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Evidence for Gene III

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    RNA Evidence

    A nucleotide BLAST search against reference RNA sequences (refseq_rna database) using the sequence for gene III gave the following:


    score 4959
    score 3986
    score 2549
    score 2471
    score 388


    The top result was:

    ref|NM_001114649.1|Zea mays starch branching enzyme III (sbe3), mRNA
    gb|EU333945.1|Zea mays starch branching enzyme III precursor, mRNA, complete cds Length=294
    GENE ID: 100136878 sbe3 | starch branching enzyme III [Zea mays]
    Score =  4959 bits (2685),  Expect = 0.0
    Identities = 2695/2700 (99%), Gaps = 0/2700 (0%)
    Strand=Plus/Plus

     

    The amino acid sequence derived from this result was:

    >gi|167860172|ref|NP_001108121.1| starch branching enzyme III [Zea mays]

    MASCPLFLLVPRPPPSLVGADRCPGSAGCSDLPTSCRPHPGRRHGYPFRCNSSSSSSAPRERPPRQRPGG

    RGDAIDPVGFLTKLGVSDRAFAQFLRDRHKALKDRRWELCSRFIDLKEASSGFELLGMHRHRQHRIDFME

    WAPGARYCSLIGDFNQWSPTENCAREGHLGHDDFGYWFIILEDKLREGQEQDEYFFQEYNYVDDYDKGDN

    GIDADEIMRRMKEEYWEPGEIRSRQSQLEMVVKLYEQMFGPNGPQTEEELGEIPDAQTRYNEFKASQKAD

    SSSQPSYDIIDNGQEFDIFSVVTDRASFEKFQAKKPPLAYWVEMRKGRIAWLEKYVPTISHKDKYRVYFN

    TPDGALERIPAWAAYVLPDAEGKQSYAVHWEPPPEEIYKWRFGRPKVKGSLRIYECHVGISGSEQKVSSF

    QEFTSKVLPHIKKAGYNAVQLIGVVEHKDYSSIGYKVTNYFAVSSRFGTPDDFKKLVDEAHGLGLVVLLD

    IIHSYASADELVGLSLFDGSNDCYFHSGKRGHHKYWGTRMFKYDDVDVLHFLLSNLNWWVTEYRIDGFQF

    HSLSSMLYTHNGFSTFTGAMEEYCNQYVDKDALIYLILANEMLHRLHPDIVTIAEDATFYPGLCEPITQG

    GLGFNYWVNLSIPEMWLWHLENVPEQEWSMNKIIKVLVSSNQNMLSYVENHNQSISGRKSFAEIILNSRE

    CSVGSVDDNLIRASSLLKLMKLITFTTSGGGYLNFMGNEFAHPEKVEFPMPSNDYSFELANRQWELLDKV

    FHKHIFNFDKDVMSLDENERIISRGPPNILHCDDTSMVIFFTRGPLFFIFNFNPQVSHQSYRVGVDEAGE

    YQLIFNPDETKYGGCETLKSSQYMRRTFDKRADGCRNSLELALPSRSAQIYKLVRILRI

     

    When we aligned this amino acid sequence to the amino acid sequence of our predicted Gene III using MultAlin we obtained the following alignment:

    geneIII_mRNAalignment.gif

    Since the mRNA and our gene model matched so closely, we felt that this was evidence that our gene III model was annotated correctly. We later realized that we could not find evidence that the mRNA sequence we used for the alignment came from a cDNA sequencing project. This means that the sequence may have come from another person who annotated part of the maize genome like we did and did not do any sequencing to confirm that their annotation was correct. We can not rely on this alignment alone.

