDNA derived from genomic crs1. For rem1 and cdc2, the primer targets lie within the translated region. Negative (no template) and positive (genomic DNA) controls are shown for each set of primers.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Analysis of Chimeric pre-mRNAs
(A) Splicing assays on RNA from cells expressing the crs1 coding region, including introns, under control of the rem1 flanking regions. Control (lane E): assay of endogenous crs1 splicing. Experimental (transformants T3 and T4): in pNSrem-crs, the crs1 sequences upstream of the translational start site and downstream of the stop codon were replaced by the corresponding regions from rem1.
(B) Test of a constitutively spliced pre-mRNA for regulation by the rem1 flanking regions. Controls (transformants T5 and T7): azr1 (encodes a predicted serine-threonine phosphatase) was expressed from its own flanking regions (pSSazr). Experimental (transformants T6 and T8): the azr1 ORF including the intron was expressed from a chimeric plasmid carrying rem1 sequences upstream of the translational start site and downstream of the stop codon (pSSrem-azr).
(C) Test of a constitutively spliced pre-mRNA for regulation by the crs1 flanking regions. Controls (transformants T9 and T11): SPAC926.07c (encodes a predicted dynein light chain) was expressed under control of its own upstream and downstream sequences (pSS926). Experimental (transformants T10 and T12): the SPAC926.07C ORF including introns was expressed from a chimeric plasmid carrying crs1 sequences upstream of the translational start site and downstream of the stop codon (pSScrs-926).
(D) Analysis of rem1/azr1 splicing over a meiotic time course. RNA was isolated from pat1–114 cells at the times indicated after shifting to the restrictive temperature. The band marked with an asterisk is most likely a PCR artifact. In all panels, splicing was assayed by RT-PCR using one flanking primer and one internal primer designed to give the same size products regardless of whether the sequences outside the ORF were native or heterologous (see Experimental Procedures and Supplemental Data). The small cytoplasmic SRP RNA was used as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions