The UGA codon was thought to have a unique meaning as the STOP signal of the genetic code. But after discovery of selenoproteins another meaning has been attributed to this codon as soon as it can incorporate a novel aminoacid called “selenocysteine”(Sec). This newly found aminoacid is not more than a cysteine having selenium instead of sulphur. The condition for selenocysteine to be incorporated is having a Sec insertion sequence (SECIS) in the near 3’ untranslated region (UTR). This sequence forms a loop that enables recruitment of several factors and enzymes that permit Sec incorporation to the nascent polypeptide chain.

The functional importance of these proteins remains poorly understood, but it seems that selenocysteine is often located in the enzyme active-sites and is usually essential for its activity. This fact confers an enhanced value to the search and characterization of these novel proteins not only in humans but also in other organisms. 25 selenoproteins have been described so far for humans but we still don’t know if similar proteins are present in insects whom genomes sequentiation is in advanced phase if not completed.

In this project we report the presence of proteins similar to those found in humans in some organisms belonging to the family arthropoda. We have focused our study in organisms having the genome sequenced enough such as Anopheles gambiae, Apis mellifera, Drosophyla pseudoscura or others such as Drosophyla melanogaster with completed genome sequentiation. Thus, we have performed similarity search (tBLASTn) followed by exonic structure analysis (exonerate), SECIS structure prediction (SeciSearch) and multiple alignment of our hits (ClustalW).

Results of BLAST (hits) showing different scores and conservation levels were submitted to exonerate to further analyse the exonic structure of our putative proteins paying special attention to location of the aminoacids matching with selenocysteine (expressed as a STOP) and its adjacencies. Considering that an in-frame TGA (STOP) signal located not at the end but between other triplets could suggest a UGA coding for selenocysteine.

SeciSearch allowed us to identify putative SECIS elements in order to emphasize the information obtained by exonerate analysis or to discard hits that even having TGA inside the exon lack the necessary SECIS to be interpreted as possible selenoproteins.

Finally, the multiple alignment was done aimed at knowing the conservation level among our putative proteins, considering that a high conservation suggests that aminoacids are part of a functional protein and keep restrictive to mutations that would possibly affect worsening the activity.

SELENOPROTEINS AND ORGANISMS


For this project, we have used the 25 selenoproteins described in mammals so far.

Among these 25 proteins only three have been found in Drosophyla melanogaster also known as "fruitfly", but the presence of homologues in other insect species is still unknown.

The aim of this study is to discover if homologues of the mammalian 25 seleproteins are present in insects, and with this purpose we have performed tblastn similarity search of the 25 selenoproteins against several insect genomes, in order to dilucidate their general distribution.

In the beginning we were suposed to perform the study with three insect genomes belonging to Drosophyla simulans, Anopheles gambiae and Appis mellifera. Even though, we considered much more interesting to extend our study to more genomes, mainly because the results would be much more emphazised if they were supported by more organisms.

Thus, we submitted all the species belonging to the family arthropoda, having a partially or completelly sequenced genome, to similarity search.

Especies from genus Drosophila:

D.melanogaster [C,G,P,D]

D.pseudoobscura [C,G,P,D

D.ananassae [S,D]

D.erecta [S,D]

D.yakuba [S,D]

D.mojavensis [S,D]

D.simulans [S,D]

D.virilis [S,D]

D.grimshawi [D]

D.persimilis [D]

D.willistoni [D]

D.sechellia [-]

C = published Chromosomes (may be partial);
S = preliminary assembly Scaffolds;
G = genome Genes;
P = genome Proteins; - = no data yet
D = Databank sequences (NT) from GenBank/EMBL, RefSeq; Databank proteins (AA) from UniProt, RefSeqp, GenPept;

Other species:

Apis mellifera

Anopheles gambiae

Armigeres subalbatus

Aedes aegypti

Glossina morsitans morsitans

Ixodes ricinus

Venturia canescens

Musca domestica

Bombyx mori

TBLASTN

Compares a protein query sequence against a nucleotide sequence database dynamically translated in all six reading frames (both strands) using the BLAST algorithm. The BLAST seeks local as opposed to global alignments and is therefore able to detect relationships among sequences which share only isolated regions of similarity (Altschul et al., 1990).
In our work, NCBI tblastn has been used with two differents databases; the insects genomes NCBI database and Drosophila Species Genomes, a service of FlyBase and Genome Sequencing Centers. This service provides a preview of drosophila genome data, with genome maps and blast sequence search for this species. It includes prepublication, as well as published, Drosophila genomes from collaborating NGHRI-funded Genome Sequencing Centers.
To run tblastn program, we have paid special attention to some parameters,assuming that they were the most significant and informative for our purpose.

Expect: The statistically signifant expectation value. If the statistical significance ascribed to a match is greater than the E value, the match will not be reported. As soon as we were comparing distant species, we avoided the established E-value to be stringent, in order to obtain more candidate hits. We used the default value(=1), but we assumed that only E-values below 0.5 would be taken into account.


Substitution matrix: As it is known, this matrix assigns a score for aligning any possible pair of residues based on the theory of amino acid substitutions. In general, different substitution matrices are tailored to detecting similarities among sequences that are diverged by differing degrees. A single matrix may nevertheless be reasonably efficient over a relatively broad range of evolutionary change. Experimentation has shown that the BLOSUM-62 matrix is among the best for detecting most weak protein similarities. For particularly long and weak alignments, the BLOSUM-45 matrix may prove superior.

In our study, we established the BLOSUM45 matrix for our similarity search which is thought to be the most appropriate for the low evolutionary kinship between our subject and query.

EXONERATE protein2genome 0.8.2

Exonerate is a general tool for sequence comparison that uses the C4 dynamic programming library. It can produce either gapped or ungapped alignments according to a variety of different alignment models. Alignments generated using these heuristics will represent a valid path through the alignment model, yet (unlike the exhaustive alignments), the results are not guaranteed to be optimal.

We have used exonerate to check the exonic structure and intron moddeling of our hits. The aim was to observe whether the -STOP codon-, candidate to be selenocystein, was located in-frame or not. Considering that a positive answer to this question would further verify the posibility of having discovered a selenoprotein.

Anyway, the candidates not showing any result after being submitted to exonerate have not been discarded. We have considered that the query ( human selenoprotein) could be so distant that the exonerate would not accept the target DNA to contain any exonic structure ( that meens protein),related to the query. In order to solve this doubt we thought that the information given by the later multiple alignment could be clarifying, thinking that the insect protein sequences could point out to be very conserved among insects ( suggesting that they are part of proteins) , even not giving any results in exonerate analysis.
We performed a gapped alignment exonerate analysis with the following parameters:


SEQUENCE INPUT OPTIONS

Pairwise comparisons will be performed between all query sequences and all target sequences. In our case, human selenoprotein sequences in fasta format are used as the query sequences(-q), and insect genomic sequences also in fasta(-t).

-q | --query
Human selenoprotein sequence in a FASTA format file.

-t | --target
The insect DNA sequence also in a FASTA format file.

GAPPED ALIGNMENT OPTIONS

-m | --model protein2genome
This model allows alignment of a protein sequence to genomic DNA. This is similar to the protein2dna model, with the addition of modelling of introns and intron phases. This model is simliar to those used by genewise.

--showtargetgff
Report GFF output for features on the target sequence. We show our exonerate results in a GFF file showing the exonic structurend intron moddeling.

FASTACHUNK

Fastachunk is a tool that enables us to cut the desired DNA fragment from a file containig a sequence. We used it when the stop"Sec" matching aminoacids hadn't been found with tblastn in order to find out the upstrean or downstrean aminoacids sequence with a later " Translate tool" program. Thus, we could realize if the stop wasn't really matching with Sec or Cys. Nevertheless, we thought that we also could find a stop codon or Cys in our subject matching with the human protein's stop,but not shown due to the penalization attributed by the program to the STOP.
Another reason why Fastachunk was used, is because the fragments added could presumably show conservation in the later multiple alignment.This could mean that even not matching with the human selenoprotein (for being distant species), they have enough conservation among insects to be considered as parts of the protein constituents.

TRANSLATE TOOL

Translate is a tool which allows the translation of a nucleotide (DNA/RNA) sequence to a protein sequence in the all six reading frames.This allowed us to recover the protein sequence of the DNA fragments submitted to Fastachunk.Thus, we could clarify the questions above, comparing the protein sequence to that obtained in tblastn and also submit them to multiple alignment.

CLUSTALW

Clustal W is a general purpose multiple sequence alignment program for DNA or proteins.It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.
This tool has enabled us to clarify the distribution of the selenoproteins in different organisms and check the conservation level of the same putative protein among them.
When running the clustalW programme, all the protein sequences matching with the human specific selenoprotein were submitted together in fasta format. The sequences were enlarged compared to the specific fragment matching with the human selenoprotein, a work that was performed using FASTACHUNK program.

All the clustalW parameters remained as they were given by default for our multiple alignment.


3.   Results

Results are given for each protein in the following order:

• tblastn hits:     blast reference    E-value      Identities
• Exonerate output in GFF format or tblastn output for the cases with no results in exonerate.
• Secis elements:    reference    sequence    score     picture
• ClustalW output and Jalview edited multiple alignment.

SelH


6 hits are given by the tblastn similarity search, but only four of them were accepted to run exonerate analysis. The four sequences submitted to this analysis showed to have their "Stop codon" outside the coding regions according to the exonerate program. Even though, SECIS structure analysis reinforced the hypothesis of classifying them as selenoproteins showing very well scored SECIS structures within the six sequences, even the two sequences that were not admitted to run exonerate.

tblastn

>gnl|Dana-agencourt-040714-asm|contig_3129					 1e-07   37%

>gnl|Dyak-washu-assembly_040407|Contig124.5					 2e-07   36%

>0gnl|Dere-agencourt-run1028-asm|contig_8152				         4e-07   35%

>gnl|Dvir-agencourt-run1029-asm|contig_2170					 0.044   25%

>gi|27645578|emb|BX072297.1|CNS09RY5 Single read from an extremity of
a full-length cDNA clone made from Anopheles gambiae total adult females.	 2e-14   37%  

>gi|21430733|gb|AY119185.1| Drosophila melanogaster SD09114 full insert cDNA	 4e-10   33% 

Gene prediction

If you want to check all the exonerate outputs in gff format, click here.

