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<p>1. Calculate molecular weight and G+C content of the following DNA sequence tryseq. (BioEdit starts automatically)<br>
(Edit>Select All sequences --- Sequence>Nucleic Acis>Nucleotide Composition)<br>
<br>
</p>
<p><b><font color="red">DNA molecule: gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I<br>
Length = 862 base pairs<br>
Molecular Weight = 263023 Daltons, single stranded<br>
Molecular Weight = 524606 Daltons, double stranded<br>
G+C content = 53,83%<br>
A+T content = 46,17%<br>
</font></b><br>
</p>
<p><b><font color="red">Nucleotide Number Mol%<br>
</font></b><b><font color="red">A 191 22,16<br>
</font></b><b><font color="red">C 255 29,58<br>
</font></b><b><font color="red">G 209 24,25<br>
</font></b><b><font color="red">T 207 24,01<br>
</font></b><br>
</p>
<p>2. Translate into six frames. How many ORFs (Open Reading Frame) are predicted which are over 100 amino acids long<br>
Copy the longest ORF into your word editor<br>
<br>
</p>
<p><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 1 to 732: Frame 1 244 aa<br>
</b></font><font color="red"><b>ATMISLVFVLLIGAAFATEDDKIVGGYECKAYSQTHQVSLNSGYHFCGGSLVNENWVVSAAHCYKSRVEVRLGEHNIKVTEGSEQFISSSRVIRHPNYSSYNIDNDIMLIKLSKPATLNTYVQPVALPTSCAPAGTMCTVSGWGNTMSSTADSNKLQCLNIPILSYSDCNNSYPGMITNAMFCAGYLEGGKDSCQGDSGGPVVCNGELQGVVSWGYGCAEPGNPGVYAKVCIFNDWLTSTMASY<br>
</b></font><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 264 to 1: Frame -2 88 aa<br>
</b></font><font color="red"><b>RDELLATFSHLDVVLAQTHLHTGLVAVSSRHNPVLIDQGATTEVVSRVQRHLVGLGVGLALIPSDDLVVLCGESGSNEQNEDQRNHGC<br>
</b></font><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 822 to 268: Frame -2 185 aa<br>
</b></font><font color="red"><b>SPVQKVDVDLERDVVELWDRAGGPKLGSDLVGGHGAGQPVIEDANLGVDTRVTGLSTSVTPGHNTLELTIAHHRATRVTLARVLASLQVSSTEHGVGDHARVGVVTVAVGQDGDVQALQLVAVSSGAHGVAPSRDGTHGASGGTAGGQSNRLHVGVEGGGFAQFDQHDVIIDVVGAVVGVTDHAG<br>
</b></font><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 529 to 356: Frame -1 58 aa<br>
</b></font><font color="red"><b>SCQGRSCYSRCRTGWGCSGTAACCCQQWSSWCCPIQRRYTWCQRGHSWWAEQQAARRC<br>
</b></font><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 530 to 709: Frame 2 60 aa<br>
</b></font><font color="red"><b>SPTPCSVLDTWREARTLARVTLVALWCAMVSSRVLCPGVTDVLSPVTLVSTPRFASSMTG<br>
</b></font><font color="red"><b>>gi|64379|emb|X70075.1|SSTRYPI S.salar mRNA for trypsin I: 697 to 533: Frame -1 55 aa<br>
</b></font><font color="red"><b>RCKPWRRHQGYRAQHIRNPRTQHPGAHHCTPQGHQSHPGKSPCLPPGIQHRTWRW<br>
</b></font><br>
</p>
<p>3. Close all BioEdit windows (except BioEdit) and create a new sequence file.(File>New Alignment --- Sequence>New sequence)<br>
Paste the longest amino acid sequence into the "New Sequence" window and give the sequence a name (e.g. test1) and select a Sequence Type (Protein). Then Apply and close.<br>
Calculate the molecular weight of the ORF and composition. Which two amino acids are most abundant?<br>
<br>
</p>
<p><font color="red"><b>Protein: test1<br>
Length = 244 amino acids<br>
Molecular Weight = 26129,17 Daltons<br>
<br>
Amino Acid Number Mol%<br>
Ala A 17 6,97<br>
Cys C 12 4,92<br>
Asp D 9 3,69<br>
Glu E 10 4,10<br>
Phe F 6 2,46<br>
Gly G 24 9,84<br>
His H 5 2,05<br>
Ile I 13 5,33<br>
Lys K 9 3,69<br>
Leu L 16 6,56<br>
Met M 7 2,87<br>
Asn N 17 6,97<br>
Pro P 10 4,10<br>
Gln Q 7 2,87<br>
Arg R 4 1,64<br>
Ser S 27 11,07<br>
Thr T 14 5,74<br>
Val V 20 8,20<br>
Trp W 4 1,64<br>
Tyr Y 13 5,33<br>
</b></font><br>
<br>
</p>
<p>4. Try one or more programs predicting hydrophobic profiles. (e.g. Kyte-Doolittle) Which regions are most hydrophobic?<br>
Try different scan-window size (2, 5, 10 & 15). What do you observe?<br>
<br>
<br>
<br>
<img src="image2" width="640" height="320" border="0"></p>
<p> <br>
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<img src="image4"width="640" height="320" border="0"><br>
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</p>
<p>5. Select amino 225 -245 (Clic and drag). Run Helical Wheel Diagram (Sequence>Protein>Helical Wheel Diagram). Which anino acids are clustered together one one side of the helix?<br>
<br>
<br>
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<br>
<br>
