Bioinformatics - protocols  C

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Basic sequence manipulation

>cDNA_C_At
CCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCCCCCGGGCTGCAGGAATTCCAAGCAACCTTTACACAT
ATAGAAGAAGAAAAACACTTCTTTGTTTCTGTCATTAATTCCCTCCCTCTATATATATATATTTAAATCT
ATTATGACAAAACAATCCAATACTGGATACTTTTTACAACAACATGCACAGACAAAGCTGAGATCTCACC
TTAAGAAACTAATGAGATTGAGTTATGTTCGTCTTCATCAGACGAAGAGGTTGAAGACGACGACGAAGAA
GAAGAAGATTGTCTTCGTCCAACGAGTCCAGGAAGAGGTTGTGGCATCATTGGATTCACTGGAACAGGAA
ACTTATGAGCAGAACTAACCATTGTTCTTTCGTTTATCATCCCTACTTCTTTGCAAACTCTGTCTACTAC
TCCAAGGAAGTCTCTAACCACCAAGAATATTCTAAACGGATGCGCTTCTTCTTTAGCCGAGTTTCCATGG
AAATACTCTGTGATTTCTTTTACAAGTGATAACGCTACGCTCTCTTGAGCTTGTACTCTGATGATCTCTT
CCTCAGCTCTTTTCAGAAACNTTTTCATCGATTCCGNNAACCTCTGACTGTTGCTTTCTTCTGTGATTGT
TGATTGGACTTGGATTGCTTCGTTGATCTTGGCAATGNNTTGAGGAAAGCTTGGAGANGTAGCTGCTTAG
TACTTCTGAGTCCATCNNAGC

• Perform restriction analysis to identify sites from the vector polylinker and remove vector sequences so that you have only clean cDNA left. You can do it easily by visual inspection (see vector sequence below, or using one of the WebCutter or Cutter sites. Check the vector-insert boundary using the map provided below and remove vector sequences. (Do this in a temporary Notepad file on your computer).

• Obtain the reverse complement sequence using the BCM sequence utilities Reverse Complement function. Save the clean cDNA sequence (in FASTA format) as a text file with extension *.nt. If you were unable to do it for server-dependent reasons, go to the SOS page.

Sequence similarity searches and domain structure analysis

• Use the DNA sequence from the previous excercise as a query for a standard nucleotide BLAST search of the non-redundant NCBI database with default parameters.  If you were unable to perform the search for server-dependent reasons, go to the SOS page.
• From the BLAST results page, retrieve the nucleotide sequence corresponding to the best hit, which is a cloned cDNA derived from the same gene (by clicking the link to the left from the gene description- see Figure). Look up also the second best hit, which corresponds to the genomic locus. If the BLAST server is down, go to the SOS page.

 
• Retrieve the cDNA sequence in FASTA format and save it with extension *.nt. Click the link to protein and save the protein sequence in FASTA format with extension *.aa (or, in the worst case, get it from the SOS page).
• Below you find the relevant portion of the (HUGE!!) genomic sequence (2nd best natch)  you'll be working with (feel free to try to obtain it yourself from the NCBI server - this is just to save you some time).


