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Find Arabidopsis protein homologous to Mimulus unigene (BLASTX)

Mimulus unigene sequences. Find Arabidopsis protein homologous to Mimulus unigene (BLASTX). Arabidopsis protein sequences. Align Mimulus EST with Arabidopsis protein (ESTWISE). Arabidopsis gene model coordinates. Calculate intron positions/phases in Arabidopsis protein.

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Find Arabidopsis protein homologous to Mimulus unigene (BLASTX)

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  1. Mimulus unigene sequences Find Arabidopsis protein homologous to Mimulus unigene (BLASTX) Arabidopsis protein sequences Align Mimulus EST with Arabidopsis protein (ESTWISE) Arabidopsis gene model coordinates Calculate intron positions/phases in Arabidopsis protein Find corresponding positions in Mimulus unigene Design primers spanning introns in Mimulus unigene (Primer3)

  2. >CONTIG5 CAGCTACGAATAATATAGCTCCACGTGTAGCCATATCCATTTTCCTGTTTCTAAGTTGTTCTTCATTTGCAGCTGCTGCAAAACATGTCTATGAATCGAAGCCTTTCAACCGCACTGATTTTCCACCTGGTTTTCTTTTTGGAGCTGCTTCTTCTGCTTATCAATTTGAAGGTGCTGCATTTGAAGGTGGGAAAGGACCTAGTATTTGGGATACTTACACTCACCAATTTCCAGAAAAGATAGCTGATCGAAGCAACGGTGACGTGGCTAACGACTTTTATCATCTGTATAAGGATGATGTGAAATTGCTGAAGGATTTAGGACTGGATGTTTTCCGGATGTCCATTGCTTGGTCACGTGTATTGCCACATGGAAAACTAAGTAGAGGAGTGAACAAAGAAGGGATTGCCTTTTACAACAATGTTATCAATGAACTCCTTGCAAATGGAATAACACCATTTGTGACACTATTTCTTTGGGACCTCCCTCAAGCACTAGAGGATGAATATAGAGGCTTCCTAAGTCCTCTAATTGTGGACGATTATCTGGATTTCGTGGAACTTTGCTTTAAGAATTTCGGAGATCGTGTTAAGAATTGGATCACATTCAACGAGCCGTTCGTGTTCACAAATGGGGGCTACGATGGGGGATTCCTCGGGACTCTAGCCCNCGGTCGGTGCTCGTCGTGGNGCAATT BLASTX of the (translated) Contig5 sequence against the Arabidopsis thaliana protein sequence database showed a best hit for At5g24550 (a beta-glucosidase) with E~10-60 Caution: in this case, 30 other hits were retrieved having E< 10-60 and At5g24550 was retrieved for three other contigs (30, 72, and 413)

  3. At5g24450 (beta-glucosidase) on Chr V complement(join(8290698..8290938, 8291041..8291149, 8291263..8291365, 8291474..8291511, 8291046..8291854, 8291967..8292085, 8292253..8292508, 8292605..8292692, 8292895..8292972, 8293084..8293159, 8293380..8293435, 8293542..8293611, 8293789..8293941) ) Exon#Exonlgth(bp)Last_ResidueIntron Phase 1 153 51 0 2 70 74 1 3 56 93 0 4 76 118 1 5 78 144 1 6 88 173 2 7 256 259 0 8 119 298 2 9 809 568 1 10 38 581 0 11 103 615 1 12 109 651 2 13 241 732 stop

  4. At5g24550 27 FSTTPLNRYSFPPHFDFGVASSAYQYEGAVEEGGRSPSIWDNFTHAFPE + + P NR FPP F FG ASSAYQ+EGA EGG+ PSIWD +TH FPE YESKPFNRTDFPPGFLFGAASSAYQFEGAAFEGGKGPSIWDTYTHQFPE CONTIG5 90 tgtactacagtccgtctgggttgtctggggtgggagcaatgatacctcg aacactagcatccgtttgcccccaatagcctaggagcgtgacacaatca taggtccctttatttttattttttatattatatgaatttgttctcataa At5g24550 76 R-TNMDNGDVAVDFYHRYKDDIKLIKEMNMDSFRFSLSWSRILPSGKLS + + NGDVA DFYH YKDD+KL+K++ +D FR S++WSR+LP GKLS KIADRSNGDVANDFYHLYKDDVKLLKDLGLDVFRMSIAWSRVLPHGKLS CONTIG5 237 aaggcaaggggagttcctagggatcagtgcggtcatagttcgtccgaca atcaggagatcaataataaaatattaatgtattgtctcgcgttcagatg gattacctcgtcctttgtgttgagggtaagttcggcttgatagataaat At5g24550 124 DGVNKEGVQFYKNLIDELIKNGIKPFVTIYHWDIPQALDDEYGSFLSPR GVNKEG+ FY N+I+EL+ NGI PFVT++ WD+PQAL+DEY FLSP RGVNKEGIAFYNNVINELLANGITPFVTLFLWDLPQALEDEYRGFLSPL CONTIG5 384 aggaaggagttaagaagccgagaactgactctgcccgcgggtagtcacc ggtaaagtctaaattaattcagtccttctttgatcactaaaaggttgct aagcaagtctccttctactataaaatgaattgcctaaagtataccatta At5g24550 173 IIDDFRNFARFCFQEFGDKVSMWTTFNEPYVYSVSGYDAG---NKAIGR I+DD+ +F +CF+ FGD+V W TFNEP+V++ GYD G A GR IVDDYLDFVELCFKNFGDRVKNWITFNEPFVFTNGGYDGGFLGTLAxGR CONTIG5 531 agggtcgtggcttaatggcgaataatagctgtaaggtgggtcgacgcgc ttaaatattatgtaatgagtaagtctaactttcaggaaggttgctcNgg tgcttgtcgatctgtcatttgtgcaccggcgcatgcctgaccgtacctg At5g24550 219 CSKWVN CS W N CSSWxN CONTIG5 678 ttttNa gccgga cgggct

  5. - Several primers are designed to span each intron. - The codon position of the 3’ nucleotide in each primer is listed Sample of two primer pairs designed for Contig5: intron product primer start lgth codonpos Tm sequence 240 285 F 92 20 1 60.2 TGAATCGAAGCCTTTCAACC R 357 20 1 60.2 AAGTCGTTAGCCACGTCACC 389 210 F 347 20 1 60.8 TGCTTGGTCACGTGTATTGC R 536 21 3 59.3 GGTGTTATTCCATTTGCAAGG

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