Figure 2.—

Analysis of Doa isoform-specific transcripts in a mutant and UAS-directed expression lines. The northern transfers in both A and B were probed with a 1.8-kb partial cDNA corresponding to the catalytic domain and the N-terminal noncatalytic domain of the 55-kDa coding transcript, which reveals all Doa isoforms (Yun et al. 1994; Kpebe and Rabinow 2008). Transfers were reprobed with rp49 as a loading control. (A) The P element inserted in l(3)s2784 primarily reduces levels of the 4.3-kb Doa transcript encoding the 105-kDa cytoplasmic kinase. Lane 1, wild type (C^S) female; lane 2, l(3)s2784/Doa^HD female. (B) GAL4-directed expression of specific Doa transcripts via EP and EY P transposons. Northern transfers characterized the isoform specificity of GAL4-directed expression of Doa^EP3080, Doa^EY11301, and Doa^EY08857on RNA from adult heads, probed with the Doa catalytic domain, revealing all isoforms. Exon-specific probes were also used (not shown). Lane 1, GMR-GAL4/+; Doa^EY11301/+; lane 2, GMR-GAL4/+; Doa ^EY08857/+; lane 3, wild type (C^S); lane 4, GMR-GAL4/+; Doa^EP3080/+. The EP3080 P element directs expression of the entire 55-kDa coding transcript and is homozygous viable, although it will not survive indefinitely without a balancer chromosome present. The EY11301 insertion expresses a transcript lacking the first (noncoding) exon of the 55-kDa coding transcript but including the entire open-reading frame (Figure 1). The P element in EY08857 is inserted immediately 5′ to the start site of the 4.3-kb transcript, which encodes the 105-kDa protein. Note the appearance of a predominant new transcript of slightly higher molecular weight than the normal 2.7-kb RNA (55-kDa coding transcript) in lanes 1 and 4, corresponding to transcripts originating in the inserted EY and EP P elements in these alleles. Amounts of the 4.3-kb transcript encoding the 105-kDa protein are unaffected in Doa^EP3080. Levels of a 4.3-kb transcript are increased when the Doa^EY11301 P element is activated, but we did not investigate its structure. It may correspond to the native 4.3-kb transcript or may be due to transcripts containing P-element sequences. In contrast, the 4.3-kb (105-kDa coding) transcript is expressed at notably higher levels compared with wild type when driven with GMR-GAL4 in Doa^EY08857, while the 55-kDa coding transcript at 2.7 kb remains unaffected (lane 2). A new transcript, slightly >4.3 kb, is also detected in RNA extracts of Doa^EY08857, again presumably due to transcription initiation within the inserted P element.