>C_gene
TTTAATAAAATAAAAATCCACTCGCATTTTTATTTTCAACATTGTGCGTA
CGGTGCAATTCAATGAACAGTGTTTACTTTCAGTGTGTACACTTCTGCGG
ACTATTACAAAGTCCACGTCTTATCCTACGTGTTATAATCTCATATGTTA
CTGTCTGAAATGGACCCCACTACGTAAAAATAAAATTAAGAATCAACCAC
TCTTCTTCCATCACCTCTTTTGGCTTTCTCTCTACTCTCTCTACTACTCT
CTCACCATCACTGAGTTAAGAGAACAAACCAAAAACAAAATTATCAAACC
ATCACCAGCAGAATCTTAGCTGGATTCATCACTCTATTCAAAAAGTTTCT
CTCTTCTCTTTTCTCAGATCTTGAACTCTTGAAGAAGAAAGAAGAAGATA
ACACAATGCTCTTCTTCTTATTCTTCTTCTACTTACTCTTATCTTCATCC
TCCGATCTAGTCTTCGCCGACCGTCGTGTACTCCACGAACCATTCTTCCC
TATAGATTCACCACCACCGTCACCACCATCACCACCACCACTTCCTAAAC
TACCATTCTCTTCAACCACTCCTCCATCTTCATCAGACCCAAATGCTTCT
CCTTTCTTCCCTTTATACCCTTCATCTCCACCACCACCTTCTCCAGCCTC
CTTCGCTTCTTTTCCGGCGAATATCTCATCTCTAATCGTCCCTCACGCCA
CTAAATCCCCACCTAACTCCAAAAAACTCCTTATCGTCGCTATCTCCGCC
GTTTCCTCCGCTGCTTTAGTCGCTCTACTTATCGCTTTACTCTATTGGCG
AAGAAGCAAACGTAACCAAGATCTTAACTTCTCCGATGATAGCAAAACAT
ACACCACCGACAGTAGCCGCCGTGTCTACCCTCCTCCTCCGGCAACGGCG
CCTCCAACACGACGCAATGCGGAGGCTAGAAGTAAACAGAGGACCACCAC
GAGCTCCACCAATAACAACAGCTCTGAGTTTCTTTACTTAGGAACAATGG
TGAATCAAAGAGGAATCGATGAACAATCTCTTAGTAATAATGGATCAAGC
TCAAGAAAACTTGAATCTCCAGATCTTCAACCACTTCCTCCATTGATGAA
ACGAAGTTTCCGTTTAAATCCAGATGTTGGTTCAATCGGAGAAGAAGATG
AAGAAGATGAGTTTTACTCTCCACGTGGCTCACAAAGCGGGCGAGAACCG
TTAAACCGGGTCGGACTTCCGGGTCAAAATCCTAGATCTGTTAACAATGA
CACTATCTCTTGCTCATCTTCAAGCTCTGGTTCACCAGGAAGATCAACAT
TTATCAGTATCTCTCCTTCAATGAGTCCTAAGAGATCTGAACCAAAACCG
CCGGTTATCTCCACACCAGAACCGGCGGAGTTAACCGATTATAGATTTGT
TCGGTCTCCGTCACTGTCGTTAGCTTCTTTATCGTCGGGATTGAAAAACT
CCGATGAAGTAGGATTGAATCAAATCTTTAGATCTCCGACGGTTACATCT
CTAACAACTTCACCGGAGAATAACAAAAAAGAGAACTCTCCATTATCATC
TACTTCAACTTCACCGGAACGACGACCAAATGATACACCAGAAGCTTACT
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

You'll later need to have this sequence as a file, so you can either save it separately or to the same file as before, just continuing after your first sequence :

>sequence1
TTTAATAAAATAAAAATCCACTCGCATTTTTATTTTCAACATTGTGCGTA
CGGTGCAATTCAATGAACAGTGTTTACTTTCAGTGTGTACAC....
>sequence2
AAAATAAAAATCCACTCGCATTTTTATTTTCAACATTGTGCGTACGGTGCA
ATTCAATGAACAGTGTTTACTTTCAGTGTGTACACTTCTGCGG...
 

• Use the genomic sequence as a query to search the Arabidopsis ESTs database on the TAIR (preferentially) or Minnesota BLAST site. Select "GenBank ESTs" on TAIR or "Ath" at the Minnesota site. Look at the outputs (could be retrieved from the SOS page), try to retrieve some sequences found, and attempt to judge from the alignment at the bottom of the BLAST output whether they come from the same gene and whether they are in the correct orientation.
• DO NOT actually retrieve the sequences - it would take quite a lot of time. Retrieved EST sequences  in the correct orientation are provided below - just save them into a FASTA file with extension *.nt.

 

 