<img src="image6"width="320" height="320" border="0"><br>
<br>
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<br>
</p>
<p>6. To try to find out what protein this may be, we will use a BLAST search. (Accessory Application>BLAST>NCBI BLAST over the Internet). Past the amino acid sequence into the window and press Search. After a while you will se a web-page from NCBI (National Center for Biotechnology Information) with the sequences in the database that is most similar to our sequence. <br>
<br>
</p>
<p>Which sequence is most similar to our sequence? From which organism? Has the structure been solved? If yes, how many disulfide bridges are the in the structure?<br>
<br>
<br>
</p>
<p>LOCUS TRY1_SALSA 242 aa linear VRT 15-JUN-2002<br>
DEFINITION <font color="red"><b>Trypsin I precursor</b></font>.<br>
ACCESSION P35031<br>
VERSION P35031 GI:464944<br>
DBSOURCE swissprot: locus TRY1_SALSA, accession P35031;<br>
class: standard.<br>
created: Feb 1, 1994.<br>
sequence updated: Feb 1, 1994.<br>
annotation updated: Jun 15, 2002.<br>
xrefs: gi: 64379, gi: 64380, gi: 64381, gi: 64382, gi: 64383, gi:<br>
64384, gi: 422581, gi: 422582, gi: 422583, gi: 515295, gi: 809221<br>
xrefs (non-sequence databases): MEROPSS01.151, InterProIPR001314,<br>
InterProIPR001254, PfamPF00089, SMARTSM00020, PROSITEPS50240,<br>
PROSITEPS00134, PROSITEPS00135<br>
KEYWORDS Hydrolase; Serine protease; Digestion; Pancreas; Zymogen; Signal;<br>
Multigene family; 3D-structure.<br>
SOURCE<b><font color="red"> Salmo salar (Atlantic salmon)</font></b><br>
ORGANISM Salmo salar<br>
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;<br>
Actinopterygii; Neopterygii; Teleostei; Euteleostei;<br>
Protacanthopterygii; Salmoniformes; Salmonidae; Salmo.<br>
REFERENCE 1 (residues 1 to 242)<br>
AUTHORS Male,R., Lorens,J.B., Smalas,A.O. and Torrissen,K.R.<br>
TITLE Molecular cloning and characterization of anionic and cationic<br>
variants of trypsin from Atlantic salmon<br>
JOURNAL Eur. J. Biochem. 232 (2), 677-685 (1995)<br>
MEDLINE 96035908<br>
PUBMED 7556223<br>
REMARK SEQUENCE FROM N.A.<br>
TISSUE=Pancreas<br>
REFERENCE 2 (residues 1 to 242)<br>
UTHORS Smalas,A.O. and Hordvik,A.<br>
TITLE <font color="red"><b>Structure determination and refinement of benzamidine- inhibited trypsin from the North Atlantic salmon (Salmo salar) at 1.82-A resolution<br>
</b></font> JOURNAL Acta Crystallogr. D 49, 318-330 (1993)<br>
REMARK X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS).<br>
REFERENCE 3 (residues 1 to 242)<br>
AUTHORS Smalas,A.O., Heimstad,E.S., Hordvik,A., Willassen,N.P. and Male,R.<br>
TITLE Cold adaption of enzymes: structural comparison between salmon and<br>
bovine trypsins<br>
JOURNAL Proteins 20 (2), 149-166 (1994)<br>
MEDLINE 95148588<br>
PUBMED 7846025<br>
REMARK X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS).<br>
COMMENT -------------------------------------------------------------------<br>
This SWISS-PROT entry is copyright. It is produced through a<br>
collaboration between the Swiss Institute of Bioinformatics and<br>
the EMBL outstation - the European Bioinformatics Institute.<br>
The original entry is available from http://www.expasy.ch/sprot<br>
and http://www.ebi.ac.uk/sprot<br>
</p>
<p> ------------------------------------------------------------------.<br>
</p>
<p> [CATALYTIC ACTIVITY] Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.<br>
</p>
<p> [SUBCELLULAR LOCATION] Extracellular.<br>
</p>
<p> [SIMILARITY] BELONGS TO PEPTIDASE FAMILY S1.<br>
</p>
<p>FEATURES Location/Qualifiers<br>
</p>
<p> source 1..242<br>
</p>
<p> /organism="Salmo salar"<br>
</p>
<p> /db_xref="taxon:8030"<br>
</p>
<p> Protein 1..242<br>
</p>
<p> /product="Trypsin I precursor"<br>
</p>
<p> /EC_number="3.4.21.4"<br>
</p>
<p> Region 1..15<br>
</p>
<p> /region_name="Signal"<br>
</p>
<p> /note="POTENTIAL."<br>
</p>
<p> Region 16..20<br>
</p>
<p> /region_name="Propeptide"<br>
</p>
<p> /note="ACTIVATION PEPTIDE."<br>
</p>
<p> Region 21..242<br>
</p>
<p> /region_name="Mature chain"<br>
</p>
<p> /note="TRYPSIN I."<br>
</p>
<p> Region 22<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 25..26<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Bond bond(27,156)<br>