     

    est Evidence

    A nucleotide BLAST search against expressed sequence tags (est database) using the sequence for gene III gave the following:

    score 1264 score 730 score 1339 score 896
    score 1192 score 1242 score 1057
    score 1190 score 691 score 1133
    score 1190 score 309 score 1070 score 974
    score 1020 score 1088 score 983 score 719
    score 1044 score 122 score 562 score 702
    score 900 score 1020 score 233 score 697
    score 785 score 202 score 671
    score 743 score 276 score 660
    score 715 score 723 score 171 score 643
    score 584 score 684 score 641
    score 222 score 206 score 104 score 634
    score 628
    score 544
    score 536
    score 527
    score 508
    score 501
    score 492
    score 65 score 424
    score 422 score 344
    score 405
    score 65 score 359
    score 65 score 268
    score 65 score 267
    score 267
    score 265
    score 154
    score 154

     

    After some exploring, we found that there were 16 sequences that matched sbe3 (starch branching enzyme III). This included 3 mRNA sequences and 13 est sequences. The following were found listed in UniGene under the id UGID:2710398.

    mRNA sequences (3)

      EU333945.1 Zea mays starch branching enzyme III precursor, mRNA, complete cds  
      NM_001114649.1 Zea mays starch branching enzyme III (sbe3), mRNA  
      BT083985.2 Zea mays full-length cDNA clone ZM_BFb0061O22 mRNA, complete cds  

    EST sequences (13)

      DR793909.1        
      DR793910.1        
      DR821942.1        
      DR958744.1        
      DV171151.1        
      EC890451.2        
      EC890452.2        
      EE022432.2        
      EE022433.2        
      EE288769.2        
      EE288770.2        
      FL163465.1        
      FL163466.1        

     

    We chose 6 est results (EE288770.2, EE022432.2, FL163465.1, DR793909.1, DR958744.1, EE288769.2) to align against the nucleotide sequence for gene III using MultAlin. The sequences used for the alignments were obtained by following the link from each result to a FASTA sequence. The sequences of the est results from the other strand were transformed into the reverse complement prior to be aligned. These particular est results were chosen for alignment because they almost completely covered gene III when combined. They all had 99-100% of sequence identity with the part of gene III that they covered. Below is the MultAlin result:
       geneIII_estalignment.gif 

    When we examined this alignment, we found that the 6 EST sequences (EE288770.2, EE022432.2, FL163465.1, DR793909.1, DR958744.1, EE288769.2) combined covered almost all of gene III (there was 1 gap). The beginning and end of gene III were covered. 

    We were concerned that the gap might mean that we had an intron included in our sequence and that gene III is actually a two exon gene instead of a one exon gene. We looked at the part of the sequence that was not supported by EST data and found that it had GT near the beginning and AG near the ending (possible splice site). We decided to try treating this part of the sequence as an intron. This gave us two exons. We translated the sequence of these two introns into amino acids and aligned the sequence to the sequence of starch branching enzyme 3 in Arabidosis thaliana. We found that the part of the gene III sequence we thought might be an intron due to lack of EST support was not an actually an intron. This means that gene III is really a one exon gene. Below is an alignment of starch branching enzyme 3 in A.thaliana, gene III as 2 exons (with the portion of the sequence not supported by EST evidence removed), and gene III as 1 exon:

     At_sbe3_alignment.gif

    Note that the portion of gene II that was not backed by EST evidence (and was therefore being considered as a possible intron) is located from position 414 to 450 in the above alignment. 

    We believe that the EST data and the alignment with starch branching enzyme III in A. thaliana is is evidence that gene III is annotated correctly, it is expressed, and it is starch branching enzyme III in maize. 

     

    The A. thaliana starch branching enzyme 3 sequence used for the above alignment is:

