Project Bio Information Technology

8/3/2019 Project Bio Information Technology

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Project Bioinformation Technology

Martijn Heddes (870313134060)

Nguyen Thi Kha Tu (860319828030)


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Introduction

In this experiment a set of bioinformatics applications were used to find out as much as possible about

a raw set of sequences obtained. The five main goals involved are gene annotation, protein

identification, determination of protein topology and localization, determination of 3D-structure model

and getting some extra information from literature. The set of sequences obtained was coded andcoded 17 different sequences.

METHOD SECTION

The 17 obtained sequences were attended to be different sequences obtained from a shotgun

sequencing reaction. Therefore, the sequences were screened on vector contamination sequences using

VecScreen[1] before constructing the contig using CAP3[2]. The contig obtained was used for a

nucleotide blast search against the Nucleotide collection (nr/nt) database in order to find similar or

related sequences with a known function. The highly similar sequences obtained were aligned to the

contig sequence and checked for differences. Some improvements were made in de contig sequence.

which was compared then to the protein sequence of the protein obtained from the blast search.Furthermore, additional literature about the specific protein was obtained searching in the medline

database using pubmed and google scholar. This in order to get some additional information about the

function of the protein, the involved working mechanism, the conserved domains and or motifs of the

protein, the evolution of the protein superfamily and the conformations of previous findings.

Signal Topology and subcellular localization

To indicate the topology and sub cellular localization of the predicted protein PSORT (version 2.0.4)

[8] and SOSUI [9] were used to search for specific motifs, which could reveal some information about

the subcellular localization of the protein. In addition, signal P was used in order to predict a terminalcleavage site.

3D protein modeling

To get a proper view of the three-dimensional structure of the predicted protein, several modeling

applications were used. Therefore, the obtained protein sequence was blasted against de PDB database

and also the predicted 3D structure together with the PDB code of the protein and other homologous

proteins were obtained from Modbase[11]. These PDB codes were used for searching the VAST[10]

database. Furthermore, the obtained predicted models were analyzed using respectively DeepView[13]

and Cn3D. Finally a check for the reliability has been done by using ProSa [12] and by making a

Ramachandran plot in DeepView.

RESULTS AND DISCUSSION

Gene annonation


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No vector contamination was detected by VecScreen in any of the 17 sequences provided. Also, no

strange sequences were detected in the file and thus all the sequences were used for the contig

alignment. Cap3 gave as outpute one contig and used 15 of the 17 sequences . Sequences 10 and 12

were determined as contamination.

