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Evidence for Gene II

    RNA Evidence

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

     


    score 1330
    score 1016
    score 966
    score 590
    score 571
    score 555
    score 263
    score 254
    score 196 score 102 score 196
    score 180
    score 135
    score 132
    score 124

    The top result was:

    >ref|NM_001157247.1| Zea mays seven-transmembrane-domain protein 1 (LOC100284352),
    mRNA
    gb|EU967998.1| Zea mays clone 312642 seven-transmembrane-domain protein 1 mRNA,
    complete cds
    Length=1136
    GENE ID: 100284352 LOC100284352 | seven-transmembrane-domain protein 1
    [Zea mays] (10 or fewer PubMed links)
    Score =  1330 bits (720),  Expect = 0.0
    Identities = 728/732 (99%), Gaps = 0/732 (0%)
    Strand=Plus/Plus
     
    When we followed the link to this result, we found out that it came from a cDNA sequencing project. The project was published in PLoS Genetics. (Soderlund C, Descour A, Kudrna D, Bomhoff M, Boyd L, et al. (2009) Sequencing, Mapping, and Analysis of 27,455 Maize Full-Length cDNAs.PLoS Genet 5(11):e1000740.doi:10.1371/journal.pgen.1000740).

    The amino acid sequence derived from this result was:
    >gi|226532940|ref|NP_001150719.1| seven-transmembrane-domain protein 1 [Zea mays]
    MISPDAARNVVGIIGNVISFGLFLSPVLTFWRICKARDVEEFKPDPYLATLLNCMLWVFYGIPVVHPNSI
    LVVTINGVGLVIEAIYLTIFFLYSDGPKRRKAFGILAVEILFMVAVVLGVILGAHTHEKRSMIVGILCVI
    FGSMMYASPLTIMSRVIKTKSVEYMPFLLSLVSFLNGCCWTAYALIRFDLYVTIPNALGAFFGLIQLILY
    FCYYKSTPKEKNVELPTVSSNAGGGNVTVSVER

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

    geneII_mRNAalignment.gif

    Since the mRNA sequence that came from cDNA sequencing matched our gene model so closely, we felt that this was evidence that our gene II model was annotated correctly and that it is expressed.

     

    est Evidence

    A nucleotide BLAST search against expressed sequence tags (est database) using the sequence for gene II gave the following when limited to maize:

     


    score 1016
    score 994
    score 872
    score 846 score 357
    score 806 score 401
    score 739 score 318
    score 736 score 178
    score 728 score 124
    score 713 score 481
    score 693
    score 664 score 342
    score 654
    score 654 score 152
    score 647
    score 641 score 523
    score 399 score 621
    score 619 score 512
    score 392 score 601
    score 599
    score 582
    score 407 score 564
    score 516 score 472
    score 490 score 508
    score 475 score 505
    score 472 score 446
    score 472 score 390
    score 470 score 124
    score 470 score 119
    score 466 score 124
    score 464 score 124
    score 460 score 124
    score 453 score 124
    score 433 score 124
    score 424 score 124
    score 407 score 124
    score 398 score 124
    score 396 score 124
    score 396 score 124
    score 342 score 124
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 135
    score 134

     

    When we looked over the list of results we found that several of the est sequences were matched to one of two different loci (LOC100282631 and LOC100284352). The est sequences matched to LOC100284352 had higher max identity to our sequence (max. ident. 98% and up) than the est sequences matched to LOC100282631 (max. ident. 93% and less). The mRNA that matched our sequence so well (ref|NM_001157247.1|Zea mays seven-transmembrane-domain protein 1) came from LOC100284352. The mRNA corresponding to LOC100282631 did not match gene II's sequence as well. We believe that the similarity between the two loci is evidence that a gene duplication occurred. We decided that we should focus on est sequences matched to LOC100284352 for our analysis.

     

    After some exploring, we found that there were 14 sequences that were matched to LOC100284352. This included 6 mRNA sequences and 8 est sequences. The following were found listed in UniGene under the id UGID:3592287.

    mRNA sequences (6)

      EU967998.1 Zea mays clone 312642 seven-transmembrane-domain protein 1 mRNA, complete cds  
      BT068874.1 Zea mays full-length cDNA clone ZM_BFc0142H15 mRNA, complete cds  
      NM_001157247.1 Zea mays seven-transmembrane-domain protein 1 (LOC100284352), mRNA  
      EZ055718.1 TSA: Zea mays contig56840, mRNA sequence  
      EZ090473.1 TSA: Zea mays contig26120, mRNA sequence  
      EZ088174.1 TSA: Zea mays contig23820, mRNA sequence  

    EST sequences (8)

      EE167925.1        
      EE167926.2        
      FK980884.1        
      FK980885.1        
      FK980886.1        
      FK980887.1        
      FK980889.1        
      FL434658.1        

     

    We chose 4 est results (FK98088.7, EE167926.2, FK980889.1, FK980885.1) to align against the nucleotide sequence for gene II using MultAlin. The sequences used for the alignments were obtained by following the link from each result to a FASTA sequence. These particular est results were chosen for alignment because they covered the majority of gene II when combined. They all had 98-99% of sequence identity with the part of gene II that they covered. Below is the MultAlin result: 

     geneII_estalignment.gifgeneII_estalignment.gif

     
    When we examined this alignment, we found that the 4 est sequences (FK98088.7, EE167926.2, FK980889.1, FK980885.1) combined covered all of gene II exons 1, 2, 3, and 4. The beginning of exon 5 was also covered indicating that the intron between exons 4 and 5 was called correctly. We were unable to find est sequences that matched to the remainder of exon 5 or exon 6. From this alignment we are able to see that at least 1 of the est sequences matches our gene II sequence at every base for exons 1, 2, 3, and 4. This is evidence that the first 4 exons and the introns in between them were called correctly. Despite the lack of est evidence for exon 5 and 6 we still believe that we annotated gene II correctly. The alignment between the amino acid sequence of gene II and the amino acid sequenced derived from the mRNA of seven-transmembrane-domain protein 1 in maize (ref|NM_001157247.1|  ) (which came from cDNA sequencing) had 100% sequence similarity at exons 5 and 6.

    We believe that gene 2 is annotated correctly and that it is expressed.

     

     

    Gene Function Evidence

    The results from our RNA and est searches indicated that our Gene II encodes a seven-transmembrane-domain protein in maize. To further investigate the possible function of this protein 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 II is pasted below:

     This image is the visual representation of the InterProScan Result

     It appears that gene II is a sugar efflux transporter based on its conserved domains.

     

     Protein Features Evidence

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

    This amino acid sequence is of a MEMBRANE PROTEIN
    which have 7 transmembrane helices.

    No.

    N terminal transmembrane region C terminal type length
    1 6 AARNVVGIINVISFGLFLSPLTF 28 SECONDARY 23
    2 43 DPYLATLLNCMLWVFYGIPVVH 64 SECONDARY 22
    3 69 LVVTINGVGLVIEAIYLTIFFL 90 PRIMARY 22
    4 101 FGILAVEILFMVAVVLGVILGA 122 PRIMARY 22
    5 130 MIVGILCVIFGSMMYASPLTIMS 152 PRIMARY 23
    6 162 YMPFLLSLVSFLNGCCWTAYALI 184 SECONDARY 23
    7 191 TIPNALGAFFGLIQLILYFCYY 212 PRIMARY 22

     

     geneII_hydropathyprofile.png

    geneII_structure.png

     Note: The SOSUI search engine can to found at http://bp.nuap.nagoya-u.ac.jp/sosui/.

     

     

    References:

    "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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