SECIS elements


gnl|Dana-agencourt-040714-asm|contig 3129: 8621 8720

AUAGAAAUCU CCGCCAG CAAUAGGCCUU AUGAA GUGUGAUCGGCC AA UCCUCUCCAGGA ACCGACCACAC UGAA CCACAAUUU UUGUUUG CCUUUGUCUG



COVE score: 27.13 (Recommended threshold for COVE: 15)



gnl|Dere-agencourt-run1028-asm|contig 8152: 905 807 (SECIS on complementary strand)

AACAAACCUU UCUACAA UAGAGCCCC AUGAU CGAUGAUUGGC AA AUCCUCUCGAGGA ACCAAUUAUUG AGAA CCUUUUUGCC UUUGUUG AUUGUAUAAU



COVE score: 21.21 (Recommended threshold for COVE: 15)



gnl|Dvir-agencourt-run1029-asm|contig 2170: 20539 20639

GCGUCGAUAG UUUUUCA GCAAUAGCGCCAU AUGAU CAUUAAACGGU AA AACCCUAACGGGA GCCGUUCACUG UGAA ACUUUUGCC UGUAAG CUGAAGAAUU



COVE score: 20.43 (Recommended threshold for COVE: 15)



gnl|Dyak-washu-assembly 040407|Contig124.5: 4158 4059 (SECIS on complementary strand)

AAACGAAUCU UUCAGCA AUAGAGCCUU AUGAU CGAUGAUUGGC AA AUCCUUUCGAGGA ACUUAUCAUUG AGAA CCCUUUUGCC UUUGUUG AUUGCUUAGU



COVE score: 18.32 (Recommended threshold for COVE: 15)

Multiple alignment


If you want to see the ClustalW alignment, click here.

Unrooted N-J tree

SPS2

SPS2 human selenoprotein matched with high homology in many of the insect genomes submitted to tblastn. Here we report the discovery of two new selenoprotein candidates belonging to the species: D.yakuba and D.simulans. All the other insect species were found to have an "Arg" aminoacid at the position of the "Sec" of humans.

tblastn

>gnl|Dvir-agencourt-run1029-asm|contig_641					e-112	  60%

>gnl|Dmov-agencourt-run0811-asm|contig_3459					e-112     60%

>gnl|Dana-agencourt-040714-asm|contig_1299					e-111	  60%

>gnl|Dere-agencourt-run1028-asm|contig_125					e-111	  59%

>gnl|Dyak-washu-assembly_040407|Contig11.8					e-111	  59%

>3 type=chromosome; loc=3:1..19738957; ID=3; release=r1.03; species=dpse	e-110	  59%

>gnl|Dana-agencourt-040714-asm|contig_30531				        2e-97	  58%

>XR_group8 type=chromosome; loc=XR_group8:1..9190824; ID=XR_group8;
release=r1.03; species=dpse							2e-73	  43%

>4_group4 type=chromosome; loc=4_group4:1..6604331; ID=4_group4;release=r1.03; 
 species=dpse									2e-68	  42%

>XL_group1e type=chromosome; loc=XL_group1e:1..12499574; ID=XL_group1e;
release=r1.03; species=dpse							2e-54     38%

>gnl|Dyak-washu-assembly_040407|Contig8.19					7e-52	  38%

>4_group3 type=chromosome; loc=4_group3:1..11635473; ID=4_group3;release=r1.03; 
 species=dpse									2e-52     36%

>gnl|Dana-agencourt-040714-asm|contig_1089					3e-51	  39%

>gnl|Dsim-washu-w501-asm|Contig27.138						3e-51	  39%

>gnl|Dvir-agencourt-run1029-asm|contig_2233					6e-31	  44%

>gnl|Dmov-agencourt-run0811-asm|contig_181					0.060	  50%

>gnl|Dsim-washu-w501-asm|Contig18.273						3e-06	  41%

>gi|24653603|ref|NM_166046.1|  Drosophila melanogaster CG8553-PB, isoform B 
(SelD) mRNA, complete cds							e-130     60%

>gi|58390119|ref|XM_317503.2|  Anopheles gambiae str. PEST ENSANGP00000010100   e-126     59%

Gene prediction

If you want to check all the exonerate outputs in gff format, click here.



For the sequences that exonerate could not have been performed, tblastn outputs are shown:

>gnl|Dmov-agencourt-run0811-asm|contig_717  Length = 57670

Score = 41.8 bits (131), Expect = 0.003
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -1
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 21133 YNKHYEKGVYRCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 21002

Score = 36.5 bits (112), Expect = 0.13
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -3
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 18638 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 18513

Score = 35.1 bits (107), Expect = 0.36
Identities = 18/37 (48%), Positives = 24/37 (64%)
Frame = -2
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
             +V C KC   +GH F +DGP P + RF I S+S+ FV
Sbjct: 20268 EVRCSKCSAHMGHVF-DDGPPPKHRRFCINSASIDFV 20161

>gnl|Dana-agencourt-040714-asm|contig_4797  Length = 61609
Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -3
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 43103 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 42972

 Score = 40.4 bits (126), Expect = 0.009
 Identities = 25/55 (45%), Positives = 34/55 (61%)
 Frame = -3
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSAS 113
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV   K+ SAS
Sbjct: 40394 PERIRTE---VRCARCSAHMGHVF-EDGPKPTRKRYCINSASIEFVTGEKDPSAS 40242

Score = 35.6 bits (109), Expect = 0.24
Identities = 27/70 (38%), Positives = 37/70 (52%), Gaps = 1/70 (1%)
Frame = -3
Query: 36    KYAHSSPWPAFTETIHADSV-AKRPEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF 94
             K+ HSS       TI +  V +K     R+E   V C +C   +GH F +DGP P + RF
Sbjct: 42236 KFRHSSHTSITVNTIKSTVVISKTLGMVRTE---VRCSRCSAHMGHVF-DDGPPPKHRRF 42069
Query: 95    *IFSSSLKFV 104
              I S+S+ FV
Sbjct: 42068 CINSASIDFV 42039

>gnl|Dyak-washu-assembly_040407|Contig7.13   Length = 111046

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +1
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 46012 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 46143

Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +1
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 48979 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 49128

Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +2
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 47174 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 47305

>gnl|Dvir-agencourt-run1029-asm|contig_386  Length = 191045
Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -1
Query: 12     FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
              +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 176732 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 176601

Score = 36.5 bits (112), Expect = 0.13
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -3
Query: 59     PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
              PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 174108 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 173983

Score = 35.6 bits (109), Expect = 0.24
Identities = 18/39 (46%), Positives = 25/39 (64%)
Frame = -3
Query: 68     KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPK 106
              +V C KC   +GH F +DGP P + RF I S+S+ FV +
Sbjct: 175752 EVRCSKCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKR 175639

>2 type=chromosome; loc=2:1..30711475; ID=2; release=r1.03; species=dpse  Length = 30711475
Score = 41.5 bits (130), Expect = 0.004    BLAST HIT on Genome Map
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -3

Query: 12       FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
                +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 28012277 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 28012146

Score = 36.5 bits (112), Expect = 0.13    BLAST HIT on Genome Map
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -1

Query: 59       PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
                PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 28009705 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 28009580

Score = 35.9 bits (110), Expect = 0.20    BLAST HIT on Genome Map
Identities = 22/46 (47%), Positives = 29/46 (63%)
Frame = -2

Query: 68       KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSAS 113
                +V C KC   +GH F +DGP P + RF I S+S+ FV K   T+AS
Sbjct: 28011345 EVRCSKCSAHMGHVF-DDGPPPKHHRFCINSASIDFV-KSAPTAAS 28011214

>gnl|Dere-agencourt-run1028-asm|contig_1017  Length = 31091

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +3
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 19356 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 19487

Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +2
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 22229 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 22378

Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +1
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 20458 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 20589

>gnl|Dsim-washu-w501-asm|Contig20.125  Length = 16361

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +3
Query: 12   FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
            +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 4344 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 4475
Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +1
Query: 59   PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
            PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 7255 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 7404
Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +2
Query: 68   KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
            +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 5453 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 5584

>gi|27605048|emb|BX031767.1|CNS08WOB  Single read from an extremity of a full-length
 cDNA clone made from Anopheles gambiae total adult females. 3-PRIME end of clone
 FK0AAA47BD04 of strain 6-9 of Anopheles gambiae (African malaria mosquito)


 Score = 75.5 bits (184), Expect = 3e-14
 Identities = 38/100 (38%), Positives = 52/100 (52%), Gaps = 2/100 (2%)
 Frame = -2

Query: 12  FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSVA--KRPEHNRSEALKV 69
           +   +E G Y+C  C  ELFSS +KY     WPAF + +    V   K P        +V
Sbjct: 865 YNKFYEKGTYICVVCSQELFSSETKYDSGCGWPAFNDVLDQGKVTLHKDPSIPGRVRTEV 686

Query: 70  SCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKE 109
            C KC   +GH F  DGP P + R+ I S+S++F+P G E
Sbjct: 685 RCSKCAAHMGHVF-EDGPPPTRKRYCINSASIEFMPAGSE 569

SECIS elements


gnl|Dsim-washu-w501-asm|Contig27.138: 1620 1527 (SECIS on complementary strand)

GAUAAGUGGU GGUAAUU AAUUAUUCAACUU AUGAA GAUUAUUUCUU AA AGGCCUCUGGCU CGGAAAUGGUC UGAA CUU UGUUGC ACACGCGAGG



COVE score: 32.12 (Recommended threshold for COVE: 15)


gnl|Dyak-washu-assembly 040407|Contig8.19: 70796 70704 (SECIS on complementary strand)

GUUAAGUGAU ACAAAUU AAUUAUUCAACUU AUGAG GACUAUUUCUU AA AGGCCUUGGCU CGGAAAUAGUC UGAA CAU UAUUGU ACAUGAGAGG



COVE score: 32.58 (Recommended threshold for COVE: 15)

Multiple alignment


If you want to see the ClustalW alignment, click here.