>Contig1

GCGGCCGAATGTGGGGGATGTCCCATTCATTACCATCCGGAATGGACACAGATTTTTCATCATATC

CATCAACGATAGGTCGGCAACACTCACAACAATCTCAAAGATATTACCAAAATAATAATTGTGGTT

TAGGTTCAGTGGGAAATATGGCAAACAGTACAAATTCCTTAAATTCAGGTACCAACAATAGTGGAA

CAAATTTGATTGTAAATTATTTGCCCCAAGATATGCAAGACCGTGAACTTTATTCATTATTTAGGAC

CATTGGTCCAATCAATACCTGCAGAATAATGCGAGATTATAAGACTGGTTACAGTTATGGATATGG

TTTTGTGGACTTTGGATCGGAAGCAGATGCATTGAGAGCCATTAATAATCTTAATGGAATCACAGT

CAGAAATAAGAGGATAAAGGTTTCATTCGCTAGGCCGGGTGGAGAACAACTGAGAGATAAATTCA

AACTTGTATGTAACAAATTTATCTAGATCAATTACTGACGAACAATTAGAAACAATTTTTGGAAAA

TATGGGCAAATTGTACAGAAAAACATTTTACGCGACAAACATACAGGAACACCGCGTGGAGTGGC

CTTTATCAGATTTAATAAGAGAGAAGAAGCTCAAGAAGCAATATCAGCTTTGAACAACGTAATACC

TGAAGGGGGAACACAACCGCTAACTGTTCGTGTTGCCGAGGAACACGGAAAGTCAAAGGGCCATG

TTTATATGGCTCCAAATCAACCACCTCACGGAAACATGGGTCATGGAAATATGGGGAATATGGGAC

ATGGAAACATGGGAATGGCCGGTGGTTCCGGAATGAATTTAAATAATATGAATGCATTCAATGGG

ATGAATCAAATGGTGCACAGAGGTAGACAAAAACATAGTTACCAACGTAAAATTCATCCATATAA

TCCAAATTTTCTTTAAACATTTAATTAAACAATATAAACAGAAACTTAGTTTTGCTTTGCTGGACAA

ATTTTAGAAAACCAATAATTAATAACACAAGTCCAAAACTAATTTTTTTTTATCGTTTTTCATATAA

AAATTCGTTGTTTATCCGATATTTTAAACTTTAAAAACGAGACACTGTAATTTAATAAGTAGTTATA

CTTAATCAATTTACTAAATTTTCAGTAGCAGTAGATGTATGTGTTAAATATCCACACTTAAAAAAGA

TGAAAACTTTTAAAACAAACAAAACACATGTAATAGTTATTCTGTATTAATATTTAAGGTATTCGAT

ATTATTCATCTTATATTTGAAAATGTCGCGAATTAATTTTAAAACAATATTAAAATTTCAATATTTA

ATGTACGACCTATTTGGGTTTGCATCAATAAACTAGATTTATGTTGTTCTCCGAAAAAAGAAAAAA

AAAAAAAAAAAAAAAAAA

Also, getting the most possible protein from ORF Finder on NCBI. The most possible open reading frame is

frame +3, producing a protein with length 307aa, starting with M (Methionine), this full possible

protein sequence is as follow:

>translation

MWGMSHSLPSGMDTDFSSYPSTIGRQHSQQSQRYYQNNNCGLGSVGNMANSTNSLNSGTNN

SGTNLIVNYLPQDMQDRELYSLFRTIGPINTCRIMRDYKTGYSYGYGFVDFGSEADALRAINN

LNGITVRNKRIKVSFARPGGEQLRDTNLYVTNLSRSITDEQLETIFGKYGQIVQKNILRDKHTG

TPRGVAFIRFNKREEAQEAISALNNVIPEGGTQPLTVRVAEEHGKSKGHVYMAPNQPPHGNM

GHGNMGNMGHGNMGMAGGSGMNLNNMNAFNGMNQMVHRGRQKHSYQRKIHPYNPNFL


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This sequence was blasted against the nr database using megablast and gave the following output.


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As shown in the Blast output our contig got a 100% query coverage with the Megaselia

scalaris mRNA for Sex-lethal orthologous protein (Megsxl), splice variant, clone pMSWc114

with a bit score of 2571. In Adition the query gave also a high similarity with Megaselia

scalaris mRNA for sex-lethal homologue (sxl gene) with a coverage of 96% and a bit score of

2479. After that a blast P search was performed for both translated sequences against the nr

database and resulted both in a 100% identity with a Sex-lethal homologous protein of the

Megaselia scalaris. Then, the taxonomy report of the Blastp output was analyzed all the

homologous and paralogous sequences were obtained in the protein database of NCBI. A

multiple sequence alignment of these sequences was been made using clustal x and of that

alignment a phylogenetic tree was build using the same program.


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Fig. the BlastP output

Fig. the BlastP output of purified predicted protein against nr database.


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Fig. the alignment output BlastP against nr database.

As the Blast output, we can confirm that our protein is Sex-lethal orthologous protein

Fig. Taxonomy output of BlastP

Protein identification

As the SuperFamily link of the BLASTP output, our protein is located in RNA/DNA binding site,

into a conserved location of RRM superfamily. RRM (RNA recognition motif), also known as RBD(RNA binding domain) or RNP (ribonucleoprotein domain), is a highly abundant domain in


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eukaryotes found in proteins involved in post-transcriptional gene expression processes including

mRNA and rRNA processing, RNA export, and RNA stability. This domain is 90 amino acids in

length and consists of a four-stranded beta-sheet packed against two alpha-helices. RRM usually

interacts with ssRNA, but is also known to interact with ssDNA as well as proteins. RRM binds a

variable number of nucleotides, ranging from two to eight. The active site includes three aromatic

side-chains located within the conserved RNP1 and RNP2 motifs of the domain. The RRM domain isfound in a variety heterogeneous nuclear ribonucleoproteins (hnRNPs), proteins implicated in

regulation of alternative splicing, and protein components of small nuclear ribonucleoproteins

(snRNPs).


