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Evidence for Gene IV

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    1. RNA Evidence

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



    score 564 score 361 score 1441
    score 499 score 339 score 1386
    score 390 score 267 score 1061
    score 217 score 167 score 1007
    score 385 score 689
    score 171 score 148 score 255
    score 178

     

     


    The top result was:

    NM_001139031.1 Zea mays LOC100193963 (si486073c04), mRNA >gb|BT036360.1|

    Total score =  2367,  E value = 0.0
    Query coverage = 89%, Identity = 96%

    This also refers to the same protein (locus LOC100193963) that was resembled in the BlastX search, made in October 2012, NP_001132503.1  (uncharacterized protein LOC100193963 [Zea mays], Query coverage: 99%; E value: 0.0; Max ident: 84%, and ) and where amino acid transporters conserved domains were detected.

     

    Other good match was:

    XM_002455881.1 Sorghum bicolor hypothetical protein, mRNA 

    Total score =  2224,  E value = 0.0
    Query coverage = 86%, Identity = 94%

    alignment geneIV and sorghum match.gif

    This match on Sorghum bicolor can suggest the feasibility of this Gene IV but we still need more evidence.

     

    The amino acid sequence derived from the first result, NM_001139031.1, was: 

    >gi|212723070|ref|NP_001132503.1| uncharacterized protein LOC100193963 [Zea mays] 

    MTWNRSAADGGGSSAGTTAGDDTGLARLRELGYKQELKRDLSVLSNFAFSFSIISVLTGITTLYNTGLNF
    GGPATMTFGWFVAGAFTMAVGASMAEICSSFPTSGGLYYWSARLSGKRWAPFASWITGWFNVVGQWAVTT
    SVDYSLAQLIQVIILLATGGKNGGGYLASKYMVIGFHAAILLSHAVINSLPITFLSFFGQFAAAWNMLGV
    FVLMVAVPTVATERASAEFVFTHFNTDNGAGIRSNLYIFVLGLLMSQYTLTGYDASAHMTEETKNADKNG
    PIGIISAIGISILVGWGYILGVTFAVKDIPYLLSPDNDAGGYAIAEVFYLAFKSRYGSGAGGIVCLGVVA
    VAVYFCGMSSVTSNSRMAYAFSRDGAMPFSSVWHKVNKQEVPINAVWLSALVALCMALPSLGSLVAFQAM
    VSIATIGLYISYALPILFRVTLARKYFVPGPFNLGRYGVLVGWVAVLWVATITVLFSLPVTYPVTKDTLN
    YTPVAVGGLLFLVLASWLLSARHWFKGPVTNLDG

     

     

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

    alignment geneIV and NO_0011325003.1.gif

    As it is visible, there is not a really close match that makes us state that gene IV is actually a gene. There are two clear gaps present in the alignment which is not helpful at all. Now we are looking at matches with expressed sequence tags.

     

    2. EST Evidence

    A nucleotide BLAST search against expressed sequence tags (EST database) limited for maize using the sequence for gene IV gave the following:


    score 656 score 361 score 113 score 1264
    score 564 score 361 score 213 score 1131
    score 564 score 202 score 1009
    score 564 score 361 score 132 score 957
    score 564 score 361 score 931
    score 564 score 361 score 161 score 863
    score 564 score 361 score 220 score 856
    score 564 score 361 score 152 score 854
    score 564 score 361 score 145 score 852
    score 564 score 361 score 850
    score 564 score 361 score 135 score 843
    score 564 score 361 score 187 score 837
    score 560 score 100 score 833
    score 558 score 355 score 828
    score 558 score 270 score 813
    score 542 score 257 score 808
    score 534 score 361 score 494 score 743
    score 532 score 361 score 137 score 730 score 163
    score 436 score 361 score 152 score 725
    score 418 score 361 score 725
    score 339 score 361 score 715
    score 324 score 361 score 708
    score 217 score 361 score 706
    score 361 score 202 score 684
    score 361 score 202 score 656 score 464
    score 361 score 656 score 460
    score 361 score 100 score 459 score 647
    score 357 score 84 score 645
    score 355 score 632
    score 340 score 623
    score 283 score 582 score 411
    score 265 score 577 score 510
    score 265 score 545 score 501
    score 252 score 545
    score 243 score 100
    score 228
    score 209
    score 171 score 148 score 102
    score 156
    score 154
    score 84

