Bioinformatics - protocols  E

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Construction and interpretation of a protein sequence alignment

>p140mDia
YKPEVQLRRPNWSKFVAEDLSQDCFWTKVKEDRFENNELFAKLTLAFSAQTKTSKAKKDQEGGEEKKSVQ
KKKVKELKVLDSKTAQNLSIFLGSFRMPYQEIKNVILEVNEAVLTESMIQNLIKQMPEPEQLKMLSELKE
EYDDLAESEQFGVVMGTVPRLRPRLNAILFKLQFSEQVENIKPEIVSVTAACEELRKSENFSSLLELTLL
VGNYMNAGSRNAGAFGFNISFLCKLRDTKSADQKMTLLHFLAELCENDHPEVLKFPDELAHVEKASRVSA
ENLQKSLDQMKKQIADVERDVQNFPAATDEKDKFVEKMTSFVKDAQEQYNKLRMMHSNMETLYKELGDYF
VFDPKKLSVEEFFMDLHNFRNMFLQAVKENQKRRETEEKMRRAKLAKEKAEKERLEKQQKREQLIDMNAE
GDETGVMDSLLEALQSGAAFRRKR
>Diaphanous Drosophila
WDVKNPMKRANWKAIVPAKMSDKAFWVKCQEDKLAQDDFLAELAVKFSSKPVKKEQKDAVDKPTTLTKKN
VDLRVLDSKTAQNLAIMLGGSLKHLSYEQIKICLLRCDTDILSSNILQQLIQYLPPPEQLKRLQEIKAKG
EPLPPIEQFAATIGEIKRLSPRLHNLNFKLTYADMVQDIKPDIVAGTAACEEIRNSKKFSKILELILLLG
NYMNSGSKNEAAFGFEISYLTKLSNTKDADNKQTLLHYLADLVEKKFPDALNFYDDLSHVNKASRVNMDA
IQKAMRQMNSAVKNLETDLQNNKVPQCDDDKFSEVMGKFAEECRQQVDVLGKMQLQMEKLYKDLSEYYAF
DPSKYTMEEFFADIKTFKDAFQAAHNDNVRVREELEKKRRLQEAREQSAREQQERQQRKKAVVDMDAPQT
QEGVMDSLLEALQTGSAFGQRNRQARRQRPAGAERRAQLSRSRSRTRVTNGQLMTREMILNEVLGSA
>Fugu formin
IKTKFRLPVFNWTALKPNQINGTVFNEIDDERELELERFEELFKTRAQGPIMDLSCTKSKVAQKAVNKVT
ILDANRSKNLAITLRKANKTFDLKTLPVDFVECLMRFLPTEMEVKALRQYERERRPLDQLAEEDRFMLLF
SKIERLTQRMNIITFIGNFSDNVAMLTPQLNAIIAASASVKSSPKLKRMLEIILALGNYMNSSKRGCVYG
FKLQSLDLLLDTKSTDRKMTLLHYIALIVKEKYPELANFYNELHFVDKAAAVSLENVLLDVRELGKGMDL
IRRECSLHDHSVLKGFLQASDTQLDKVQRDAKTAEEAFNNVVNYFGESAKTAPPSVFFPVFVRFLKAYKD
AVEENELRKKQEQAMREKLLAEEAKQQDPKVQAQKKRQQQHELIAELRKRQAKDHRPVYEGKDGTIEDII
TVLK
>Cappuccino Drosophila
PPTAPPATKEIWTEIEETPLDNIDEFTELFSRQAIAPVSKPKELKVKRAKSIKVLDPERSRNVGIIWRSL
HVPSSEIEHAIYHIDTSVVSLEALQHMSNIQATEDELQRIKEAAGGDIPLDHPEQFLLDISLISMASERI
SCIVFQAEFEESVTLLFRKLETVSQLSQQLIESEDLKLVFSIILTLGNYMNGGNRQRGQADGFNLDILGK
LKDVKSKESHTTLLHFIVRTYIAQRRKEGVHPLEIRLPIPEPADVERAAQMDFEEVQQQIFDLNKKFLGC
KRTTAKVLAASRPEIMEPFKSKMEEFVEGADKSMAKLHQSLDECRDLFLETMRFYHFSPKACTLTLAQCT
PDQFFEYWTNFTNDFKDIWKKEITSLLNELMKKSKQAQIESRRNVSTKVEKSGRISLKERMLMRRSKN
>Bni1 yeast
PRPHKKLKQLHWEKLDCTDNSIWGTGKAEKFADDLYEKGVLADLEKAFA
AREIKSLASKRKEDLQKITFLSRDISQQFGINLHMYSSLSVADLVKKILN
CDRDFLQTPSVVEFLSKSEIIEVSVNLARNYAPYSTDWEGVRNLEDAKPP
EKDPNDLQRADQIYLQLMVNLESYWGSRMRALTVVTSYEREYNELLAKLR
KVDKAVSALQESDNLRNVFNVILAVGNFMNDTSKQAQGFKLSTLQRLTFI
KDTTNSMTFLNYVEKIVRLNYPSFNDFLSELEPVLDVVKVSIEQLVNDCK
DFSQSIVNVERSVEIGNLSDSSKFHPLDKVLIKTLPVLPEARKKGDLLED
EVKLTIMEFESLMHTYGEDSGDKFAKISFFKKFADFINEYKKAQAQNLAA
EEEERLYIKHKKIVEEQQKRAQEKEKQKENSNSPSSEGNEEDEAEDRRAV
MDKLLEQLKNA
 