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And from output of Structure Search in NCBI, we can predict very highly that our protein have a

homodimer, with 2 domains.

As the below result of ClustalX to find the similarity of our protein with its orthologs, a multiple sequence

alignment of the proteins from different nineteen eukaryotes which have more than 95% identity with our protein

has been done (Appendix 1 and 2). Expectedly, from the phylogenetic tree shows clear relationships with our

protein, and the protein of Megaselia_scalaris is nearest with our protein as the Blast output. Hence, the

phylogenetic tree built was very highly reliable.


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Fig 3. Phylogeny relationship of our protein with other homolog proteins from other organisms

The active sites (large conserved region) consist of two RNA-binding domains (Fig. 2, RBD1, RBD2).

(Bopp et al. 1996)


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Fig: the location ofRBD1, RBD2


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Two main active sites of our protein is totally (100% identity) conserved comparing with the active sites RBD1,

RBD2 of the RNA binding domains of Sex-lethal protein (Bopp et al. 1996), so we can say that our protein is a

sex-lethal protein containing two RNA binding domains and can bind RNA well.

Protein Signal Topology & subcellular localization (Martijn do this part!)

According to SignalP, no signal peptide was detected from our protein sequence, indicating that it was

not a secretory protein. This result was in line with the one from SOSUI that our protein is a soluble

protein.


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In addition, by using PSORT Prediction the protein was predicted to be localized in the cytoplasm

with a high certainty while there is no clear prediction whether it could localize in outer or inner

membranes. This is quite logical as prokaryotes themselves, containing a single plasma membrane and

cell wall, have no organelles within their cells and lack a periplasmic space.

3D protein modeling

We got a predicted model with two domains, a fair sequence identity (49%), a very significant E-

Value (6e-28) and a Model Score 1.00, which is very good. Therefore, we have a high prediction that

our 3D protein model is very similar to this model

As the output of the Ramachandran Plot of PDB Viewer programme, almost all

residues of our protein were in the allowed region. Although there are four residues

outside the allowed regions which are acceptable because proteins are not perfect, theyare flexible.


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Overall structure quality was showed by Z-score (-6.7) which was quite low, indicating high

stableness and less probability for erroneous structures. Residue scores of our protein and template

were also negative which showed usual energy level

Apendix 1:

>Megaselia_scalaris_gi|6456838|emb|CAA04179.2| Sex-lethal orthologous protein [Megaselia scalaris]

MWGMSHSLPSGMDTDFSSYPSTIGRQHSQQSQRYYQNNNCGLGSVGNMANSTNSLNSGTNNSGTNLIVNY

LPQDMQDRELYSLFRTIGPINTCRIMRDYKTGYSYGYGFVDFGSEADALRAINNLNGITVRNKRIKVSFA

RPGGEQLRDTNLYVTNLSRSITDEQLETIFGKYGQIVQKNILRDKHTGTPRGVAFIRFNKREEAQEAISA

LNNVIPEGGTQPLTVRVAEEHGKSKGHVYMAPNQPPHGNMGHGNMGNMGHGNMGMAGGSGMNLNNMNAFN

GMNQMVHRGRQKHSYQRKIHPYNPNFL

>Ceratitis_capitata_gi|2981305|gb|AAC38968.1| sex-lethal homolog CcSXL [Ceratitis capitata]

MYGNMNNGGHAPYGYNGYRPSGGRMWGMSHSLPSGMDTDFTSSYPGPSMNRRGGYNDFSGGGGGGGGTMG

SMNNMVNAASTNSLNCGGGGGRDGHGGGSNGTNLIVNYLPQDMTDRELYALFRTIGPINTCRIMRDYKTG

YSFGYAFVDFAAETDSQRAIKSLNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDQLDTIFGK

YGMIVQKNILRDKLTGKPRGVAFVRFNKREEAQEAISALNNVIPEGASQPLTVRLAEEHGKAKAQHYMSQ

LGLIGGGGGGGGGGGGGGGGMGGPPPPPMNMGYNNMVHRGRQNKSRFQKMHPYHNAQKFI

>Lucilia_cuprina_gi|13357168|gb|AAK20025.1|AF234183_1 sex-lethal protein SXL1 [Lucilia cuprina]

