Identification and Characterization of Syntaxin 1 Antisense Variants in Limulus polyphemus
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A Limulus SMARTTM cDNA library screening resulted in the cloning of four syntaxin 1 homologs (referred to as Limulus syntaxin [Lim-syn] 1A, 1B, 1C, and 1D) (Wang, Y., Cao, Z., Xu, W., Kemp, M. D., McAdory, B. S., Newkirk, R. F., Ivy, M. T., and Townsel, J. G. (2004). Gene326:189–199) and two novel intron-retaining syntaxin 1-like variants, designated Limulus syntaxin variant [Lim-synV] 1A/1C and Lim-synV 1B/1D.
The variants exhibited high amino acid sequence identity with the four syntaxin 1 homologs. Specifically, Lim-synV 1A/1C and Lim-synV 1B/1D were homologous to Lim-syn 1A/1C and Lim-syn 1B/1D, respectively. Surprisingly, both Lim-synV 1A/1C and 1B/1D are unusual in that each has a poly A+ tail, an intron, and the common splice motif “GT–AG” at the intron–exon boundary. Exons one and two on the complementary transcript of Lim-synV 1B/1D are separated by a 150 bp intron beginning at #95/96 of the predicted sequences for Lim-syn 1B and 1D, respectively.
In contrast, examination of the approximately 3.17 kb Lim-synV 1A/1C clone indicated the inclusion of an insert of 1120 base pairs (bp) beginning at codon #37/38 of the predicted Lim-syn 1A and 1C cDNAs’ open reading frames (ORFs). Further, the intron sequence of Lim-synV 1A/C contained multiple stop codons and showed no significant homology to other known sequences as determined by a search of the GenBank database. Thus, the focus of this paper will be Lim-synV 1B/D exclusively.
To substantiate that an intron is retained in the full-length mRNA, two types of syntaxin cDNA fragments for Lim-syn 1B/D were generated by RT-PCR and analyzed on Northern blots. The products generated were a mixture of intron-retaining, as well as intron-spliced products. The syntaxin-like variants that retained the intron presumably are derived from a mRNA molecule that has not undergone splicing.
Although the significance of such intron-containing mRNAs in Limulus has not yet been elucidated, future studies of such variants may serve to broaden our knowledge concerning established splicing mechanisms as well as to focus attention on nonconventional concepts about gene product regulation.
KeywordsLimulus syntaxin cloning SNARE proteins splice variants
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