    >gi|374110657|sp|D2WL32.1|GLGB3_ARATH RecName: Full=1,4-alpha-glucan-branching enzyme 3, chloroplastic/amyloplastic; Short=AtSBE III; AltName: Full=Branching enzyme 1; Short=AtBE1; AltName: Full=Protein EMBRYO DEFECTIVE 2729; AltName: Full=Starch-branching enzyme 3; Flags: Precursor
    MVSLSNQTRFSFHPNNLVVSEKRRLGISGVNFPRKIKLKITCFAAERPRQEKQKKKSQSQSTSDAEAGVD
    PVGFLTRLGIADRIFAQFLRERHKALKDLKDEIFKRHFDFRDFASGFELLGMHRHMEHRVDFMDWGPGSR
    YGAIIGDFNGWSPTENAAREGLFGHDDYGYWFIILEDKLREGEEPDELYFQQYNYVDDYDKGDSGVSAEE
    IFQKANDEYWEPGEDRFIKNRFEVPAKLYEQMFGPNSPQTLEELGDIPDAETRYKQWKEEHKDDPPSNLP
    PCDIIDKGQGKPYDIFNVVTSPEWTKKFYEKEPPIPYWLETRKGRKAWLQKYIPAVPHGSKYRLYFNTPD
    GPLERVPAWATYVQPEDEGKQAYAIHWEPSPEAAYKWKYSKPKVPESLRIYECHVGISGSEPKVSTFEEF
    TKKVLPHVKRAGYNAIQLIGVPEHKDYFTVGYRVTNFFAASSRYGTPDDFKRLVDEAHGLGLLVFLDIVH
    SYAAADQMVGLSLFDGSNDCYFHYGKRGHHKHWGTRMFKYGDLDVLHFLISNLNWWITEYQVDGYQFHSL
    ASMIYTHNGFASFNNDLDDYCNQYVDRDALMYLILANEILHVQHPNIITIAEDATYYPGLCEPVSQGGLG
    FDYYVNLSASEMWVSLLDNVPDNEWSMSKIVSTLVANKEYADKMLSYAENHNQSISGGRSFAEILFGGVD
    NGSPGGKELLDRGISLHKMIRLITFTSGGRAYLNFMGNEFGHPERVEFPTQSNNFSFSLANRRWDLLESG
    VHHHLFSFDKELMDLDKSKGILSRGLPSIHHVNDANMVISFSRGPFLFIFNFHPSNSYEKYDVGVEEAGE
    YTMILNSDEVKYGGQGIVTEDHYLQRSISKRIDGQRNCLEVFLPSRTAQVYKLTRILRI
     

     

    Gene Function Evidence

    The results from our RNA and est searches indicated that our Gene III encodes a protein known as starch branching enzyme III in maize. To further investigate the possible function of Gene III we translated our annotated nucleotide sequence to an amino acid sequence using the ExPASY translate tool and used InterPro Scan (one of the tools that can be found at ExPASy Bioinformatics Resource Portal http://www.expasy.ch/tools/). The visual representation of the InterPro Scan results for Gene III is pasted below:

    This image is the visual representation of the InterProScan Result

     Note: 1,4-alpha-glucan-branching enzyme is a synonym for starch branching enzyme.

    We also ran our amino acid sequence for gene III using HHpred, which is another program for protein homology detection. It can be accessed at http://toolkit.tuebingen.mpg.de/hhpred. The top result from our HHpred search was "OS06G0726400 protein; starch-branching, transferase" (http://pdb.rcsb.org/pdb/explore.do?structureId=3AML) which is Starch Branching Enzyme I (BEI) from Oryza sativa. The next two results were both 1,4-alpha-glucan-branching enzyme.

     

    We also took a look back at the results of our initial BLASTx comparisons of our sequence to multiple plant genomes. We found five plant genomes (not including Zea mays) that gave a "starch branching enzyme" or "alpha-amylase protein" as a top result (starch branching enzyme has a alpha-amylase domain). These plants were Oryza sativa, Triticum aestivum, Sorghum sp., Arabidopsis thaliana, and Hordeum vulgare. These results can be viewed here.

     

    All of this evidence combined confirmed that gene III is a starch branching enzyme in maize.

     

    Protein Features Evidence

    When we submitted the amino acid sequence derived from gene III to SOSUI (engine version 1.11) to predict the secondary structure we obtained the following result:

    This amino acid sequence is of a SOLUBLE PROTEIN.

    geneIII_hydropathyprofile.png

     

     

     

     

     

    References:

    HHpred: Söding, J. (2005) Protein homology detection by HMM-HMM comparison. Bioinformatics 21: 951-960.

    "Multiple sequence alignment with hierarchical clustering"
    F. CORPET, 1988, Nucl. Acids Res., 16 (22), 10881-10890.

    Hirokawa T., Boon-Chieng S., and Mitaku S., Bioinformatics, 14 378-9 (1998)
    SOSUI: classification and secondary structure prediction system for membrane proteins.


    Zdobnov E.M., Apweiler R. InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics. 2001;17:847–848.

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