MYGQNIRNVTYAPYGYNGYRQSGERMWRMSHSLPSGMDTDFTSSYPGPSAMNHRGGRGGGYNDFSGGGSA

MGSMCNMAPAISTNSVNSGGGDCGDTQGCNGTNLIVNYLPQDMTDRELYALFRTCGPINTCRIMKDYKTG

YSFGYAFVDFASEIDAQNAIKSLNGVTVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDELEKIFGK

YGNIVQKNILRDKLTGKPRGVAFVRFNKREEAQEAISALNNVIPEGASQPLTVRLAEEHGKMKANHFMNQ

LGMGPPAAPIPAAGPGYNNMVHRGRHNKNRNQKSHPYHNPQKFI


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>Musca_domestica_gi|6226777|sp|O17310.1|SXL_MUSDO RecName: Full=Sex-lethal homolog

MYGQNVRNVSYYPPYGYNGYKQSGERMWRMSHSLPSGMDTDFTSSYPGPSAMNPRGRGGYNDFSGGGSAM

GSMCNMAPAQSTNSLNSGGDGGGGDTQAVNGTNLIVNYLPQDMTDRELYALFRTCGPINTCRIMKDYKTG

YSFGYAFVDFASEIDAQNAIKTVNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDELEKIFGKYGNIVQKNILRDKLTGRPRGVAFVRFNKREEAQEAISALNNVIPEGASQPLTVRLAEEHGKMKAHHFMNQ

LGMGPPAAPIPAAGPGYNNMVHRGRQNKMRNHKVHPYHNPQKFI

>Chrysomya_rufifacies_gi|6226775|sp|O97018.2|SXL_CHRRU RecName: Full=Sex-lethal homolog

MWRMSHSLPSGMSRYAFSPQDTDFTSSYPGPSAMNHRGGRGGGYNDFSGGGSAMGSMCNMAPAISTNSVN

SGGGDCGDNQGCNGTNLIVNYLPQDMTDRELYALFRTCGPINTCRIMKDYKTGYSFGYAFVDFASEIDAQ

NAIKSLNGVTVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDELEKIFGKYGNIVQKNILRDKLTGK

PRGVAFVRFNKREEAQEAISALNNVIPEGASQPLTVRLAEEHGKMKAHHFMNQLGMGPPAAPIPAAGPGY

NSMVHRGRHNKNRNQKPHPYHNPQKFI

>Bactrocera_oleae_gi|52075416|emb|CAG29242.1| sex-lethal protein [Bactrocera oleae]

MYGNMNNGGHVPYGFNGYRPSGGRRWGMSHSLPSGMDTDFTSSYPGPSMNRRGYNDFSGGGGGGSGGGGG

AMGSMNNAPAISTNSLNCGSGGGGGDGHGGGSNGTNLIVNYLPQDMTDRELYALFRTIGPINTCRIMRDY

KTGYSFGYAFVDFASETDSQRAIKSLNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDQLDTI

FGKYGMIVQKNILRDKLTGKPRGVAFVRFNKREEAQEAISALNNVIPEGGSQPLTVRLAEEHGKAKAQQY

MSQLGLIGGGGGGGGGGGGMGGPPPPPMNMGYNNMVHRGRQNKSRFQKMHPYHNAQKFI

>Drosophila_grimshawi_gi|195048229|ref|XP_001992493.1| GH24172 [Drosophila grimshawi]

MYGNNNPGSNNNNGGYPPYGYNNKSSGGRVFGMSHSLPSGMSRYAFSPQDTEFTFPSSSSRRGYNEFAGV

NGGSANSLGGLCNMPMASNNSLNNLCGLSIGSGGSDDLMNDPRNSNTNLIVNYLPQDMTDRELYALFRSI

GTINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNL

PRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVRL

AEEHGKAKAAHFMTQMGMGPPQVPPPPPPPPPHMASFNMVHRDGAMEKLRSLFDAICDAIFGLDSDNFAD

LLDGLYRRKYHYPYL

>Drosophila_willistoni_gi|195439114|ref|XP_002067476.1| GK16445 [Drosophila willistoni]

