Bp f

5' fragment 3' fragment

Figure 2, RNase H Digestion of RNA. Samples of 20jig of total RNA were incubated separately with an oligonucleotide that anneals to the exon 5 <E5r) for 30nun at 37°C and then Ih at 30 °C after addition of 0.8 U of RNase H (Boehringer Mannheim). The digested RNA was analysed by Northern Blot. Lane C denotes control non-digested RNA. The ,:P-labelled cDNA probe contained the fragment corresponding to positions 31-1,700bp in the cDNA sequence from COT.14 RNA molccular size markers (M) are indicated to the right. B. The diagram shows the products formed after binding the primer E5r to RNA and digesting with RNAsc H.

exon2-exon3... etc. mRNA, since the first in-frame ATG is located in the second exon 2. The second protein, translated from the trans-spliced mRNA composed by exons 1-2-3-23-4 etc. should also appear. The theoretical Mr of these translated proteins are 70,302 Da and 80,572 Da, respectively. The occurrence of the two forms of the COT enzyme in peroxisomes may be a direct consequence of this trans-splicing mechanism in the COT gene.

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