Unrooted N-J tree

SelT

tblastn

>gnl|Dana-agencourt-040714-asm|contig_1097					3e-30     43%

>gnl|Dvir-agencourt-run1029-asm|contig_992					5e-30	  42%

>4_group4 type=chromosome; loc=4_group4:1..6604331;ID=4_group4;release=r1.03; 
 species=dpse									6e-30	  40%

>gi|24581818|ref|NM_135053.2| Drosophila melanogaster CG3887-PA (CG3887) 
mRNA, complete cds							 	2e-37	  43%

>gi|42765836|gb|AY440807.1| Armigeres subalbatus ASAP ID: 42399 putative:
 selenoprotein T mRNA.							        1e-36	  44% 

>gi|42761900|gb|AY433028.1| Aedes aegypti ASAP ID: 36405 putative:
 selenoprotein T mRNA.							        7e-34	  48%

>gi|58393304|ref|XM_319979.2| Anopheles gambiae str.PEST ENSANGP00000016703	8e-33     45% 

Gene prediction

If you want to check all the exonerate outputs in gff format, click here.

>gi|42765836|gb|AY440807.1| Armigeres subalbatus ASAP ID:42399 putative:selenoprotein T mRNA


 Score =  151 bits (381), Expect = 1e-36
 Identities = 69/154 (44%), Positives = 101/154 (65%)
 Frame = +2

Query: 25  GPLLKFQICVS*GYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASFLSVFKLVLI 84
           G  + F  C S GYR+ F+EY  +I ++YP+I I G NY P     +++  L V KL++I
Sbjct: 218 GATMTFMYCYSCGYRKAFDEYYNIIHEKYPEITIRGGNYDPPGFNMYLSKILLVTKLLMI 397

Query: 85  GLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGAFEITLNDVP 144
             ++   D F F     PS W+W  +NK+YACM+VFFL NM+E Q +S+GAFEI LNDVP
Sbjct: 398 IALVSNFDLFGFLRQPMPSWWRWCTDNKIYACMLVFFLGNMLEAQLISSGAFEIALNDVP 577

Query: 145 VWSKLESGHLPSMQQLVQILDNEMKLNVHMDSIP 178
           VW KLE+G +P+ Q+L QI+D+ ++ +  ++  P
Sbjct: 578 VWPKLETGRIPAPQELFQIIDSHLQFSDKIEQNP 679

>gi|42761900|gb|AY433028.1|  Aedes aegypti ASAP ID: 36405 putative: selenoprotein T mRNA


 Score =  140 bits (353), Expect = 2e-33
 Identities = 63/132 (47%), Positives = 90/132 (68%)
 Frame = +3

Query: 25  GPLLKFQICVS*GYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASFLSVFKLVLI 84
           G  + F  C S GYR+ ++EY  +I ++YP+I I G NY P     +++    V KL +I
Sbjct: 324 GATMTFMYCYSCGYRKAYDEYYNIIHEKYPEITIRGANYDPPGFNMYLSKIXLVAKLAMI 503

Query: 85  GLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGAFEITLNDVP 144
            +++   + F F  ++ PS WQW  +NK+YACMMVFFL NM+E Q +S+GAFEI+LNDVP
Sbjct: 504 MVLMSNFNLFGFLNLRIPSWWQWCTDNKMYACMMVFFLGNMLEAQLISSGAFEISLNDVP 683

Query: 145 VWSKLESGHLPS 156
           VWSKLE+G +P+
Sbjct: 684 VWSKLETGRIPA 719

>gi|58393304|ref|XM_319979.2|  Anopheles gambiae str. PEST ENSANGP00000016703, partial mRNA

 Score =  138 bits (348), Expect = 8e-33
 Identities = 66/145 (45%), Positives = 93/145 (64%)
 Frame = +1

Query: 25  GPLLKFQICVS*GYRRVFEEYMRVISQRYPDIRIEGENYLPQPIYRHIASFLSVFKLVLI 84
           G  + F  C S GYR+ F++Y  +I ++YP+I I G NY P  +   ++  L V KL+LI
Sbjct: 130 GATMTFLYCYSCGYRKAFDDYHNLILEKYPEITIRGSNYDPSGVNMLLSKVLLVTKLLLI 309

Query: 85  GLIIVGKDPFAFFGMQAPSIWQWGQENKVYACMMVFFLSNMIENQCMSTGAFEITLNDVP 144
             ++   D   + G      WQW   NK+YA MM+FFL N +E Q +S+GAFEITLNDVP
Sbjct: 310 AALMSNYDIGRYIGNPFAGWWQWCFNNKLYASMMIFFLGNTLEAQLISSGAFEITLNDVP 489

Query: 145 VWSKLESGHLPSMQQLVQILDNEMK 169
           VWSKLE+G  P+ Q++ QI+DN ++
Sbjct: 490 VWSKLETGRFPAPQEMFQIIDNHLQ 564

SECIS elements


No SECIS elements were found by SeciSearch.

Multiple alignment


If you want to see the ClustalW alignment, click here.

Unrooted N-J tree

SelR

tblastn

>gnl|Dmov-agencourt-run0811-asm|contig_717			        	0.003	  40%

>gnl|Dana-agencourt-040714-asm|contig_4797					0.004     40%

>gnl|Dyak-washu-assembly_040407|Contig7.13                              	0.004	  40%

>gnl|Dvir-agencourt-run1029-asm|contig_386					0.004     40%

>2 type=chromosome; loc=2:1..30711475; ID=2; release=r1.03; species=dpse	0.004	  40%

>gnl|Dere-agencourt-run1028-asm|contig_1017					0.004     40%

>gnl|Dsim-washu-w501-asm|Contig20.125						0.004	  40%

>gi|27605048|emb|BX031767.1|CNS08WOB Single read from an extremity of a 
full-length cDNA clone made from Anopheles gambiae total adult females.3-PRIME  3e-14     38% 

Gene prediction

For the sequences that exonerate could not have been performed, tblastn outputs are shown:

>gnl|Dmov-agencourt-run0811-asm|contig_717  Length = 57670

Score = 41.8 bits (131), Expect = 0.003
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -1
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 21133 YNKHYEKGVYRCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 21002

Score = 36.5 bits (112), Expect = 0.13
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -3
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 18638 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 18513

Score = 35.1 bits (107), Expect = 0.36
Identities = 18/37 (48%), Positives = 24/37 (64%)
Frame = -2
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
             +V C KC   +GH F +DGP P + RF I S+S+ FV
Sbjct: 20268 EVRCSKCSAHMGHVF-DDGPPPKHRRFCINSASIDFV 20161

>gnl|Dana-agencourt-040714-asm|contig_4797  Length = 61609
Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -3
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 43103 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 42972

 Score = 40.4 bits (126), Expect = 0.009
 Identities = 25/55 (45%), Positives = 34/55 (61%)
 Frame = -3
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSAS 113
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV   K+ SAS
Sbjct: 40394 PERIRTE---VRCARCSAHMGHVF-EDGPKPTRKRYCINSASIEFVTGEKDPSAS 40242

Score = 35.6 bits (109), Expect = 0.24
Identities = 27/70 (38%), Positives = 37/70 (52%), Gaps = 1/70 (1%)
Frame = -3
Query: 36    KYAHSSPWPAFTETIHADSV-AKRPEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF 94
             K+ HSS       TI +  V +K     R+E   V C +C   +GH F +DGP P + RF
Sbjct: 42236 KFRHSSHTSITVNTIKSTVVISKTLGMVRTE---VRCSRCSAHMGHVF-DDGPPPKHRRF 42069
Query: 95    *IFSSSLKFV 104
              I S+S+ FV
Sbjct: 42068 CINSASIDFV 42039

>gnl|Dyak-washu-assembly_040407|Contig7.13   Length = 111046

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +1
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 46012 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 46143

Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +1
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 48979 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 49128

Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +2
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 47174 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 47305

>gnl|Dvir-agencourt-run1029-asm|contig_386    Length = 191045
Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -1
Query: 12     FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
              +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 176732 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 176601

Score = 36.5 bits (112), Expect = 0.13
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -3
Query: 59     PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
              PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 174108 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 173983

Score = 35.6 bits (109), Expect = 0.24
Identities = 18/39 (46%), Positives = 25/39 (64%)
Frame = -3
Query: 68     KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPK 106
              +V C KC   +GH F +DGP P + RF I S+S+ FV +
Sbjct: 175752 EVRCSKCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKR 175639

>2 type=chromosome; loc=2:1..30711475; ID=2; release=r1.03; species=dpse  Length = 30711475
Score = 41.5 bits (130), Expect = 0.004    BLAST HIT on Genome Map
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = -3

Query: 12       FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
                +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 28012277 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 28012146

Score = 36.5 bits (112), Expect = 0.13    BLAST HIT on Genome Map
Identities = 21/46 (45%), Positives = 29/46 (63%)
Frame = -1

Query: 59       PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFV 104
                PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV
Sbjct: 28009705 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFV 28009580

Score = 35.9 bits (110), Expect = 0.20    BLAST HIT on Genome Map
Identities = 22/46 (47%), Positives = 29/46 (63%)
Frame = -2

Query: 68       KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSAS 113
                +V C KC   +GH F +DGP P + RF I S+S+ FV K   T+AS
Sbjct: 28011345 EVRCSKCSAHMGHVF-DDGPPPKHHRFCINSASIDFV-KSAPTAAS 28011214

>gnl|Dere-agencourt-run1028-asm|contig_1017   Length = 31091

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +3
Query: 12    FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
             +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 19356 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 19487

Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +2
Query: 59    PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 22229 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 22378

Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +1
Query: 68    KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
             +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 20458 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 20589

