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Correction of an amontillado (amon) cDNA artifact and identification of single nucleotide polymorphisms in the amon gene.

Rayburn, Lowell Y.M., Semil P. Choksi, and Michael Bender. Department of Genetics, Life Sciences Building, University of Georgia, Athens, GA 30602.

      Comparing genomic sequence (Adams et al., 2000) with an amon cDNA sequence (Siekhaus and Fuller, 1999) showed that the first 43 nucleotides of this cDNA do not align with upstream amon genomic sequences. They do, however, match sequences within more 3' areas of the cDNA (see Figure 1A), suggesting that the first 43 nucleotides may be an artifact of cDNA cloning.  The 43 nucleotides can be separated into two components. Nucleotides 3-21 (bold type) match those at positions 3268-3286 (bold type) of the cDNA, and nucleotides 18-47 (underlined) reverse complement the sequence from 3225-3254 (underlined) in the 3’ end of the cDNA. The Berkeley Drosophila Genome Project (BDGP) database contains 12 amon EST sequences, eleven of which extend further 5’ than the cDNA reported by Siekhaus and Fuller (1999) and do match genomic sequences. The two amon ESTs with the greatest 5' extent (BDGP clone ID #RE06156 and #RE58333) lengthen amon cDNA sequences by 64 nucleotides (Figure 1B). Good matches (4/6 nucleotides, underlined) to a consensus Drosophila Initiator sequence at the 5' end of the cDNA and to the Drosophila Downstream Promoter Element functional range set (Kutach and Kadonaga, 2000) suggest that these two amon ESTs may mark the 5' end of the amon message.

Figure 1. Correction of an amon cDNA artifact. A. Sequence of an amon cDNA (Siekhaus and Fuller, 1999). Dashes represent sequences not reproduced here. Sequences 5' to the arrow do not match upstream amon genomic sequences. The sequence in bold near the 5' end of the cDNA matches the bold sequence near the 3' end in the forward direction. The underlined sequence near the 5' end of the cDNA matches the underlined sequence near the 3' end in reverse complement orientation. Numbering is that of Siekhaus and Fuller (1999). B. Genomic sequence upstream of amon. Sequence in bold represents the extent of the two longest amon ESTs. Underlined sequences represent matches to consensus Initiator and Downstream Promoter Elements.

      While sequencing EMS induced mutations in amon, we identified 19 single nucleotide polymorphisms in amon coding sequences. Table 1 shows polymorphisms between amon genomic sequence (Adams et al., 2000) , an amon cDNA (Siekhaus and Fuller, 1999) , the red e parental chromosome used in our screen (Rayburn et al., in preparation), and two third chromosome balancers (TM3, Sb and TM3, Sb y+) obtained from the Bloomington stock center. Because we sequenced genomic DNA amplified from red e/TM3 heterozygotes, polymorphisms between these chromosomes served as useful positive controls. One of these polymorphisms (a vs. c at position 2110) is predicted to result in a Lysine to Glutamine change at position 562, suggesting that Amon is functional with either Lysine or Glutamine at this position.  The remaining 18 polymorphisms are not predicted to result in an amino acid change.

     Acknowledgments:  This work was supported by an NIH grant (GM53681) to M.B. and an NIH training grant (GM07103) to L.Y.M.R.

      References:  Adams, M.D., S.E. Celniker, R.A. Holt, C.A. Evans, J.D. Gocayne, et al. 2000, Science. 287: 2185-2195;  Kutach, A.K., and J.T. Kadonaga 2000, Mol Cell Biol. 20: 4754-4764;  Siekhaus, D.E., and R.S. Fuller 1999, J Neurosci. 19: 6942-6954.

 

Table 1: Polymorphisms in the coding regions of amon. amonExon cDNA bp * amon genomic amon  cDNA red e genomic TM3 genomic Codon Change 2 664 t t t c gat --> gac 2 721 t t c t agt -->agc 3 952 g a g g agg -->aga 8 1432 c a a c atc --> ata 8 1456 c g c c ggc --> ggg 8 1477 t c c t aat --> aac 8 1489 c t t c tac --> tat 9 1726 t t c t cct --> ccg 10 2038 g a g g ttg --> tta 10 2092 a a g g gaa --> gag 10 2109 a a c c aca --> acc 10 2110 a c a a aaa --> caa 11 2125 c c g c tcc --> tcg 11 2132 c c t t ctg --> ttg 11 2176 c c t t acc --> act 11 2188 t t c c ttt --> ttc 11 2234 t t c c ttg --> ctg 12 2320 c c g g ccc --> ccg 12 2347 g g a a ccg --> cca