• Examine the results, keeping the results window open (If you were unable to perform the search for server-dependent reasons or accidentally closed the window, go to the SOS page.).
• Open a new window, go to the same server as before, and repeat the alignment with varying the multiple alignment gap opening penalty: try e.g. values of 0.1 and 10 and observe the effect on alignment results.

Sequence similarity searches and domain structure analysis

• Return to the ClustalW results window and find the best conserved motif in your sequence.Try to create a Prosite-style pattern corresponding to this motif.

The syntax of a Prosite pattern is as follows: A-P-X-X-[MLI]-H means Ala; Pro; 2 x any amino acid; any of Met, Leu, Ile; His (more on Prosite patterns can be found here).
• Use the pattern to search either the predicted Arabidopsis protein sequences using the TAIR Pattern Matching or  the non-redundant database using the ISREC PatternFind program.Compare the results of both searches, looking specifically for Arabidopsis sequences in the PatternFind output. (If any server is down, go to the SOS page).
• Optional: Return to your pattern and try to develop it, trying to specify the varying position or loosening the stringency by allowing related amino acids. Look at the effect on the TAIR Pattern Matching search.

Basic sequence manipulation

   1 TTTAATAAAA TAAAAATCCA CTCGCATTTT TATTTTCAAC ATTGTGCGTA    50
  51 CGGTGCAATT CAATGAACAG TGTTTACTTT CAGTGTGTAC ACTTCTGCGG   100
 101 ACTATTACAA AGTCCACGTC TTATCCTACG TGTTATAATC TCATATGTTA   150
 151 CTGTCTGAAA TGGACCCCAC TACGTAAAAA TAAAATTAAG AATCAACCAC   200
 201 TCTTCTTCCA TCACCTCTTT TGGCTTTCTC TCTACTCTCT CTACTACTCT   250
 251 CTCACCATCA CTGAGTTAAG AGAACAAACC AAAAACAAAA TTATCAAACC   300
 301 ATCACCAGCA GAATCTTAGC TGGATTCATC ACTCTATTCA AAAAGTTTCT   350
 351 CTCTTCTCTT TTCTCAGATC TTGAACTCTT GAAGAAGAAA GAAGAAGATA   400
 401 ACACAATGCT CTTCTTCTTA TTCTTCTTCT ACTTACTCTT ATCTTCATCC   450
 451 TCCGATCTAG TCTTCGCCGA CCGTCGTGTA CTCCACGAAC CATTCTTCCC   500
 501 TATAGATTCA CCACCACCGT CACCACCATC ACCACCACCA CTTCCTAAAC   550
 551 TACCATTCTC TTCAACCACT CCTCCATCTT CATCAGACCC AAATGCTTCT   600
 601 CCTTTCTTCC CTTTATACCC TTCATCTCCA CCACCACCTT CTCCAGCCTC   650
 651 CTTCGCTTCT TTTCCGGCGA ATATCTCATC TCTAATCGTC CCTCACGCCA   700
 701 CTAAATCCCC ACCTAACTCC AAAAAACTCC TTATCGTCGC TATCTCCGCC   750
 751 GTTTCCTCCG CTGCTTTAGT CGCTCTACTT ATCGCTTTAC TCTATTGGCG   800
 801 AAGAAGCAAA CGTAACCAAG ATCTTAACTT CTCCGATGAT AGCAAAACAT   850
 851 ACACCACCGA CAGTAGCCGC CGTGTCTACC CTCCTCCTCC GGCAACGGCG   900
