1.A Limulus SMART(TM) 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). Gene 326:189-199) and two novel intron-retaining syntaxin 1-like variants, designated Limulus syntaxin variant [Lim-synV] 1A/1C and Lim-synV 1B/1D. 2.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. 3. 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. 4. 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.5. 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.
"Even if the phylogenetic analysis of the SNARE domains was quite clear, there could also have been a misannotation of one of the Qa members, or it might be so divergent that it was not found in the genome database. The syntaxin 1 repertoire seems to be increased by alternative splicing in other organisms [55,56] with tissue-specific expression profiles and functions. Thus, another hypothesis is that Leishmania could produce both LmjF28.1470 "
[Show abstract][Hide abstract] ABSTRACT: Leishmania major is a protozoan parasite with a highly polarised cell shape that depends upon endocytosis and exocytosis from a single area of the plasma membrane, the flagellar pocket. SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors) are key components of the intracellular vesicle-mediated transports that take place in all eukaryotic cells. They are membrane-bound proteins that facilitate the docking and fusion of vesicles with organelles. The recent availability of the genome sequence of L. major has allowed us to assess the complement of SNAREs in the parasite and to investigate their location in comparison with metazoans.
Bioinformatic searches of the L. major genome revealed a total of 27 SNARE domain-containing proteins that could be classified in structural groups by phylogenetic analysis. 25 of these possessed the expected features of functional SNAREs, whereas the other two could represent kinetoplastid-specific proteins that might act as regulators of the SNARE complexes. Other differences of Leishmania SNAREs were the absence of double SNARE domain-containing and of the brevin classes of these proteins. Members of the Qa group of Leishmania SNAREs showed differential expressions profiles in the two main parasite forms whereas their GFP-tagging and in vivo expression revealed localisations in the Golgi, late endosome/lysosome and near the flagellar pocket.
The early-branching eukaryote L. major apparently possess a SNARE repertoire that equals in number the one of metazoans such as Drosophila, showing that the machinery for vesicle fusion is well conserved throughout the eukaryotes. However, the analysis revealed the absence of certain types of SNAREs found in metazoans and yeast, while suggesting the presence of original SNAREs as well as others with unusual localisation. This study also presented the intracellular localisation of the L. major SNAREs from the Qa group and reveals that these proteins could be useful as organelle markers in this parasitic protozoon.
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