>gnl|Dsim-washu-w501-asm|Contig20.125  Length = 16361

Score = 41.5 bits (130), Expect = 0.004
Identities = 18/44 (40%), Positives = 24/44 (54%)
Frame = +3
Query: 12   FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSV 55
            +  H+E GVY C  C  +LFSS +KY     WPAF + +    V
Sbjct: 4344 YNKHYEKGVYQCIVCHQDLFSSDTKYDSGCGWPAFNDVLDKGKV 4475
Score = 37.6 bits (116), Expect = 0.062
Identities = 23/54 (42%), Positives = 32/54 (59%)
Frame = +1
Query: 59   PEHNRSEALKVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
            PE  R+E   V C +C   +GH F  DGPKP + R+ I S+S++FV     TS+
Sbjct: 7255 PERIRTE---VRCARCNAHMGHVF-EDGPKPTRKRYCINSASIEFVNADPATSS 7404
Score = 35.3 bits (108), Expect = 0.29
Identities = 18/45 (40%), Positives = 26/45 (57%)
Frame = +2
Query: 68   KVSCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKETSA 112
            +V C +C   +GH F +DGP P + RF I S+S+ FV     + A
Sbjct: 5453 EVRCSRCSAHMGHVF-DDGPPPKHRRFCINSASIDFVKSATPSKA 5584

>gi|27605048|emb|BX031767.1|CNS08WOB  Single read from an extremity of a full-length
 cDNA clone made from Anopheles gambiae total adult females. 3-PRIME end of clone
 FK0AAA47BD04 of strain 6-9 of Anopheles gambiae (African malaria mosquito)


 Score = 75.5 bits (184), Expect = 3e-14
 Identities = 38/100 (38%), Positives = 52/100 (52%), Gaps = 2/100 (2%)
 Frame = -2

Query: 12  FQNHFEPGVYVCAKCGYELFSSRSKYAHSSPWPAFTETIHADSVA--KRPEHNRSEALKV 69
           +   +E G Y+C  C  ELFSS +KY     WPAF + +    V   K P        +V
Sbjct: 865 YNKFYEKGTYICVVCSQELFSSETKYDSGCGWPAFNDVLDQGKVTLHKDPSIPGRVRTEV 686

Query: 70  SCGKCGNGLGHEFLNDGPKPGQSRF*IFSSSLKFVPKGKE 109
            C KC   +GH F  DGP P + R+ I S+S++F+P G E
Sbjct: 685 RCSKCAAHMGHVF-EDGPPPTRKRYCINSASIEFMPAGSE 569

SECIS elements


No SECIS elements were found by SeciSearch.

Multiple alignment


If you want to see the ClustalW alignment, click here.

Unrooted N-J tree

GPx4

After tblastn similarity search,the all five human GPX selenoproteins, were found to match with the same DNA sequence in each of the species. This fact was probably due to the high similarity of the human GPx selenoproteins. Thus, we decided to determine, for each species, to which of the human selenoproteins was our common DNA sequence more closely related. To find out our best candidate we could have based on e-values and homology percentage shown in tblastn results, but in order to make our choice more confident we performed a multiple sequence aligment followed by an unrooted N-J tree, where we included all human GPx proteins and the protein we found for each of the species.

tblastn

As we can notice in the previous trees, all the proteins that we have studied are closer to GPx4 than to any other human GPx selenoprotein. Although this fact doesn't mean that all the proteins found in insect genomes correspond with human GPx4 function. Anyway, we will use this protein for gene prediction and multiple alingment and only GPx4 tblastn results will be shown.

>gnl|Dyak-washu-assembly_040407|Contig5.61					2e-25	  46%

>gnl|Dere-agencourt-run1028-asm|contig_2920					1e-24	  39%

>XR_group8 type=chromosome; loc=XR_group8:1..9190824 ID=XR_group  
release=r1.03; species=dpse							6e-23	  38%

>3 type=chromosome; loc=3:1..19738957;ID=3; release=r1.03; species=dps		1e-20	  39%

>gnl|Dsim-washu-w501-asm|Contig12.100						5e-14	  39% 

>gnl|Dana-agencourt-040714-asm|contig_263					7e-14	  49%

>gnl|Dana-agencourt-040714-asm|contig_4443					3e-13	  48%

>gnl|Dana-agencourt-040714-asm|contig_1256					3e-13	  39%

>gnl|Dvir-agencourt-run1029-asm|contig_1983					1e-13     48%

>gnl|Dere-agencourt-run1028-asm|contig_2847					1e-13	  37%

>gnl|Dsim-washu-w501-asm|Contig0.495						1e-13	  46%

>gnl|Dyak-washu-assembly_040407|Contig15.28					2e-12	  38%

>gnl|Dmov-agencourt-run0811-asm|contig_5053					3e-06	  40%

>gi|48117434|ref|XM_396418.1| Apis mellifera similar to putative thioredoxin 
perxidase									1e-47     55%

>gi|33306812|gb|AF394234.1| Aedes aegypti glutathione peroxidase (GPx) mRNA,	2e-44     56%

>gi|58384275|ref|XM_313166.2| Anopheles gambiae str. PEST ENSANGP00000024750	1e-34     46%

>gi|50897528|gb|AY625510.1| Glossina morsitans morsitans clone Gmm0092 
putative glutathione								6e-33     45%

>gi|42765409|gb|AY440380.1| Armigeres subalbatus ASAP ID: 42545 glutathione 
peroxidase mRNA									5e-29     48%

>gi|28564458|emb|AJ547804.1|IRI547804 Ixodes ricinus mRNA for glutathione 
peroxidase (gluper1)								1e-17     48%
 
>gi|12958610|gb|AF321612.1| Venturia canescens virus-like particle protein mRNA 7e-18     33%

>gi|40882422|gb|BT011331.1| Drosophila melanogaster SD18370 full insert cDNA	2e-34     44%

Gene prediction

If you want to check all the exonerate outputs in gff format, click here.

For the sequences that exonerate could not have been performed, tblastn outputs are shown:

>gnl|Dyak-washu-assembly_040407|Contig5.61(contig is not correct, exonerate can not
 be performed)    Length = 146236

Score = 76.1 bits (253), Expect(2) = 2e-25
Identities = 40/86 (46%), Positives = 60/86 (69%)
Frame = +1
Query: 30    ASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHAR 89
             ++  D++ A S++EF+ KD  G+ ++L+KY+G V +V N+AS+ G T+ NY +L DL  +
Sbjct: 63946 SANGDYKNAASIYEFTVKDTHGNDISLEKYKGKVVLVVNIASKCGLTKNNYQKLTDLKEK 64125
Query: 90    YAECGLRILAFPCNQFGKQEPGSNEE 115
             Y E GL IL FPCNQFG Q P ++ E
Sbjct: 64126 YGERGLVILNFPCNQFGSQMPEADGE 64203

Score = 60.1 bits (196), Expect(2) = 2e-25
Identities = 32/62 (51%), Positives = 44/62 (70%)
Frame = +2
Query: 132   KICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 191
             ++ VNGDDA PL+K++K   K  G LG+ IKWNFTKFL++K G  + RY P  +P+ I K
Sbjct: 64322 QVDVNGDDAAPLYKYLKA--KQTGTLGSGIKWNFTKFLVNKEGIPINRYAPTTDPMDIAK 64495
Query: 192   DL 193
             D+
Sbjct: 64496 DI 64501

>gnl|Dere-agencourt-run1028-asm|contig_2920    Length = 10481

Score =  113 bits (385), Expect = 1e-24
Identities = 74/188 (39%), Positives = 110/188 (58%), Gaps = 24/188 (12%)
Frame = +3
Query: 30   ASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHAR 89
            ++  D++ A S++EF+ KD  G+ ++L+KY+G V +V N+AS+ G T+ NY +L DL  +
Sbjct: 7311 SANGDYKNAASIYEFTVKDTHGNDISLEKYKGKVVLVVNIASKCGLTKNNYQKLTDLKEK 7490
Query: 90   YAECGLRILAFPCNQFGKQEPGSNEE----------------------IKEFAAGYNVKF 127
            Y E GL IL FPCNQFG Q P ++ E                      +K        +F
Sbjct: 7491 YGERGLVILNFPCNQFGSQMPEADGEAMVCHLRDSKADIGEVFAKVRLLKLVVGCSRPRF 7670
Query: 128  D--MFSKICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEE 185
            +   F ++ VNGD+A PL+K++K   K  G LG+ IKWNFTKFL++K G  + RY P  +
Sbjct: 7671 NNLRFLQVDVNGDNAAPLYKYLK--AKQTGTLGSGIKWNFTKFLVNKEGVPINRYAPTTD 7844
Query: 186  PLVIEKDL 193
            P+ I KD+
Sbjct: 7845 PMDISKDI 7868

>gnl|Dsim-washu-w501-asm|Contig12.100     Length = 22061

Score = 71.1 bits (235), Expect(2) = 5e-14
Identities = 48/122 (39%), Positives = 64/122 (52%), Gaps = 2/122 (1%)
Frame = -2
Query: 74   GKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSN--EEIKEFAAGYNVKFDMFS 131
            G T   Y  L  L   Y E GLRIL FPCNQFG Q P S+  E +  +         +F+
Sbjct: 8890 GLTLSQYNGLRYLLEEYEEQGLRILNFPCNQFGGQMPESDGQEMLDHLRREGANIGHLFA 8711
Query: 132  KICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 191
            KI V G  A PL+K +  +        + I+WNF KFL+D+ G + KRYG   EP+ +
Sbjct: 8710 KIDVKGAQADPLYKLLTRHQ-------HDIEWNFVKFLVDRKGNIHKRYGAELEPVALTD 8552
Query: 192  DL 193
            D+
Sbjct: 8551 DI 8546
Score = 26.9 bits (78), Expect(2) = 5e-14
Identities = 13/40 (32%), Positives = 24/40 (60%)
Frame = -3
Query: 34   DWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQX 73
            D R   ++H ++ +D  G+ V LD + G V ++ N+AS+
Sbjct: 9072 DMRWRLTIHALTVRDTFGNPVQLDIFAGHVMLIVNIASR* 8953