MYGNNNPGSNNNNGGYPPYGYKQSSGGRGFGMSHSLPSGMSRYAFSPQDTEFTFPSSSSRRGYNDFPGGG

GNGGSANSLGGGNMCNMPPMASNNSLNNLCGLSLGSGGSDDLMNDHRPSNTNLIVNYLPQDMTDRELYAL

FRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLY

VTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPL

SVRLAEEHGKAKAAHFMSQMGMGPSPPNVPPPPPPPPPHMAAGFNNMVHRDGAMEKLRSLFDAICDAIFG

LDSDNFADLLDGLYRRKYHYPYL

>Drosophila_virilis_gi|195396313|ref|XP_002056776.1| Sex lethal [Drosophila virilis]

MYGNNNPGSNNNNGGYPPYGYNKSSGGRVFGMSHSLPSGMSRYAFSPQDTEFTFPSSSSRRGYNDFPGGN

GGSANSLGGNICNMPPMASNNSLNNLCGLSIGSGGSDDHMNDQRNSNTNLIVNYLPQDMTDRELYALFRA

IGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTN

LPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVR

LAEEHGKAKAAHFMSQMGMGPPQAPPPPPPPPPHMAAGFNNMVHRDGAMEKLRSLFDAICDAIFGLDSDN


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FADLLDGLYRRKYHYPYL

>Drosophila_mojavensis_gi|195132500|ref|XP_002010681.1| GI21573 [Drosophila mojavensis]

MYGNNNPGSNNNNGGYPPYGYNKSSGGRVFGMSHSLPSGMSRYAFSPQDTEFTFPSSSSRRGYNDFPGGN

GGSANSLGGNICNMPMASNNSLNNLCGLSIGSGGSDDLMNDQRTSNTNLIVNYLPQDMTDRELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNL


AEEHGKAKAAHFMSQMGMGPPQAPPPPPPPPPHMAAGFNNMVHRDGAMEKLRSLFDAICDAIFGLDSDNF

ADLLDGLYRRKYHYPYL

>Drosophila_melanogaster_gi|78706524|ref|NP_001027063.1| Sex lethal, isoform N [Drosophila

melanogaster]

MDFNFDTVTPCSTMSSYYNFKMASGGRGFGMSHSLPSGMDTEFSFPSSSSRRGYNDFPGCGGSGGNGGSA

NNLGGGNMCHLPPMASNNSLNNLCGLSLGSGGSDDLMNDPRASNTNLIVNYLPQDMTDRELYALFRAIGP

INTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPR

TITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVRLAE

EHGKAKAAHFMSQMGVVPANVPPPPPQPPAHMAAAFNMMHRGRSIKSQQRFQNSHPYFDAKKFI

>Drosophila_subobscura_gi|1403308|emb|CAA67016.1| sex-lethal [Drosophila subobscura]

MYGNNNPGSNNNNGGYPPYGYNKSSGGRGFGMSHSLPSGMDTEFSFPSSSSRRGYNEFPGGGGIGIGANG

GSANNLGGNMCNLLPMTSNNSLSNLCGLSLGSGGSDDHMMMHDQRSSNTNLIVNYLPQDMTDRELYALFR

AIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVT

NLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSV

RLAQEHGKAKAAHFMSQIGVPSANAPPPPPPPPHMAFNNMVHRGRSIKSQQRFQKTHPYFDAQKFI

>Drosophila_sechellia_gi|195353431|ref|XP_002043208.1| Sxl [Drosophila sechellia]

MYGNNNPGSNNNNGGYPPYGYNNKSSGGRGFGMSHSLPSGMDTEFSFPSSSSRRGYNDFPGCGGSGGNGG

SANNLGGGNMCHLPPMASNNSLNNLCGLSLGSGGSDDLMNDPRASNTNLIVNYLPQDMTDRELYALFRAI

GPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNL


AEEHGQGEGCPLYVADGRGSS

>Drosophila_simulans_gi|195565544|ref|XP_002106359.1| Sex lethal [Drosophila simulans]

MYGNNNPGSNNNNGGYPPYGYNNKSSGGRGFGMSHSLPSGMDTEFSFPSSSSRRGYNDFPGCGGSGGNGG

SANNLGGGNMCHLPPMASNNSLNNLCGLSLGSGGSDDLMNDPRASNTNLIVNYLPQDMTDRELYALFRAI

GPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNL

PRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRFSESNILFYGAMEKLRSLLDGIWDAIFGLD

