Fig. 6. Regulation of ix transcription. (A) Northern hybridization of female and male polyA+ RNA with probes from ix and ninaE. The arrow points to the position to which a 750 nucleotide molecule would migrate, as determined by a size marker run on the gel that was blotted (not shown). The relative abundance of female and male ix transcripts is given at the bottom, normalized to the amounts of ninaE transcript in each lane. (B) Scheme for RNase protection assay. A restriction map of the genomic region surrounding the putative ix translation start site (indicated by arrow labeled ‘Met...’) is shown, with the locations indicated of the putative transcription start site (as identified by 5' RACE, labeled ‘RACE start’), of the polyadenylation signal sequence (as identified by 3' RACE, labeled ‘polyA’), and of the potential intron from a transcript originating 5' to the RACE start (as identified by RT-PCR analysis, labeled ‘RT-PCR intron’). Aligned below the map is the full-length, 527 nucleotide probe used for the assay, which stretches from the MscI site to the 5'-most PstI site, and includes sequences from the T7-promoter-containing vector used to produce it (dashed region of arrow). Below the probe are the predicted protected fragments corresponding to the different potential ix transcripts. An unspliced transcript originating 5' to the MscI site would protect a probe fragment of 457 nucleotides, whereas an unspliced transcript originating at the site identified by 5' RACE would protect a probe fragment of 203 nucleotides. If a transcript originating 5' to the MscI site were spliced at the donor and acceptor sites identified by RT-PCR, this processed transcript would protect two probe fragments, 46 nucleotides and 132 nucleotides in length. (C) RNase protection assay. Female and male polyA+ RNA samples were each hybridized in solution with the probe shown in B, then digested with RNase and electrophoresed. Yeast RNA controls were also performed, either with (‘Y+’) or without (‘Y–’) RNase. Size markers are in lanes marked ‘M’ and the sizes of marker bands are indicated at right. The arrow points to the position to which a 203 nucleotide molecule would migrate.