>gnl|Dere-agencourt-run1028-asm|contig_2847     Length = 53404

Score = 70.2 bits (232), Expect(2) = 1e-13
Identities = 46/122 (37%), Positives = 64/122 (52%), Gaps = 2/122 (1%)
Frame = +1
Query: 74   GKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSN--EEIKEFAAGYNVKFDMFS 131
            G T   Y  L  L   Y + GLRIL FPCNQFG Q P S+  E +  +         +F+
Sbjct: 4213 GLTSSQYNGLRYLLEEYEDRGLRILNFPCNQFGGQMPESDGQEMLDHLRREGANIGHIFA 4392
Query: 132  KICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 191
            K+ V G  A PL+K +  + +        I+WNF KFL+D+ G + KRYG   EP+ +
Sbjct: 4393 KVDVKGAQADPLYKLLTRHQQD-------IEWNFVKFLVDRKGNIHKRYGAELEPVALTD 4551
Query: 192  DL 193
            D+
Sbjct: 4552 DI 4557
Score = 26.3 bits (76), Expect(2) = 1e-13
Identities = 13/42 (30%), Positives = 25/42 (59%)
Frame = +3
Query: 34   DWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGK 75
            D R   +++ ++ +D  G  V LDK+ G V ++ N+AS+  +
Sbjct: 4038 DMRWRLTIQALTVRDTFGKPVQLDKFAGHVMLIVNIASK*ER 4163

>gnl|Dana-agencourt-040714-asm|contig_4443     Length = 7909

Score = 46.9 bits (149), Expect(2) = 3e-13
Identities = 27/56 (48%), Positives = 35/56 (62%)
Frame = +1
Query: 60   RGFVCIVTNVASQXGKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSNEE 115
            +G V +V N+ASQ G T+ NY  L DL  +Y + G  IL FPCNQFG Q   ++ E
Sbjct: 1363 KGQVFLVVNIASQCGLTKNNYQTLTDLKEKYGDIG*IILNFPCNQFGSQMLETDGE 1530

>gnl|Dmov-agencourt-run0811-asm|contig_5053     Length = 2228

Score = 51.7 bits (166), Expect = 3e-06
Identities = 29/71 (40%), Positives = 46/71 (64%)
Frame = -2
Query: 31   SRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHARY 90
            S  D++ A S++EF+ KD  G+ V+L+KY+G V ++ N+AS+ G T+ NY +L DL  +Y
Sbjct: 1636 SDGDYKNAASIYEFNVKDTHGNDVSLEKYKGQVILIVNIASKCGLTKNNYKKLTDLKEKY 1457
Query: 91   AECGLRILAFP 101
             E G     +P
Sbjct: 1456 GERGTDHPELP 1424

>gnl|Dana-agencourt-040714-asm|contig_1256     Length = 119871

Score = 74.2 bits (246), Expect = 3e-13
Identities = 50/126 (39%), Positives = 67/126 (53%), Gaps = 2/126 (1%)
Frame = -1

Query: 74    GKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSN-EEIKEFAAGYNVKF-DMFS 131
             G T   Y  L +L  +Y E GL IL FPCNQFG Q P S+  EI E           +F+
Sbjct: 23997 GLTSSQYAGLHELREKYEERGLSILNFPCNQFGAQMPESDGREILEHLRQKKANIGHIFA 23818

Query: 132   KICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 191
             KI VNG +A PL+K +  +          I+WNF KFLID+ G +  RYG  ++P V+
Sbjct: 23817 KIKVNGRNADPLYKLLTRK-------APRIEWNFVKFLIDRKGNIYGRYGAEKKPAVLVN 23659

Query: 192   DLPHYF 197
             D+   +
Sbjct: 23658 DIERLL 23641

>gnl|Dyak-washu-assembly_040407|Contig15.28    Length = 56780

Score = 71.6 bits (237), Expect = 2e-12
Identities = 47/122 (38%), Positives = 64/122 (52%), Gaps = 2/122 (1%)
Frame = -1

Query: 74    GKTEVNYTQLVDLHARYAECGLRILAFPCNQFGKQEPGSN--EEIKEFAAGYNVKFDMFS 131
             G T   Y  L  L   Y + GLRIL FPCNQFG Q P S+  E +  +         +F+
Sbjct: 32579 GLTSTQYNGLRYLLEEYEDRGLRILNFPCNQFGAQMPESDGQEMLDHLRREGANIGQLFA 32400

Query: 132   KICVNGDDAHPLWKWMKIQPKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 191
             KI V G  A PL+K +  +        + I+WNF KFL+D+ G + KRYG   EP+ +
Sbjct: 32399 KIDVKGAQADPLYKLLTRHQ-------HDIEWNFVKFLVDRRGNIYKRYGAELEPVALTD 32241

Query: 192   DL 193
             D+
Sbjct: 32240 DI 32235

>gi|48117434|ref|XM_396418.1| Apis mellifera similar to putative thioredoxin
 perxidase (LOC412967), mRNA

Score =  187 bits (476), Expect = 1e-47
Identities = 91/165 (55%), Positives = 120/165 (72%), Gaps = 1/165 (0%)
Frame = +1

Query: 34  DWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHARYAEC 93
           +W+ A ++++F AKDI G+ V+L+KYRG VCI+ NVAS  G T+ NY +LV L+ +Y E
Sbjct: 163 NWKSASTIYDFHAKDIHGNDVSLNKYRGHVCIIVNVASNCGLTDTNYRELVQLYEKYNEK 342

Query: 94  -GLRILAFPCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPK 152
            GLRILAFP N+FG QEPG++ EI EF   YNV FD+F KI VNGD+AHPLWKW+K Q
Sbjct: 343 EGLRILAFPSNEFGGQEPGTSVEILEFVKKYNVTFDLFEKINVNGDNAHPLWKWLKTQ-- 516

Query: 153 GKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF 197
             G + + IKWNF+KF+I+K G VV R+ P  +PL +E +L  YF
Sbjct: 517 ANGFITDDIKWNFSKFIINKEGKVVSRFAPTVDPLQMESELKKYF 651

>gi|33306812|gb|AF394234.1| Aedes aegypti glutathione peroxidase (GPx) mRNA, complete cds

Score =  177 bits (448), Expect = 2e-44
Identities = 91/160 (56%), Positives = 119/160 (74%), Gaps = 2/160 (1%)
Frame = +1

Query: 40  SMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHARYAEC-GLRIL 98
           S+++FSA DIDG+ V+ ++YRG V I+ NVAS+ G T  +Y +L +L+  Y E  GLRIL
Sbjct: 274 SVYDFSAVDIDGNKVDFERYRGHVLIIVNVASKCGYTAGHYKELNELYEEYGETEGLRIL 453

Query: 99  AFPCNQFGKQEPGSNEEIKEFA-AGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGIL 157
           AFPCNQFG QEPG+NEEIK FA      KFD+F+KI VNGD+AHPLW+++K Q +G G L
Sbjct: 454 AFPCNQFGNQEPGTNEEIKHFARVEKGAKFDLFAKIYVNGDEAHPLWQFLK-QRQG-GTL 627

Query: 158 GNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF 197
            +AIKWNFTKF++DKNG  V+R+GP   PL +  +L  YF
Sbjct: 628 FDAIKWNFTKFIVDKNGQPVERHGPQTSPLQLRDNLKKYF 747

>gi|58384275|ref|XM_313166.2| Anopheles gambiae str. PEST ENSANGP00000024750 
(ENSANGG00000011473), mRNA

Score =  144 bits (364), Expect = 1e-34
Identities = 76/163 (46%), Positives = 109/163 (66%), Gaps = 2/163 (1%)
Frame = +1

Query: 33  DDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHARYAE 92
           +D++ A+S+++F+ KD  G  V+L+KYRG V ++ N+ASQ G T+ NY +L +L  +YA+
Sbjct: 118 EDYKNAKSVYDFTVKDSQGADVSLEKYRGKVLLIVNIASQCGLTKGNYAELTELSQKYAD 297

Query: 93  CGLRILAFPCNQFGKQEP-GSNEEIKEFAAGYNVKF-DMFSKICVNGDDAHPLWKWMKIQ 150
              +IL+FPCNQFG Q P G  EE+         +  D+F+KI VNGD AHPL+K++K
Sbjct: 298 KDFKILSFPCNQFGGQMPEGDGEEMVCHLRSAKAEVGDVFAKIDVNGDGAHPLYKYLK-- 471

Query: 151 PKGKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDL 193
            K  G LG++IKWNF KFL++K+G  V RY P   P  I KD+
Sbjct: 472 HKQGGTLGDSIKWNFAKFLVNKDGQPVDRYAPTTSPSSIVKDI 600

>gi|50897528|gb|AY625510.1 Glossina morsitans morsitans clone Gmm0092 putative 
glutathione peroxidase mRNA

Score =  139 bits (350), Expect = 6e-33
Identities = 74/161 (45%), Positives = 107/161 (66%), Gaps = 5/161 (3%)
Frame = +3

Query: 38  ARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDLHARYAECGLRI 97
           A S+++F+ KD  G+ V+L++YRG V ++ N+ASQ G T+ NY +L DL  +Y + GL+I
Sbjct: 312 ASSIYDFTVKDTYGNDVSLEQYRGHVVLIVNIASQCGLTKNNYKKLTDLREKYGDKGLKI 491

Query: 98  LAFPCNQFGKQEPGSNEE-----IKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPK 152
           L FPCNQFG Q P S+ E     +++  A      D+F K+ VNG +A PL++++K   K
Sbjct: 492 LNFPCNQFGSQMPESDGEPMVCHLRDAKADIG---DVFQKVDVNGANAAPLYQYLK--AK 656

Query: 153 GKGILGNAIKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDL 193
             G L +AIKWNFTKFL++K G  VKRY P  +P+ I KD+
Sbjct: 657 QGGTLVSAIKWNFTKFLVNKEGIPVKRYAPTTDPMDIAKDI 779

>gi|28564458|emb|AJ547804.1|IRI547804 Ixodes ricinus mRNA for glutathione peroxidase (gluper1 
gene) Length = 914

 Score = 88.6 bits (218), Expect = 1e-17
 Identities = 46/94 (48%), Positives = 65/94 (69%), Gaps = 1/94 (1%)
 Frame = +1