SENFADLLDGLYRRKYHYPYL

>Drosophila_erecta_gi|194896665|ref|XP_001978518.1| GG17637 [Drosophila erecta]

MSHSLPSGMDTEFSFPSSSSRRGYNDFPGCGGSGGNGGSANNLGGGNMCHLPPMASNNSLNNLCGLSLGS

GGSDDLMNDPRASNTNLIVNYLPQDMTDRELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQ

RAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGR

PRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVRLAEEHGKAKAAHFMSQMGVAAPNVPPPPPPPPP


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HMAAGFNNMVHRDGAMEKLRSLFDAICDAIFGLDSDNFADLLDGLYRRKYHYPYL

>Drosophila_yakuba_gi|195480535|ref|XP_002101294.1| GE17544 [Drosophila yakuba]

MSHSLPSGMDTEFSFPSSSSRRGYNDFPGCGGSGGNGGSANNLGGGNMCHLPPMASNNSLNNLCGLSLGS

GGSDDLMNDPRASNTNLIVNYLPQDMTDRELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESIKDTNLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGR

PRGVAFVRYNKREEAQEAISALNNVIPEGGSQPLSVRLAEEHGKAKAAHFMSQMGVAAPNVPPPPPPPPP

HMAAGFNNMVHRDGAMEKLRSLFDAICDAIFGLDSDNFADLLDGLYRRKYHYPYL

>Drosophila_ananassae_gi|194763735|ref|XP_001963988.1| GF20968 [Drosophila ananassae]

MYGNNNPGSNNNNGGYPPYGYNNKSSGGRGFGMSHSLPSGMSRYAFSPQDTEFSFPSSSSRRGYNDFPGC

GIGGNGGSANSLGGGGGGNMCNLPPMTSNNSLNNLCGLSLGSGGSDDHMLNDQRPSNTNLIVNYLPQDMT

DRELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGES

IKDTNLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIP

EGGSQPLSVRLAEEHGKAKAAHFMSQMGVPAPNAPQPPPPPPLPHMAAGFNSMVHRDGAMEKLRSLFDAI

CDAIFGLDSDNFADLLDGLYRRKYHYPYL

>Drosophila_persimilis_gi|195168643|ref|XP_002025140.1| GL26885 [Drosophila persimilis]

MYGNNNPGSNNNNGGYPPYGYNKSSGGRGFGMSHSLPSGMSRYAFSPQDTEFSFPSSSSRRGYNEFPGGG

GIGIGANGGSANNLGGNMCNLLPMTSNNSLSNLCGLSLGSGGSDDHMMMHDQRSSNTNLIVNYLPQDMTD

RELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESI

KDTNLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPE

GGSQPLSVRLAEEHGKAKAAHFMSQMGVPAPNAPPPPPPPPPHMAAGFNNMVHRDGAMEKLRSLFDAICD

AIFGLDSDNFADLLDGLYRRKYHYPYL

>Drosophila_pseudoobscura_gi|198471287|ref|XP_002133706.1| GA22653 [Drosophila pseudoobscura

pseudoobscura]

MYGNNNPGSNNNNGGYPPYGYNKSSGGRGFGMSHSLPSGMSRYAFSPQDTEFSFPSSSSRRGYNEFPGGG

GIGIGANGGSANNLGGNMCNLLPMTSNNSLSNLCGLSLGSGGSDDHMMMHDQRSSNTNLIVNYLPQDMTD

RELYALFRAIGPINTCRIMRDYKTGYSFGYAFVDFTSEMDSQRAIKVLNGITVRNKRLKVSYARPGGESI

KDTNLYVTNLPRTITDDQLDTIFGKYGSIVQKNILRDKLTGRPRGVAFVRYNKREEAQEAISALNNVIPE

GGSQPLSVRLAEEHGKAKAAHFMSQMGVPAPNAPPPPPPPPPHMAAGFNNMVHRDGAMEKLRSLFDAICD

AIFGLDSDNFADLLDGLYRRKYHYPYL

Appendix 2:

Bopp, D., Calhoun, G., Horabin, J.I., Samuels, M., and Schedl, P. 1996. Sex-specific control of Sex-lethal is a

conserved mechanism for sex determination in the genus Drosophila. Development, 122: 971982.

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