>R30345 gi|937008|gb|R30345.1|R30345 12950 Lambda-PRL2 Arabidopsis thaliana cDNA clone 159O16T7, mRNA sequence
AGTTGATGCAATGCTCTATGTAGCCAACTTTGAGTCCGAGGTTGAATACTTGAAGAAATCTTTTGAGACT
CTTGAGGCTGCTTGTNAAGAACTGAGGAACAGTAGGATGTTCTTAAAGCTTCTTGAAGCGGTTCTAAAGA
CAGGAAACCGTATGAACGTTGGAACAAACCGAGGAGATGCACATGCGTTCAAGCTTGATACACTTCTCAA
GCTAGTCGATGTCAAAGGCGCTGATGGGAAAACAACTCTCTTGCATTTCGTTGTACAAGAGATAATCCGA
GCAGAAGGCACACGTCTCTCCAGGTAACAATACACAANCAGNTGACATTAAATGCCGGGAAACTAGGGTC
TCCAAGGTTGTATCCAAGGTCTCCTGTTTCTGNGCTTTNGGTAANGTCAAAGAAAAGCTTCTTCCGNTGG
CNTNAGNAGGTNCTTTGNNNGCTTANGGNNNCAAAGGTTTTTT
>Z18512 gi|17638|gb|Z18512.1|Z18512 ATTS0888 AC16H Arabidopsis thaliana cDNA clone TAY026 5', mRNA sequence
GAAACTCCGAAACCGAAGCTAAAGGCGTTACATTGGGATAAAGTTAGAGCAAGTTCGGATCGTGAGATGG
TTTGGGATCATCTTCGATCAAGCTCTTTCAAATTAGATGAGGAGATGATTGAGACGTTGTTTGTGGCGAA
GTCGTTAAACAACAAACCAAATCAGAGTCAGACAACTCCAAGATGTGTTCTCCCGAGCCCGAACCAAGAG
AACAGAGTCCTGGACCCGAAGAAGGCTCAGAATATTGCCATCTTGCTTCGTGCACTTAATGTCACTATAG
AAGAAGTT
>rAV544190 gi|8715604|gb|AV544190.1|AV544190 AV544190 Arabidopsis thaliana roots Columbia cDNA clone RZ35g12F
GCTGCGATGGACTCAGAAGTACTAAGCAGCTACGTCTCCAAGCTTTCTCA
AGGCATTGCCAAGATCAACGAAGCAATCCAAGTCCAATCAACAATCACAG
AAGAAAGCAACAGTCAGAGGTTTTCGGAATCGATGAAAACGTTTCTGAAA
AGAGCTGAGGAAGAGATCATCAGAGTACAAGCTCAAGAGAGCGTAGCGTT
ATCACTTGTAAAAGAAATCACAGAGTATTTCCATGGAAACTCGGCTAAAG
AAGAAGCGCATCCGTTTAGAATATTCTTGGTGGTTAGAGACTTCCTTGGA
GTAGTAGACAGAGTTTGCAAAGAAGTAGGGATGATAAACGAAAGAACAAT
GGTTAGTTCTGCTCATAAGTTTCCTGTTCCAGTGAATCCAATGATGCCAC
AACCTCTTCCTGGACTCGTTGGACGAAGACAATCTTCTTCTTCTTCGTCG
TCGTCTTCAACCTCTTCGTCTGATGAAGACGAACATAACTCAATCTCATT
AGTTTCTTAAGGTGAGATCTCAGCTTTGTCTGTGCATGTTGTTGTAAAAA
GTATCCAGTATTGGATTGTTTTGTCATAATAGATTTAAATATATATATAT
AGAGGGAGGGAATTAATGACAGAAACAAAGAAGTGTTTTTCTTCTTCTAT
ATGTGTAAAGGTTGCTTG
>rAV546860 gi|8718274|gb|AV546860.1|AV546860 AV546860 Arabidopsis thaliana roots Columbia cDNA clone RZL20h01
GAGATCATCAGAGTACAAGCTCAAGAGAGCGTAGCGTTATCACTTGTAAA
AGAAATCACAGAGTATTTCCATGGAAACTCGGCTAAAGAAGAAGCGCATC
CGTTTAGAATATTCTTGGTGGTTAGAGACTTCCTTGGAGTAGTAGACAGA
GTTTGCAAAGAAGTAGGGATGATAAACGAAAGAACAATGGTTAGTTCTGC
TCATAAGTTTCCTGTTCCAGTGAATCCAATGATGCCACAACCTCTTCCTG
GACTCGTTGGACGAAGACAATCTTCTTCTTCTTCGTCGTCGTCTTCAACC
TCTTCGTCTGATGAAGACGAACATAACTCAATCTCATTAGTTTCTTAAGG
TGAGATCTCAGCTTTGTCTGTGCATGTTGTTGTAAAAAGTATCCAGTATT
GGATTGTTTTGTCATAATAGATTTAAATATATATAT
>rAV567287 gi|8738741|gb|AV567287.1|AV567287 AV567287 Arabidopsis thaliana green siliques Columbia cDNA clone
AGAATATTCTTGGTGGTTAGAGACTTCCTTGGAGTAGTAGACAGAGTTTG
CAAAGAAGTAGGGATGATAAACGAAAGAACAATGGTTAGTTCTGCTCATA
AGTTTCCTGTTCCAGTGAATCCAATGATGCCACAACCTCTTCCTGGACTC
GTTGGACGAAGACAATCTTCTTCTTCTTCGTCGTCGTCTTCAACCTCTTC
GTCTGATGAAGACGAACATAACTCAATCTCATTAGTTTCTTAAGGTGAGA
TCTCAGCTTTGTCTGTGCATGTTGTTGTAAAAAGTATCCAGTATTGGATT
GTTTTGTCATAATAGATTTAAATATATATAT
>AV548463 gi|8719876|gb|AV548463.1|AV548463 AV548463 Arabidopsis thaliana roots Columbia cDNA clone RZL54g03F 3', mRNA sequence
CAAAAAACTCCTTATCGTCGCTATCTCCGCCGTTTCCTCCGCTGCTTTAGTCGCTCTACTTATCGCTTTA