 901 CCTCCAACAC GACGCAATGC GGAGGCTAGA AGTAAACAGA GGACCACCAC   950
 951 GAGCTCCACC AATAACAACA GCTCTGAGTT TCTTTACTTA GGAACAATGG  1000
1001 TGAATCAAAG AGGAATCGAT GAACAATCTC TTAGTAATAA TGGATCAAGC  1050
1051 TCAAGAAAAC TTGAATCTCC AGATCTTCAA CCACTTCCTC CATTGATGAA  1100
1101 ACGAAGTTTC CGTTTAAATC CAGATGTTGG TTCAATCGGA GAAGAAGATG  1150
1151 AAGAAGATGA GTTTTACTCT CCACGTGGCT CACAAAGCGG GCGAGAACCG  1200
1201 TTAAACCGGG TCGGACTTCC GGGTCAAAAT CCTAGATCTG TTAACAATGA  1250
1251 CACTATCTCT TGCTCATCTT CAAGCTCTGG TTCACCAGGA AGATCAACAT  1300
1301 TTATCAGTAT CTCTCCTTCA ATGAGTCCTA AGAGATCTGA ACCAAAACCG  1350
1351 CCGGTTATCT CCACACCAGA ACCGGCGGAG TTAACCGATT ATAGATTTGT  1400
1401 TCGGTCTCCG TCACTGTCGT TAGCTTCTTT ATCGTCGGGA TTGAAAAACT  1450
1451 CCGATGAAGT AGGATTGAAT CAAATCTTTA GATCTCCGAC GGTTACATCT  1500
1501 CTAACAACTT CACCGGAGAA TAACAAAAAA GAGAACTCTC CATTATCATC  1550
1551 TACTTCAACT TCACCGGAAC GACGACCAAA TGATACACCA GAAGCTTACT  1600
TGAGATCTCCGTCGCATTCTTCTGCTTCTACATCACCGTATAGATGTTTT
CAGAAATCTCCGGAGGTCTTACCGGCGTTTATGAGTAATCTCCGGCAAGG
TTTGCAATCTCAGTTACTATCTTCTCCTTCTAACTCTCATGGAGGACAAG
GTTTCCTTAAGCAGTTAGATGCATTACGTTCTCGTTCACCGTCGTCGTCT
TCTTCTTCTGTTTGTTCTTCACCGGAGAAAGCTTCTCATAAGTCACCAGT
TACATCTCCTAAGTTATCTTCCCGGAATTCGCAGTCTCTATCATCTTCTC
CGGATAGAGATTTTAGTCATAGCTTAGATGTATCACCACGGATATCGAAC
ATTTCACCTCAAATTTTACAGTCTCGTGTGCCTCCGCCTCCTCCTCCTCC
CCCACCGTTGCCGTTGTGGGGACGACGGAGTCAGGTGACTACTAAAGCGG
ACACAATCTCGAGACCGCCTTCTCTTACACCGCCTTCACATCCTTTTGTG
ATCCCATCTGAAAACTTACCAGTGACTTCGTCTCCTATGGAGACTCCAGA
GACGGTTTGTGCGAGTGAGGCGGCGGAGGAAACTCCGAAACCGAAGCTAA
AGGCGTTACATTGGGATAAAGTTAGAGCAAGTTCGGATCGTGAGATGGTT
TGGGATCATCTTCGATCAAGCTCTTTCAAGTGAGTTAATGTGACATACTC
GTTTATATGATACTATATGCTTTTAGTGAGAATGTGGTTGTTGAGATTAT
GAATGTGGTTTGCAGATTAGATGAGGAGATGATTGAGACGTTGTTTGTGG
CGAAGTCGTTAAACAACAAACCAAATCAGAGTCAGACAACTCCAAGATGT
GTTCTCCCGAGCCCGAACCAAGAGAACAGAGTCCTGGACCCGAAGAAGGC
TCAGAATATTGCCATCTTGCTTCGTGCACTTAATGTCACTATAGAAGAAG
TTTGTGAGGCTCTTCTTGAAGGTAAACTATGCTGTCACATACATAGTTTC
TCATTTTCTTCTCCTTTGATCTCCAGAATTAGAGTTCTTATGCATTTGTT
AATGGTTTTTCGATGATATGGTTGAGTTATTCTGAAAGCTTTGCTTCTTT
GATGGTGTGGAGATTCTTGGTTACATTGATGTTCTTAGTTATGCTTTTTC
AGGCAATGCTGATACACTGGGGACTGAACTTCTTGAGAGCTTACTGAAGA
TGGCACCGACAAAAGAAGAAGAGCGCAAGTTGAAAGCGTACAATGATGAT
TCGCCTGTTAAGCTTGGACATGCTGAGAAATTCCTTAAGGCAATGTTGGA
CATCCCTTTCGCCTTTAAAAGAGTTGATGCAATGCTCTATGTAGCCAACT
TTGAGTCCGAGGTTGAATACTTGAAGAAATCTTTTGAGACTCTTGAGGTA
TATATTACAAGCTATTCTCTCTCTTTTTACCATATGGTTGTATTGTAACA
GATTATGACTTCATTTCTATTGTTTGTGTAGGCTGCTTGTGAAGAACTGA
GGAACAGTAGGATGTTCTTAAAGCTTCTTGAAGCGGTTCTAAAGACAGGA
AACCGTATGAACGTTGGAACAAACCGAGGAGATGCACATGCGTTCAAGCT