Query: 27  TMCASRDDWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNVASQXGKTEVNYTQLVDL 88
           T+  S + W+ A+S++EFSA DIDG+ V+ +KYRG V  + NVA +   T+ +Y +L  L
Sbjct: 298 TLANSDEGWKNAKSIYEFSALDIDGNKVDFNKYRGHVTQIVNVACKCLLTQEHYKKLSAL 477


Query: 87  HARYAEC-GLRILAFPCNQFGKQEPGSNEEIKEF 119
             + +Y+E GLRI+AFP N F KQEP + EIKEF
Sbjct: 478 YHKYSESKGLRIMAFPTNDFAKQEPWAEPEIKEF 579


 Score = 107 bits (268), Expect = 2e-23
 Identities = 54/97 (55%), Positives = 63/97 (64%)
 Frame = +2


Query: 101 PCNQFGKQEPGSNEEIKEFAAGYNVKFDMFSKICVNGDDAHPLWKWMKIQPKGKGILGNA 160
           P      + PG N   + F   ++V FDMFSKI VNGD+AHPLWK++K   K  G L NA
Sbjct: 524 PPTTLPSRNPGPNPRSRSFVKQFDVTFDMFSKISVNGDNAHPLWKYLK--EKQPGFLFNA 697


Query: 161 IKWNFTKFLIDKNGCVVKRYGPMEEPLVIEKDLPHYF 197 
           IKWNFTKFL+DKNG VKRYGP + IE DL YF
Sbjct: 698 IKWNFTKFLVDKNGQPVKRYGPTDSFETIEADLLKYF 808

>gi|12958610|gb|AF321612.1|AF321612 Venturia canescens virus-like particle protein mRNA, 
complete cds    Length = 861

 Score = 89.4 bits (220), Expect = 7e-18
 Identities = 57/168 (33%), Positives = 99/168 (58%), Gaps = 10/168 (5%)
 Frame = +1


Query: 34  DWRCARSMHEFSAKDIDGHMVNLDKYRGFVCIVTNV---ASQXGKTEVNYTQLVDLHARY 90
           DW+ A+S+++F+A +IDG ++NL+KY+G I+ N A+Q G +Y +L +L+ + 
Sbjct: 340 DWKTAKSLYQFTATNIDGDLINLNKYKGRPLIILNASSKANQLGTDMDHYEELKELYDKL 519


Query: 91  --AECGLRILAFPCNQF--GKQEPGSNEEIKEF-AAGYNVKFDMFSKICVNGDDAHPLWK 145
              ++ L+ILAF CNQF    ++  +N + KEF      ++ D+F+K+ V G+ A PLWK
Sbjct: 520 KGSKNELKILAFLCNQFDDSDKKDETNVDFKEFITTDKKLEADLFTKVEVTGEGAQPLWK 699


Query: 146 WMKIQPKGKGILGNA--IKWNFTKFLIDKNGCVVKRYGPMEEPLVIEK 19
           W+  Q      + +   I  +FT F++DK G  + R+   ++   IE+
Sbjct: 700 WLYEQYCTDIDVTDCKEINHDFTIFVVDKMGHYLGRFEYTKDLSSIER 843

SECIS elements


gnl|Dana-agencourt-040714-asm|contig 1256: 769 868
AAAUUCGUUA UCGCCAG UUCGUAAUAUAU AUGAA GUGAUCCAGGA AA UCCCAUAGCUGUAGCU UUCUGGGCCAU UGAG GUGGA CUAGCUA CCGCAAUUUA

COVE score: 13.44 (Recommended threshold for COVE: 15



gnl|Dvir-agencourt-run1029-asm|contig 1983: 22898 22990
UGGGGCGUUG CCAUACU AUGUGAAAGCACU AUGAU UCGCACAUAUGC AA AUGCUAU GUGUGUGUGUGU GUGAA CAU GUUCAUG AAUUUGCAUA

COVE score: 6.42 (Recommended threshold for COVE: 15)

Multiple alignment


After checking GPx4 tblastn results we noticed some problematic cases in the following genomic fragments:

>gnl|Dana-agencourt-040714-asm|contig_1256(ana-1)

>gnl|Dyak-washu-assembly_040407|Contig15.28(yak-1)

>gnl|Dere-agencourt-run1028-asm|contig_2847(ere1)

>gnl|Dsim-washu-w501-asm|Contig12.100(sim-2)

As seen in the tblastn results related to the sequences above,although having good E-values, there is no evidence of matching the human"sec"( Stop codon).Even though, we realized that the flanking aminoacids were matching but in different frames.After performing exonerate analysis, no results were obtained.In order to overcome this problems, we proceded to find protein sequences using FASTACHUNK and TRANSLATE TOOL.

Regarding to the common aminoacid sequence found in the rest of genomic fragments, we concluded that our protein sequence was divided in two frames, so we adapt our candidate proteins with fragments coming from different frames and we submit them to multiple alignment analysis. Arguments explaining this fact will be given in discussion section.

Unrooted N-J tree

15 KDa

tblasn

>gnl|Dmov-agencourt-run0811-asm|contig_1424					   2e-22  42%

>gnl|Dvir-agencourt-run1029-asm|contig_3670					   1e-21  39%

>gnl|Dana-agencourt-040714-asm|contig_267					   2e-21  51%

>gnl|Dere-agencourt-run1028-asm|contig_376					   2e-21  51%

>gnl|Dyak-washu-assembly_040407|Contig17.16					   6e-21  53%

>gnl|Dsim-washu-w501-asm|Contig3419.1						   6e-21  53%

>XR_group6 type=chromosome; loc=XR_group6:1..6604477; ID=XR_group6;release=r1.03; 
 species=dpse									   6e-20  42%

>gi|24666044|ref|NM_140743.1| Drosophila melanogaster CG7484-PB(CG7484)mRNA	   5e-27  45%
					
>gi|58386839|ref|XM_315090.2| Anopheles gambiae str.PEST ENSANGP00000011457	   2e-32  44% 
 
>gi|18389880|gb|AF457547.1| Anopheles gambiae selenoprotein mRNA,partial cds	   1e-31  44%

>gi|42765457|gb|AY440428.1| Armigeres subalbatus ASAP ID:40327 selenoprotein mRNA  3e-31  43%

>gi|42763702|gb|AY431560.1| Aedes aegypti ASAP ID:35705 selenoprotein mRNA	   8e-30  45%

>gi|48094317|ref|XM_394140.1| Apis mellifera similar to CG7484-PB(LOC410663),mRNA  3e-20  38%

Gene prediction

If you want to check all the exonerate outputs in gff format, click here.

For the sequences that exonerate could not have been performed, tblastn outputs are shown:

>gi|18389880|gb|AF457547.1| Anopheles gambiae selenoprotein mRNA, partial cds


 Score =  134 bits (337), Expect = 1e-31
 Identities = 67/152 (44%), Positives = 95/152 (62%), Gaps = 4/152 (2%)
 Frame = +1

Query: 15  LRLLLATVL--QAVSAFGAEFSSEACRELGFXXXXXXXXX-XXXGQFNLLQLDPDCRGCC 71
           +RL   T L    V+  GAEFS+E CRELG                + L++L   C  CC
Sbjct: 1   MRLFAITCLLFSIVTVIGAEFSAEDCRELGLIKSQLFCSACSSLSDYGLIELKEHCLECC 180

Query: 72  QEEAQFETK-KLYAGAILEVCGXKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKL 130
           Q++ + ++K K+Y  A+LEVC  K G +PQ+QAF++SD+P  F  L IKYVRG DP++KL
Sbjct: 181 QKDTEADSKLKVYPAAVLEVCTCKFGAYPQIQAFIKSDRPAKFPNLTIKYVRGLDPIVKL 360

Query: 131 LDDNGNIAEELSILKWNTDSVEEFLSEKLERI 162
           +D+ G + E LSI KWNTD+V+EF   +L ++
Sbjct: 361 MDEQGTVKETLSINKWNTDTVQEFFETRLAKV 456

>gi|42765457|gb|AY440428.1| Armigeres subalbatus ASAP ID: 40327 selenoprotein mRNA


 Score =  132 bits (333), Expect = 3e-31
 Identities = 68/157 (43%), Positives = 100/157 (63%), Gaps = 2/157 (1%)
 Frame = +3

Query: 8   CLVPAFGLRLLLATVLQAVSAFGAEFSSEACRELGFXXXXXXXXX-XXXGQFNLLQLDPD 66
           C++  F L L+   V+       AEF+++ CRELGF             G++ L +L   
Sbjct: 105 CIMNKFLLSLIPVLVI-VFQHTKAEFTTKDCRELGFIESQLFCSSCDTLGEYGLDELKDH 281

Query: 67  CRGCCQEEAQFETKKL-YAGAILEVCGXKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSD 125
           CR CCQ++A+   K + Y  A+LEVC  K G +PQ+QAF++SD+P+ F  L IKYVRG D
Sbjct: 282 CRECCQKDAESSGKLMVYPKAVLEVCTCKFGVYPQIQAFIKSDRPQKFPNLTIKYVRGLD 461

Query: 126 PVLKLLDDNGNIAEELSILKWNTDSVEEFLSEKLERI 162
           P++KL+D++GN+ E LSI KWNTD+V+EF   +L ++
Sbjct: 462 PIVKLMDESGNVKETLSITKWNTDTVQEFFETRLTKV 572

>gi|42763702|gb|AY431560.1| Aedes aegypti ASAP ID: 35705 selenoprotein mRNA


 Score =  128 bits (321), Expect = 8e-30
 Identities = 61/134 (45%), Positives = 89/134 (66%), Gaps = 2/134 (1%)
 Frame = +1

Query: 31  AEFSSEACRELGFXXXXXXXXX-XXXGQFNLLQLDPDCRGCCQEEAQFETKKL-YAGAIL 88
           AEF+++ CR+LGF             G++ L +L   CR CCQ++ +   K + Y  A+L
Sbjct: 196 AEFTAKDCRDLGFIKSQLYCSSCGTLGEYGLDELKDHCRECCQKDVESTGKLMVYPKAVL 375