CTCTATTGGCGAAGAAGCAAACGTAACCAAGATCTTAACTTCTCCGATGATAGCAAAACATACACCACCG
ACAGTAGCCGCCGTGTCTACCCTCCTCCTCCGGCAACGGCGCCTCCAACACGACGCAATGCGGAGGCTAG
AAGTAAACAGAGGACCACCACGAGCTCCACCAATAACAACAGCTCTGAGTTTCTTTACTTAGGAACAATG
GTGAATCAAAGAGGAATCGATGAACAATCTCTTAGTAATAATGGATCAAGCTCAAGAAAACTTGAATCTC
CAGATCTTCAACCACTTCCTCCATTGATGAAACGAAGTTTCCGTTTAAATCCAGATGTTGGTTCAATCGG
AGAAGAAGATGAAGAAGATGAGTTTTACTCTCCACGTGGCTCACAAAGCGGGCGAGAACCGTTAAACCGG
GTCGGACTTCCGGGTCAAAATCCTAGATCTGTTAACAATGACACTATCTCTTGCTCATCTTCAAGCTCTG
GTTCACCAGGAAGATCAACATTTATCAGTATCTCTCCTTC
>AV528187 gi|8687715|gb|AV528187.1|AV528187 AV528187 Arabidopsis thaliana aboveground organs two to six-week old
CTCTGAGTTTCTTTACTTAGGAACAATGGTGAATCAAAGAGGAATCGATGAACAATCTCTTAGTAATAAT
GGATCAAGCTCAAGAAAACTTGAATCTCCAGATCTTCAACCACTTCCTCCATTGATGAAACGAAGTTTCC
GTTTAAATCCAGATGTTGGTTCAATCGGAGAAGAAGATGAAGAAGATGAGTTTTACTCTCCACGTGGCTC
ACAAAGCGGGCGAGAACCGTTAAACCGGGTCGGACTTCCGGGTCAAAATCCTAGATCTGTTAACAATGAC
ACTATCTCTTGCTCATCTTCAAGCTCTGGTTCACCAGGAAGATCAACATTTATCAGTATCTCTCCTTCAA
TGAGTCCTAAGAGATCNTGAACAAAACCGCCG
>AV552669 gi|8724082|gb|AV552669.1|AV552669 AV552669 Arabidopsis thaliana roots Columbia
TTCAATCGGAGAAGAAGATGAAGAAGATGAGTTTTACTCTCCACGTGGCTCACAAAGCGGGCGAGAACCG
TTAAACCGGGTCGGACTTCCGGGTCAAAATCCTAGATCTGTTAACAATGACACTATCTCTTGCTCATCTT
CAAGCTCTGGTTCACCAGGAAGATCAACATTTATCAGTATCTCTCCTTCAATGAGTCCTAAGAGATCTGA
ACCAAAACCGCCGGTTATCTCCACACCAGAACCGGCGGAGTTAACCGATTATAGATTTGTTCGGTCTCCG
TCACTGTCGTTAGCTTCTTTATCGTCGGGATTGAAAAACTCCGATGAAGTAGGATTGAATCAAATCTTTA
GATCTCCGACGGTTACATCTCTAAC
>rAV522684 gi|8682211|gb|AV522684.1|AV522684 AV522684 Arabidopsis thaliana aboveground organs two to six-week old
AAACTCGGCTAAAGAAGAAGCGCATCAGTTTAGAATATTCTTGGTGCTTA
GAGACTTCCTTGGAGTAGTCGACAGAGTTTGCAAAGAAGTAGGGATGATA
AACGAACAGAACAATGGTTCGTTCTGCTCATAAGTTTCCTGTTCCAGTGA
ATCCACTGATGCCACAACCTCTTCATGGACTCGTTGGACGAAGACAATCC
TTCTTCTTCTTCGTCGTCGTCTTCAACCTCTTCGTCTGATGAAGACGAAC
ATAACTCAATCTCATTAGTCTCTTAAGGTGAGATCTCAGCCTTGTCTGTG
CATCTTCTTCTAAAACGTATCCAGTATTGGATTGTTCTGTCATAATAGAT
TTAAATATATATATATAGAGGGAGGGAATTAATGACAGAAACAAAGAAGT
GT
>rAV546377 gi|8717791|gb|AV546377.1|AV546377 AV546377 Arabidopsis thaliana roots Columbia
GGTTAGTTCTGCTCATAAGTTTCCTGTTCCAGTGAATCCAATGATGCCAC
AACCTCTTCCTGGACTCGTTGGACGAAGACAATCTTCTTCTTCTTCGTCG
TCGTCTTCAACCTCTTCGTCTGATGAAGACGAACATAACTCAATCTCATT
AGTTTCTTAAGGTGAGATCTCAGCTTTGTCTGTGCATGTTGTTGTAAAAA
GTATCCAGTATTGGATTGTTTTGTCATAATAGATTTAAATATATATATAT
AGAGGGAGGGAATTAATGACAGAAACAAAGAAGTGTTTTTCTTTTCTGCA
TTTGTGTAAAAAAAATAATATAGGTTTACCTTAAAATTTGTTCATCTTAA
ATTAATAATTTAAGAATCAAATAAATTTGTTT
>Z17974 gi|17102|gb|Z17974.1|Z17974 ATTS0458 AC16H Arabidopsis thaliana cDNA clone TASF009R 5', mRNA sequence
ACACTCTCCTGACTCGTGACGAAGACAATCTTCTTCTTCTTCGTCGTCGTCTTCAACCACTTCGTCTGAT
GAAGACGAACATAACTCAATCTCATTAGTTTCTTAAGGTGAGATCTCAGCTTGTCTGTGCATCTTGTTGT
AAAAAGTATCCAGTATTGGATTGTTTTGTCATAATAGATTTAAATATATATATATAGAGGGAGGGAATTA
ATGACAGAAACAAAGAAGTGTTTTTCTTTTCTGCATTTGAGTAAAAAAAATAATATAGGTTTACCTTAAA
AAAAAAA
 

