Worm Breeder's Gazette 9(3): 57
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have made gene-specific oligonucleotides to the 5' untranslated regions of actin mRNAs in order to map the transcriptional start sites of the four genes by primer extension. Primer extension dideoxy sequencing of actin gene 4 (X) mRNA confirmed earlier Sl-mapping results of a -41 to -43 transcriptional start site relative to the initiator AUG. The primer extension sequence was identical to the genomic sequence found upstream of actin gene 4. Primer extensions using actin gene 1 and/or 3 (V) and gene 2 (V) oligonucleotides yielded a unique 20 nucleotide sequence. This 20 nucleotide sequence is not the same as any of the genomic sequences adjacent to the primers used suggesting a splicing event during message maturation. This was not unexpected as each of these genespecific oligonucleotides abuts a consensus (genes 1 and 3) or near consensus (gene 2) TnCAG splice acceptor site. Actin genes 1 and 2 are oriented in opposite directions with their 5' ends toward each other. m e 20 nucleotide sequence which was derived from primer extensions is not in the 6 kb region containing actin genes 1 and 2. In order to find the source of this unique 5' sequence on the actin mRNAs, an oligonucleotide homologous to the 20 nucleotide sequence was synthesized and used to probe Southern blots of genomic DNA digested with several enzymes. m e results suggest that the 20 nucleotide sequence is part of a 900 bp tandem repeat with a repeat copy number greater than 10. We have screened a C. MBL4 library (provided by Chris Link) confirming the repetitive nature of the 20 nucleotide sequence and identifying phage of interest. One possible interpretation of these results is that the 5' ends of actin 1,2 and 3 mRNAs arise by splicing leaders that are transcribed from a tandem repeat elsewhere in the genome. Precedents for splicing mRNA leader sequences derived from a separate RNA molecule exist in trypanosomes, mouse hepatitis virus and flu virus. Alternatively, the primer entension sequence from actin 1,2 and 3 mRNAs is a giant artifact. We are currently trying to decide between these possibilities.