TGATACACTTCTCAAGCTAGTCGATGTCAAAGGCGCTGATGGGAAAACAA
CTCTCTTGCATTTCGTTGTACAAGAGATAATCCGAGCAGAAGGCACACGT
CTCTCAGGTAACAATACACAAACAGATGACATTAAATGCCGGAAACTAGG
TCTCCAAGTTGTATCAAGTCTCTGTTCTGAGCTTAGTAACGTCAAGAAAG
CTGCTGCGATGGACTCAGAAGTACTAAGCAGCTACGTCTCCAAGCTTTCT
CAAGGCATTGCCAAGATCAACGAAGCAATCCAAGTCCAATCAACAATCAC
AGAAGAAAGCAACAGTCAGAGGTTTTCGGAATCGATGAAAACGTTTCTGA
AAAGAGCTGAGGAAGAGATCATCAGAGTACAAGCTCAAGAGAGCGTAGCG
TTATCACTTGTAAAAGAAATCACAGAGTATTTCCATGGAAACTCGGCTAA
AGAAGAAGCGCATCCGTTTAGAATATTCTTGGTGGTTAGAGACTTCCTTG
GAGTAGTAGACAGAGTTTGCAAAGAAGTAGGGATGATAAACGAAAGAACA
ATGGTTAGTTCTGCTCATAAGTTTCCTGTTCCAGTGAATCCAATGATGCC
ACAACCTCTTCCTGGACTCGTTGGACGAAGACAATCTTCTTCTTCTTCGT
CGTCGTCTTCAACCTCTTCGTCTGATGAAGACGAACATAACTCAATCTCA
TTAGTTTCTTAAGGTGAGATCTCAGCTTTGTCTGTGCATGTTGTTGTAAA
AAGTATCCAGTATTGGATTGTTTTGTCATAATAGATTTAAATATATATAT
ATAGAGGGAGGGAATTAATGACAGAAACAAAGAAGTGTTTTTCTTTTCTG
CATTTGTGTAAAAAAAATAATATAGGTTTACCTTAAAATTTGTTCATCTT
AAATTAATAATTTAAGAATCAAATAAATTTGTTTATCTGAACCGTGTGTA
CCACGAAAGAATGTGAGAGCAAACATATTACTTACTTACCCTTCGTTGCT
GAATATAATGATCATTATAAATCACTACCTCCAGTACCTTCTACCTTCTT
CAAAGAACCTTGTTGGATTTGAACCAAAGTTGGAACATAATTGACGAGAG
GTGAGCATCTAGATTCTGCATCGTGATGATGATCCACTTTTATCTATTTA

• Convert the sequence to FASTA using  the BCM sequence utilities ReadSeq function. (If server down, the sequence can be found on the  SOS page.)
• Perform restriction analysis of the resulting sequence using either the BCM sequence utilitiesWebCutter interface or one of the other WebCutter or Cutter sites.

Gene building: searching for coding sequences in chromosomal DNA

• Try to predict the exon-intron structure of the sequence from the previous excercise using the NetGene2 server (chose Arabidopsis, and take care to load only sequence, not the FASTA header, into the bottom box). (If server down, the results can be found on the  SOS page.)
• (Optional): try the same using the Web Gene Gene Builder interface. Take into account the results of the previous excercise (the simplest way is using the best protein match, provided for your convenience below, as key protein). Chose Direct strand, Gene Model, Sequence error report, Use EST mapping and Complete gene model; switch off the Protein homology search. Don`t forget to select organism = Arabidopsis, and the plant scoring matrix! (NOTE: there are currently problems at the server, but look at least at the interface to get an idea what it should do).
• Look up results of other prediction methods - GenScan and GeneFinder (use links or contact group D) and compare the results.