Query: 89  EVCGXKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNT 148
           EVC  K G +PQ+QAF++SD+P+ F  L IKYVRG DP++KL+D+ GN+ E LSI KWNT
Sbjct: 376 EVCTCKFGAYPQIQAFIKSDRPQKFPNLTIKYVRGLDPIVKLMDEAGNVKETLSITKWNT 555

Query: 149 DSVEEFLSEKLERI 162
           D+V+EF   +L ++
Sbjct: 556 DTVQEFFETRLTKV 597

>gi|48094317|ref|XM_394140.1| Apis mellifera similar to CG7484-PB (LOC410663), mRNA

 Score = 96.7 bits (239), Expect = 3e-20
 Identities = 53/136 (38%), Positives = 77/136 (56%), Gaps = 2/136 (1%)
 Frame = +1

Query: 26  VSAFGAEFSSEACRELGFXXXXXXXXXXXXGQFNLLQLDPDCRGCCQEEAQFETK--KLY 83
           V+    EFS++ C+ LGF             + NLL         C     ++    K Y
Sbjct: 22  VNIVSTEFSADDCKSLGF------------NKANLL---------CSTYDDYDASGLKRY 138

Query: 84  AGAILEVCGXKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSI 143
             A+LEVC  K G +PQ+QAF++S++P  ++ LQIKYVRG DP++KL D +  + + L I
Sbjct: 139 PRAVLEVCTCKFGAYPQIQAFIKSNRPNKYKNLQIKYVRGLDPIIKLFDADNKVEDILDI 318

Query: 144 LKWNTDSVEEFLSEKL 159
            KW+TDSV+EFL+  L
Sbjct: 319 HKWDTDSVDEFLATHL 366

SECIS elements


gnl|Dvir-agencourt-run1029-asm|contig 3670: 11575 11669

UAGUACGAAG GCAUGCG CGUCUGGGAGAGC AUGAU GGCUGACUUUA AA GACGGCAGUCGA GAAGUCACGC UGAA AGGA UCACAGU GGAACAUAGA



COVE score: 18.15 (Recommended threshold for COVE: 15)

Multiple alignment


If you want to see the clustalW alignment, click here.

Unrooted N-J tree

TR3

As in GPx family, after doing tblastn similarity search,all three TR proteins were found to match with the same DNA sequence in each of the species. Consequently, we decided to perform a multiple sequence aligment followed by an unrooted N-J tree as we did with GPx family.Inthis case we included all human TR proteins and the protein we found for each of the species.

As we can notice in the previous figures all the proteins that we have studied are closer to TR3 than to any other human TR selenoprotein.Even though, this fact doesn't mean that all proteins found in insect genomes correspond with human TR3 function. Anyway, we will use this protein for gene prediction and multiple alingment, only TR3 tblastn results will be shown.

tblastn

>XR_group8 type=chromosome; loc=XR_group8:1..9190824; ID=XR_group8; 
release=r1.03; species=dpse							e-149	  54%

>gnl|Dvir-agencourt-run1029-asm|contig_2338					e-147	  53%

>gnl|Dmov-agencourt-run0811-asm|contig_5619					e-146	  53%

>gnl|Dana-agencourt-040714-asm|contig_394					e-146	  53%

>gnl|Dyak-washu-assembly_040407|Contig131.3					e-145	  53%

>gnl|Dana-agencourt-040714-asm|contig_2492					e-142	  53%

>XL_group3a type=chromosome; loc=XL_group3a:1..2686958; ID=XL_group3a; 
release=r1.03; species=dpse							e-140	  50%

>gnl|Dmov-agencourt-run0811-asm|contig_2707					e-139	  53%

>gnl|Dvir-agencourt-run1029-asm|contig_3163					e-138	  53%

>gnl|Dyak-washu-assembly_040407|Contig34.25					e-137	  52%

>gnl|Dere-agencourt-run1028-asm|contig_5904					e-101	  52%

>gnl|Dere-agencourt-run1028-asm|contig_278					1e-62	  52%

>gnl|Dsim-washu-w501-asm|Contig11.144						1e-29	  46%

>gnl|Dsim-washu-w501-asm|Contig2066.1						1e-19	  50%

>gi|33089107|gb|AY329357.1| Apis mellifera ligustica thioredoxin reductase 
(Trxr-1) mRNA,									e-153     54%

>gi|58380391|ref|XM_310514.2| Anopheles gambiae str. PEST ENSANGP00000017329	e-153     56%

>gi|1848293|gb|U88187.1|MDU88187 Musca domestica glutathione reductase 
family member mRNA,								e-150     53%

>gi|27819972|gb|BT003266.1| Drosophila melanogaster LD21729 full insert cDNA	e-147     53%

>gi|42764077|gb|AY431890.1| Aedes aegypti ASAP ID: 42805 thioredoxin 
reductase mRNA sequence								e-131     55%

>gi|50897530|gb|AY625511.1| Glossina morsitans morsitans clone Gmm2366 
putative thioredoxin								e-101     50%




>gnl|Dana-agencourt-040714-asm|contig_1097					3e-30     43%

>gnl|Dvir-agencourt-run1029-asm|contig_992					5e-30	  42%

>4_group4 type=chromosome; loc=4_group4:1..6604331;ID=4_group4;release=r1.03; 
 species=dpse									6e-30	  40%

>gi|24581818|ref|NM_135053.2| Drosophila melanogaster CG3887-PA (CG3887) 
mRNA, complete cds							 	2e-37	  43%

>gi|42765836|gb|AY440807.1| Armigeres subalbatus ASAP ID: 42399 putative:
 selenoprotein T mRNA.							        1e-36	  44% 

>gi|42761900|gb|AY433028.1| Aedes aegypti ASAP ID: 36405 putative:
 selenoprotein T mRNA.							        7e-34	  48%

>gi|58393304|ref|XM_319979.2| Anopheles gambiae str.PEST ENSANGP00000016703	8e-33     45% 

Gene prediction

gnl|Dyak-washu-assembly_040407|Contig131.3(contig is not correct, exonerate can not be performed)
          Length = 18324
Score =  518 bits (1333), Expect = e-145
Identities = 261/486 (53%), Positives = 333/486 (68%), Gaps = 2/486 (0%)
Frame = -1

Query: 38    DYDXXXXXXXXXXXACAKEAAQLGRKVAVVDYVEPSPQGTRWGLGGTCVNVGCIPKKLMH 97
             DYD           ACAKEAA  G +V   DYV+P+P GT+WG+GGTCVNVGCIPKKLMH
Sbjct: 14415 DYDLVVLGGGSAGLACAKEAAGCGARVLCFDYVKPTPVGTKWGIGGTCVNVGCIPKKLMH 14236

Query: 98    QAALLGGLIQDAPNYGWEVA-QPVPHDWRKMAEAVQNHVKSLNWGHRVQLQDRKVKYFNI 156
             QA+LLG  + +A  YGW V  Q +  DWRK+  +VQNH+KS+NW  RV L+D+KV+Y N
Sbjct: 14235 QASLLGEAVHEAVAYGWNVDDQNLRPDWRKLVRSVQNHIKSVNWVTRVDLRDKKVEYVNS 14056

Query: 157   KASFVDEHTVCGVAKGGKEIL-LSADHIIIATGGRPRYPTHIEGALEYGITSDDIFWLKE 215
               SF D HT+  VA  G E   ++++++++A GGRPRYP  I GA+E GITSDDIF  +
Sbjct: 14055 MCSFRDSHTIEYVAMPGAENRQVTSEYVVVAVGGRPRYPD-IPGAVELGITSDDIFSYER 13879

Query: 216   SPGKTLVVGASYVAWECAGFLTGIGLDTTIMMRTSPLRGFDQQMSSMVIEHMASHGTRFL 275
              PG+TLVVGA YV  ECA FL G+G + T+M+R+  LRGFD+QMS ++   M   G  FL
Sbjct: 13878 EPGRTLVVGAGYVGLECACFLKGLGYEPTVMVRSIVLRGFDRQMSELLAAMMTERGIPFL 13699

Query: 276   RGCAPSRVRRLPDGQLQVTWEDSTTGKEDTGTFDTVLWAIGRVPDTRSLNLEKAGVDTSP 335
                 P  V R  DG+L V + ++TT K+ +  FDTVLWAIGR      LNLE AGV T
Sbjct: 13698 GTTIPKAVERQADGRLLVRYHNTTTQKDGSDVFDTVLWAIGRKGLIEDLNLEAAGVKTHD 13519

Query: 336   DTQKILVDSREATSVPHIYAIGDVVEGRPELTPTAIMAGRLLVQRLFGGSSDLMDYDNVP 395
             D  KI+VD  EATSVPHI+A+GD++ GRPELTP AI++GRLL +RLF GS+ LMDY +V
Sbjct: 13518 D--KIVVDGAEATSVPHIFAVGDIIYGRPELTPVAILSGRLLARRLFAGSTQLMDYADVA 13345

Query: 396   TTVFTPLEYGCVGLSEEEAVARHGQEHVEVYHAHYKPLEFTVAGRDASQCYVKMVCLREP 455
             TTVFTPLEY CVG+SEE A+   G +++EV+H +YKP EF +  +    CY+K V
Sbjct: 13344 TTVFTPLEYSCVGMSEETAIELRGADNIEVFHGYYKPTEFFIPQKSVRHCYLKAVAEVSG 13165

Query: 456   PQLVLGLHFLGPNAGEVTQGFALGIKCGASYAQVMRTVGIHPTCSEEVVKLRISKRSGLD 515
              Q +LGLH++GP AGEV QGFA  +K G +   ++ TVGIHPT +EE  +L I+KRSG D
Sbjct: 13164 DQKILGLHYIGPVAGEVIQGFAAALKSGLTVKTLLNTVGIHPTTAEEFTRLSITKRSGRD 12985

Query: 516   PTVTGC 521
             PT   C
Sbjct: 12984 PTPASC 12967

The computational analysis as an attempt to decipher genomic contents emerges as a powerful tool, permiting new genes to be found. New programmes for gene prediction, similarity search systems and other tools are being developped and continously improved in a way that we can combine the ones that best fit to solve our specific problem. Our project was aimed at determining the presence or absence of selenoproteins in different insect genomes by using the various computational analysis resources that are nowadays available. After having performed an exhaustive analysis by the methods mentioned above, we report evidence of candidate selenoproteins existing in insect genomes.