• Return to the protein sequence coresponding to your cDNA.  Perform a domain structure analysis of the protein corresponding to the best match to your cDNA using the SMART Sequence Analysis utility. Paste only clean single-letter code sequence into the sequence box (no FASTA header) and include also Outlier homologues, PFAM domains, signal peptides and internal repeats (for explanation what this means see the SMART page).
• If you found any domains within the sequence, look up the domain description (available by clicking the domain in the SMART output). (If server was down, you can get the SMART output from the SOS page). Discuss the results with group D who analyzed the same sequence by different means.

Gene building: searching for coding sequences in chromosomal DNA

• Open MACAW on your computer (should be OK over the browser). Go File... New Project..., select Sequence type= Nucleotide and Significance=number.Chose the Nucleotide scoring matrix and import sequences (Sequence... Import...) from one of the *.nt files created previously. Continue until you imported the genomic and cDNA sequence and all the ESTs. Save the Macaw file on your computer before proceeding further in order to decrease the likelihood of crash.
• Use Segment pair overlap to align the sequences (start with threshold 1000 and go down if necessary). You might try Gibbs sampler to find out why it is NOT suitable for this task.

Construction and interpretation of a protein sequence alignment

• Below you find partial sequences of a number of proteins related to the one you are working with. Use CMBI CLUSTAL W to align them with your sequence. Use default parameters, leaving out the phylogenetic tree option. Why shouldn't you build the tree at this stage?

