FIG. 4.

PRY3 5′ cap analysis by RNA ligase-mediated RACE. Transcript-specific RACE PCR products were analyzed by agarose gel electrophoresis. Data shown are representative of three independent experiments each using two independent, gene-specific primer sets demonstrating transcript specificity. For both PRY3- and STE2-specific RACE reactions, amplification products in the “no cap” lanes indicate the RNA adaptor ligated to the exposed 5′ phosphates of uncapped transcripts. “Control” lanes indicate completeness of the CIP reaction. Amplification products in the “cap” lanes indicate the transcripts in which the RNA adaptor ligated to the newly exposed 5′ phosphate groups of capped transcripts following the removal of the 5′ cap by TAP. “NT” represents no-template control for PCRs. Lanes 1, 16, 17, and 25 are loaded with a 100-bp ladder. PCR products were isolated from lanes 3, 6, 7, 10, 13, 14, 22, and 23 and sequenced. (A) PRY3 RLM-RACE results for both primer sets used. Sequencing results from PCR products from lanes 3, 7, 10, and 14 demonstrated 5′ termini at +452 relative to the AUG translation initiation codon. Sequencing results from lanes 6 and 13 demonstrated 5′ termini at −76. (B) STE2, an α-factor-induced transcript which has a known 5′ transcriptional start site of −31 relative to the initiating AUG, was used as a control (results shown for primer set 1).