The results show presence of human selenoprotein orthologues in some insect species (on one hand D.ananassae, D.erecta, D.virilis and D.yakuba for selenoproteinH on the other hand D.simulans and D.yakuba for seleprotein SPS2).In addition to the mentioned results, we found many "Cys" orthologues and also many other proteins that even not having "Sec" (stop codon) or "Cys" their conservation level is extremely high.

THE SPS2 FAMILY

The overall conservation level of the obtained candidates was very high. We found that the majority of our candidates had an "Arg" residue matching with the human "Sec"(Stop codon) but two of the candidates had a Stop codon (candidate "Sec") matching to the human one. The later studies emphasized our hypothesis after finding that the Stop was in-frame according to the exonerate analysis and in addition, the Secis structures were present in the RNA sequence. We concluded that the proteins with "Arg" are other proteins belonging to the wide SPS family, and the other two with Stop codon are selenoproteins. The neighboor joinning tree associates these two proteins more closely to the human SPS2 selenoprotein than to the others even if they come from phylogenetically closer organisms.

THE H SELENOPROTEIN

Some candidate selenoproteins were found among the hits. 6 organisms had a stop codon matching with the human "Sec" and SECIS STRUCTURES with significant score were found in all of them. The stop codons were not in-frame according to the exonerate analysis, but we think that this could be due to the phylogenetic distance between the query human protein and the submitted genomes. It is likely that using the human selenoprotein as the template for the exonic structure moddeling of the insect genomic sequences not the most accurate approximation towards the real model.Moreover, in the multiple alignment, the Stop codon flanking aminoacids remain conserved among the species making us think that they are part of the protein what means part of the exonic structure.

THE R SELENOPROTEINS

No selenoprotein candidates were found among the hits. All the organisms were Cys homologous to the human selenoprotein and the region flanking this position seems to be the most conserved. Even though, with exception to this region conservation among sequences is barely found. This strongly suggests the presence of a common domain among all the submitted sequences. This is the probable reason why exonerate could not have been performed. The sequences just share a motif rather than being orthologous proteins. Nevertheless, if we focus in lower level empairing each sequence with the other in all the possible combinations, clusters of different similarity arise. Thus, the possibility of having orthology among some of them can not be discarded. No SECIS structures were found.

THE T SELENOPROTEINS

Orthologues of the human T selenoprotein were found in a wide variety of insect genomes. The multiple alignment showed that there are very conserved regions, and that the most of the protein's positions are identycal for all the sequences submitted. Eventhough, all the candidates had a Cys in the position that a "Sec" is found in the human selenoprotein, making us think that the the "Sec" was adquired in a later step of the evolution. The fact that the exonic model coming from the exonerate analysis does not include the Cys matching to the human "Sec" is probably due to the query used but not a real exclusion of this position from the real exonic region. No Secis structures were found.

In this family 21 cys-homologous proteins in 15 different species were reported, all of them, according to the phylogenetic analysis, related to the human Gpx4.

Results coming from tblastn were quite significant, and some of them were further confirmed in the exonerate analysis were the human "Sec"matching Cys residues were placed in-frame. The multiple alignment showed a high conservation level, even in those sequences that could not have been submitted to exonerate.

As it was mentioned in the results section, although four of the genomic fragments had no reliable results after doing tblastn and exonerate, we reconstructed the proteins from two different frame sequences and submitted them to multiple aligment analysis. This revealed that our proteins are very well conserved among them and also towards the other sequences, what is more significant. This fact suggests that we are in front of four genes needing a frameshift to encode cys-homologous proteins. Thus, we speculate that these genes could have suffered an alternative 3' splice site. Even though, we could be facing a simple sequentiation mistake.

Our results also revealed another significant fact. Most of Drosophila species showed more than one cys-homologous protein:
D.ananassae : 3 cys-homologous

D.yakuba : 2 cys-homologous (one could not be confirmed due to a incorrect contig)

D.erecta: 2 cys-homologous

D.pseudoobscura: 2 cys-homologous

D.simulans: 2 cys-homologous

D.virilis: 1 cys-homologous

D.mojavensis: 1 cys-homologous

D.melanogaster: 1cys-homologous

Although all of these proteins are more closed to Gpx4 than to any other Gpx human protein, we can speculate that different cys-homologous proteins found for each species performe different functions knowing that at least, they are encoded by different genomic sequences.

Multiple sequence aligment followed by N-J unrooted tree suggested that the different cys-homologous proteins found in most of Drosophila species come from a duplication process. This is proved by the fact that in those species having more than one homologous protein, each of them is located in a different tree cluster. D.ananassae is supossed to have suffered two duplication process, while species showing just one cys-homologous protein are supossed not to have suffered any, and they are clustered together.

Independently of our results we have to bear in mind that not all the genomes that we have worked with are completely sequenced but D.melanogaster and D.pseudoobscura, so these results can not be considered as definitives.

A duplication event could drive to two different functional proteins as it has been mentioned above, but not necessarily. As it is known a duplication could yield a pseudogene, so unless performing more exhaustive and even experimental analysis, it will be impossible to confirm whether our cys-homologous candidates are functional or not.

Only two SECIS structures were found by SeciSearch and due to obtained scores, only the one corresponding to D.ananassae can be considered significant. Presence of SECIS in cys-homologous proteins represents an unusual case that is more deeply discussed at the end of this section..

THE 15KDA SELENOPROTEIN


We found 15Kda selenoprotein orthologues in almost every insect having a sequenced genome. The hits went up to 11 organisms in the similarity search, and it was obeserved that all of them had a Cys aminoacid at the position of the human "Sec". This fact made us think that non of them could be selenoproteins, but we found a significant Secis structure at the sequence belonging to D.virilisis. The score(18.5) is good enough to to think that a real Secis could be formed by this sequence, but the existence of a SECIS structure with no target codon to place a possible "Sec" would represent a not usual case. This could be explained from a model were an ancient selenoprotein with both SECIS and Sec substituted its Sec for a Cys but it has not passed time enough to make the SECIS dissapear.

THE TR FAMILY

In this family 20 cys-homologous proteins in 13 different species were reported, all of them, according to the phylogenetic analysis, related to the human TR3.
Results coming from tblastn were really significant, and most of them were further confirmed in the exonerate analysis were the human "Sec" matching Cys residues were placed in-frame. The multiple alignment showed a high conservation level, even in those sequences that could not have been submitted to exonerate.

As it has been observed with the GPX family, Drosophila species showed more than one cys-homologous candidate proteins. In this case all species but D.melanogaster showed 2 cys-homologous proteins.
Multiple sequence aligment followed by N-J unrooted tree suggested that the different cys-homologous proteins found in most of Drosophila species come from a duplication process. This is proved by the fact that in those species having more than one homologous protein, each ofthem is located in a different tree cluster.
As it was commented previuosly, we have to bear in mind that not all the genomes that we have worked with are completely sequenced but D.melanogaster and D.pseudoobscura, so these results can not be considered as definitives.
Although, as we have already commented, a duplication event could yield to a pseudogene expression, in this case, due to the high conservation among all sequences and the equal number of cys-homologous candidate proteins found, for most of the species, we can suggest, more consistently, that for Gpx family that we could be in front of two differents proteins. Anyway, unless performing more exhaustive and even experimental analysis, it will be impossible to confirm whether our cys-homologous candidates are functional or not.
Only one SECIS structure was found. Its score was not really significant.

The candidate selenoproteins found in this project represent probably a small sample of all the ones widespread in different organisms, so further genomic analysis need to be performed in order to establish their real importance. Appart from the obvious conclusions mentioned above, the unusual cases found make some evolutionary questions arise. The most likely is that proteins having Cys aminoacids suffered mutations that enabled their downstream 3'UTR region to form a SECIS structure.
Once having a SECIS structure a substitution of Cys by Sec would be affordable but not before, because that would lead to loose the translation of a larger or smaller part of the mRNA probably producing an unfunctional protein. Obviously, a change leading to an unfunctional protein would not be permitted by the natural selection.Nevertheless, formation of a SECIS element without any Sec to target seems difficult too. It is true that the UTR region is much less restrictive to mutations than the coding ones. This makes this region more given to changes, including those needed to form a SECIS element, but it is difficult to believe that this kind of neutral changes would be accumulated and conserved until that point were the Cys would change into Sec and the SECIS would turn useful. Thus,two opposite logics make the evolutionary question of selenoprotein emergence look like the tale of the hen and the egg represented by the STOP codon (Sec) and the SECIS element.

ARTICLES:

• Castellano S, Morozova N, Morey M, Berry M, Serras F, Corominas M, Guigó R. In silico identification of novel selenoproteins in the Drosophila melanogaster genome. EMBO reports 2, 697-702 (2001).
  
  
• Hatfield D, Gladyshev V. How selenium has altered our understanding of the genetic code. Molecular and Cellular Biology 22. 3565-3576 (2002)
  
  
• S. Castellano, S.V. Novoselov, G.V. Kryukov, A. Lescure, E. Blanco, A. Krol. V.N. Gladyshev and R. Guigó. Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution. EMBO reports, 5(1):71-77 (2004)
  
  
• G.V. Kryukov, S. Castellano, S.V. Novoselov, A.V. Lobanov, O. Zehtab, R. Guigó and V.N. Gladyshev Characterization of mammalian selenoproteomes. Science, 300(5624):1439-1443 (2003)
  
  

We thank greatfully all our lecturers for making the world of Bioinformatics more accessible to us, specially Charles Chapple for supervising our job and having the patience of bearing us. We greatly thank the opportunity of discovering this world, a world that we will be pleased not to visit again to the end of our days. Thank you for making us work so hard, to the point that our girlfriends and boyfriends broke up for feeling abandoned. Thank you for making us smoke again, a habit that we managed to give up but we went back to, during this project. We greatly thank the greasy sandwiches from "la cafeteria del hospìtal" for feeding us with high vitaminic content food, essential for the highly demanding brain work that we had to perform. Finally, we thank our families for their unconditional support.