>Bprotein
SGGETSKQVKLKPLHWDKVNPDSDHSMVWDKIDRGSFSFDGDLMEALFGYVAVGKKSPEQ
GDEKNPKSTQIFILDPRKSQNTAIVLKSLGMTREELVESLIEGNDFVPDTLERLARIAPT
KEEQSAILEFDGDTAKLADAETFLFHLLKSVPTAFTRLNAFLFRANYYPEMAHHSKCLQT
LDLACKELRSRGLFVKLLEAILKAGNRMNAGTARGNAQAFNLTALLKLSDVKSVDGKTSL
LNFVVEEVVRSEGKRCVMNRRSHSLTRSGSSNYNGGNSSLQVMSKEEQEKEYLKLGLPVV
GGLSSEFSNVKKAACVDYETVVATCSALAVRAKDAKTVIGECEDGEGGRFVKTMMTFLDS
VEEEVKIAKGEERKVMELVKRTTDYYQAGAVTKGKNPLHLFVIVRDFLAMVDKVCLDIMR
NMQRRK
>gi|6691125 Nicotiana tabacum NFH2
EKSEEILKPKLKTLHWDKVRASSDCEMVWDQLKSSSFKLNEEMIETLFVVKNPTLNTSAT
AKHFVVSSMSQENRVLDPKKSQNIAILLRVLNGTTEEICEAFLEGNAENIGTELLEILLK
MAPSKEEERKLKEYKDDSPFKLGPAEKFLKAVLDIPFAFKRIDAMLYISNFDYEVDYLGN
SFETLEAACEELRSSRMFLKLLEAVLKTGNRMNVGTNRGDAHAFKLDTLLKLVDVKGADG
KTTLLHFVVQEIIKSEGARLSGGNQNHQQSTTNDDAKCKKLGLQVVSNISSELINVKKSA
AMDSEVLHNDVLKLSKGIQNIAEVVRSIEAVGLEESSIKRFSESMNRFMKVAEEKILRLQ
AQETLAMSLVKEITEYVHGDSAREEAHPFRIFMVVKDFLMILDCVCKEVGTINERTI
>gi|6041849 Arabidopsis BAC F21O3
EGTTDRPKPKLKPLPWDKVRPSSRRTNTWDRLPYNSSNANSKQRSLSCDLPMLNQESKVL
DPRKSQNVAVLLTTLKLTTNDVCQALRDGHYDALGVELLESLARVAPSEEEEKKLISYSD
DSVIKLAPSERFLKELLNVPFVFKRVDALLSVASFDSKVKHLKRSFSVIQAACEALRNSR
MLLRLVGATLEAGMKSGNAHDFKLEALLGLVDIKSSDGRTSILDSVVQKITESEGIKGLQ
VVRNLSSVLNDAKKSAELDYGVVRMNVSKLYEEVQKISEVLRLCEETGHSEEHQWWKFRE
SVTRFLETAAEEIKKIEREEGSTLFAVKKITEYFHVDPAKEEAQLLKVFVIVRDFLKILE
GVCKKMEVTSSLA
>gi|6225268 DIAPHANOUS PROTEIN HOMOLOG 1
PKKLYKPEVQLRRPNWSKLVAEDLSQDCFWTKVKEDRFENNELFAKLTLTFSAQTKTKKD
QEGGEEKKSVQKKKVKELKVLDSKTAQNLSIFLGSFRMPYQEIKNVILEVNEAVLTESMI
QNLIKQMPEPEQLKMLSELKDEYDDLAESEQFGVVMGTVPRLRPRLNAILFKLQFSEQVE
NIKPEIVSVTAACEELRKSESFSNLLEITLLVGNYMNAGSRNAGAFGFNISFLCKLRDTK
STDQKMTLLHFLAELCENDYPDVLKFPDELAHVEKASRVSAENLQKNLDQMKKQISDVER
DVQNFPAATDEKDKFVEKMTSFVKDAQEQYNKLRMMHSNMETLYKELGEYFLFDPKKLSV
EEFFMDLHNFRNMFLQAVKENQKRRKTEEKMRRAKLAKEKAEKERLEKQQKREQLIDMNA
EGDETGVMDSLLEALQSGAAFRR
>gi|544344 FORMIN 4 (LIMB DEFORMITY PROTEIN).
RKPAIEPSCPMKPLYWTRIQINDKSQDAAPTLWDSLEEPHIRDTSEFEYLFSKDTTQQKK