     

    The best results were:

    EE185959.2ZM_BFc0171C07.r ZM_BFc Zea mays cDNA clone ZM_BFc0171C07 5', mRNA sequence

    Total score =  1264,  E value = 0.0
    Query coverage = 46%, Identity = 95%

     

    EC895705.2 ZM_BFc0038P24.r ZM_BFc Zea mays cDNA clone ZM_BFc0038P24 5', mRNA sequence

    Total score = 1146,  E value = 2e-158
    Query coverage = 44%, Identity = 96%

    These particular EST results were chosen for alignment because they had the major coverage when combined. So we used both RNA sequences to make the alignment with the sequence of gene IV using MultiAlin:

     

    alignment geneIV, EE185959, EC895705.gif

    We can see two big gaps (one around position 500 and the other one around position 830) and some small mismatches, which is consistent with the previous amino acid alignment. Thereby, so far we cannot tell if gene IV is actually an amino acid transporter gene because of the gaps

    After some exploring, we found that there were 104 sequences that matched locus LOC100193963.This included 10 mRNA sequences and 94 est sequences. The following were found listed in UniGene under the id UGID:654651. However, the best hits are:

    Best Hits and Hits from model organisms

    Species

    Id(%)

    Len(aa)

    NP_001132503.1

    uncharacterized protein LOC100193963

    Z. mays

    100.0

    523

    NP_565254.1

    bidirectional amino acid transporter 1

    A. thaliana

    79.4

    504

    XP_957400.2

    hypothetical protein NCU07175

    N. crassa

    49.8

    524

    Other hits (2 of 9) [Show all]

    Species

    Id(%)

    Len(aa)

    XP_002455926.1

    hypothetical protein SORBIDRAFT_03g027370

    S. bicolor

    96.4

    533

    XP_003569326.1

    PREDICTED: LOW QUALITY PROTEIN: uncharacterized amino-acid permease C15C4.04c-like

    B. distachyon

    90.7

    613

     Again, we found that a hypothetical protein in Sorghum bicolor is 96% to the protein that our gene IV resemebled the best. So we need a little more research.

    Our last BlastX search was made on Octuber, 2012. But we realized now on November that some new proteins have been submitted to NCBI. So the current results for gene IV is:

    Accesion

    Description

    Total score

    Query coverage

    E value

    Max ident

    AFW83200.1

    hypothetical protein ZEAMMB73_325000 [Zea mays]

    1118

    99%

    0.0

    100%

    DAA58905.1

    TPA: hypothetical protein ZEAMMB73_147770 [Zea mays]

    887

    99%

    0.0

    82%

    NP_001132503.1

    uncharacterized protein LOC100193963 [Zea mays]

    885

    99%

    0.0

    82%

     

    The first two (AFW83200.1and DAA58905.1) where submitted to NCBI on November 2012. So there are more options for making alignments with gene IV, especially with the first one because has 100% identity. Then, the MultiAlin alignment is:

    alignment geneIV and AFW83200.1.gif

     Now we have a perfect match. Therefore, we can state that gene IV is an actual gene with much more confidence, although there is not RNA sequences or EST that support those two previous gaps.

     

    3. Gene Function Evidence

    The results from our RNA and est searches indicated that our Gene IV encodes a membrane amino acid transporter protein. To further investigate the possible function of Gene IV we translated our annotated nucleotide sequence to an amino acid sequence using the ExPASY translate tool and used InterPro Scan for looking conserved domains. The visual representation of the InterPro Scan results for Gene III is pasted below:

     

    CONSERVED DOMAINS GENEIV.png

    Clearly, amino acid transporter proteins coserved domains have been resembled. Therefore the function of our gene IV presumably is to encode a membrane protein  involved in the transportation of amino acids into the cell.
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