KPLSEAYEKKNKVKKIIKLLDGKRSQTVGILISSLHLEMKDIQQAIFTVDDSVVDLETLA
ALYENRAQEDELTKIRKYYETSKEEDLKLLDKPEQFLHELAQIPNFAERAQCIIFRAVFS
EGITSLHRKVEIVTRASKGLLHMKSVKDILALILAFGNYMNGGNRTRGQADGYSLEILPK
LKDVKSRDNGMNLVDYVVKYYLRYYDQEAGTDKSVFPLPEPQDFFLASQVKFEDLLKDLR
KLKRQLEASEQQMKLVCKESPREYLQPFKDKLEEFFKKAKKEHKMEESHLENAQKSFETT
VGYFGMKPKTGEKEVTPSYVFMVWFEFCSDFKTIWKRESKNISKER
>gi|2281090 unknown protein [Arabidopsis thaliana]
EKKVETMKPKLKTLHWDKVRASSSRVMVWDQIKSNSFQVNEEMIETLFKVNDPTSRTRDG
VVQSVSQENRFLDPRKSHNIAILLRALNVTADEVCEALIEGNSDTLGPELLECLLKMAPT
KEEEDKLKELKDDDDGSPSKIGPAEKFLKALLNIPFAFKRIDAMLYIVKFESEIEYLNRS
FDTLEAATGELKNTRMFLKLLEAVLKTGNRMNIGTNRGDAHAFKLDTLLKLVDIKGADGK
TTLLHFVVQEIIKFEGARVPFTPSQSHIGDNMAEQSAFQDDLELKKLGLQVVSGLSSQLI
NVKKAAAMDSNSLINETAEIARGIAKVKEVITELKQETGVERFLESMNSFLNKGEKEITE
LQSHGDNVMKMVKEVTEYFHGNSETHPFRIFAVVRDFLTILDQVCKEVGRVNERTV
>gi|1061334 Drosophila melanogaster cappuccino
RKSAVNPPKPMRPLYWTRIVTSAPPAPRPPSVANSTDSTENSGSSPDEPPAANGADAPPT
APPATKEIWTEIEETPLDNIDEFTELFSRQAIAPVSKPKELKVKRAKSIKVLDPERSRNV
GIIWRSLHVPSSEIEHAIYHIDTSVVSLEALQHMSNIQATEDELQRIKEAAGGDIPLDHP
EQFLLDISLISMASERISCIVFQAEFEESVTLLFRKLETVSQLSQQLIESEDLKLVFSII
LTLGNYMNGGNRQRGQADGFNLDILGKLKDVKSKESHTTLLHFIVRTYIAQRRKEGVHPL
EIRLPIPEPADVERAAQMDFEEVQQQIFDLNKKFLGCKRTTAKVLAASRPEIMEPFKSKM
EEFVEGADKSMAKLHQSLDECRDLFLETMRFYHFSPKACTLTLAQCTPDQFFEYWTNFTN
DFKDIWKKEITSLLNEL
>gi|5080823 Hypothetical protein [Arabidopsis thaliana]
GKTEDPTQPKLKPLHWDKMNPDASRSMVWHKIDGGSFNFDGDLMEALFGYVARKPSESNS
VPQNQTVSNSVPHNQTYILDPRKSQNKAIVLKSLGMTKEEIIDLLTEGHDAESDTLEKLA
GIAPTPEEQTEIIDFDGEPMTLAYADSLLFHILKAVPSAFNRFNVMLFKINYGSEVAQQK
GSLLTLESACNELRARGLFMKLLEAILKAGNRMNAGTARGNAQAFNLTALRKLSDVKSVD
AKTTLLHFVVEEVVRSEGKRAAMNKNMMSSDNGSGENADMSREEQEIEFIKMGLPIIGGL
SSEFTNVKKAAGIDYDSFVATTLALGTRVKETKRLLDQSKGKEDGCLTKLRSFFESAEEE
LKVITEEQLRIMELVKKTTNYYQAGALKERNLFQLFVIIRDFLGMVDNACSEIARNQRKQ
Q
 
• (Optional:) select a portion of your protein sequence suitable for the construction of the tree, and rerun the analysis with tree ON.