</p>
<p>/bond_type="<font color="red"><b>disulfide</b></font>"<br>
</p>
<p> Region 29..34<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 33<br>
</p>
<p> /region_name="Variant"<br>
</p>
<p> /note="T -> A (IN TRYPSINS IA/IB)."<br>
</p>
<p> Region 35..39<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 43..49<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Bond bond(45,61)<br>
</p>
<p>/bond_type="<b><font color="red">disulfide</font></b>"<br>
</p>
<p> Region 54..57<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 59..61<br>
</p>
<p> /region_name="Helical region"<br>
</p>
<p> Site 60<br>
</p>
<p> /site_type="active"<br>
</p>
<p> /note="CHARGE RELAY SYSTEM."<br>
</p>
<p> Region 67..70<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 74<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 75..76<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 83..92<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 94..95<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 97<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 98..101<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 102<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 103<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Site 104<br>
</p>
<p> /site_type="active"<br>
</p>
<p> /note="CHARGE RELAY SYSTEM."<br>
</p>
<p> Region 106..110<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 124<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Bond bond(129,229)<br>
</p>
<p>/bond_type="<font color="red"><b>disulfide</b></font>"<br>
</p>
<p> Region 132..133<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 135..140<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Bond bond(136,202)<br>
</p>
<p>/bond_type="<font color="red"><b>disulfide</b></font>"<br>
</p>
<p> Region 151..152<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 153<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 155..161<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 164..170<br>
</p>
<p> /region_name="Helical region"<br>
</p>
<p> Bond bond(167,181)<br>
</p>
<p>/bond_type="<font color="red"><b>disulfide</b></font>"<br>
</p>
<p> Region 172..174<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 177..178<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 179..182<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 185..186<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 190<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Site 190<br>
</p>
<p> /site_type="unclassified"<br>
</p>
<p> /note="REQUIRED FOR SPECIFICITY (BY SIMILARITY)."<br>
</p>
<p> Bond bond(192,216)<br>
</p>
<p>/bond_type="<font color="red"><b>disulfide</b></font>"<br>
</p>
<p> Region 193..194<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 196..197<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Site 196<br>
</p>
<p> /site_type="active"<br>
</p>
<p> /note="CHARGE RELAY SYSTEM."<br>
</p>
<p> Region 199..202<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 203..204<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 205..212<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 219..220<br>
</p>
<p> /region_name="Hydrogen bonded turn"<br>
</p>
<p> Region 223..227<br>
</p>
<p> /region_name="Beta-strand region"<br>
</p>
<p> Region 228..230<br>
</p>
<p> /region_name="Helical region"<br>
</p>
<p> Region 232..240<br>
</p>
<p> /region_name="Helical region"<br>
</p>
<p>ORIGIN <br>
</p>
<p> 1 mislvfvlli gaafateddk ivggyeckay sqthqvslns gyhfcggslv nenwvvsaah<br>
</p>
<p> 61 cyksrvevrl gehnikvteg seqfisssrv irhpnyssyn idndimlikl skpatlntyv<br>
</p>
<p> 121 qpvalptsca pagtmctvsg wgntmsstad snklqclnip ilsysdcnns ypgmitnamf<br>
</p>
<p> 181 cagyleggkd scqgdsggpv vcngelqgvv swgygcaepg npgvyakvci fndwltstma<br>
</p>
<p> 241 sy<br>
</p>
<p>//<br>
<br>
</p>
<p>7. Close all BioEdit windows and press the following file, which starts BioEdit again. You will se several sequences.<br>
</p>
<p>Select all sequences (Edit>Select All sequences). We will now try to align all these sequences (Accessory Application>ClustalW Multiple alignment). In the ClustalW Multiple Alignment window, clic for Full Multiple alignment, Calculate NJ tree and then press Run ClustalW.<br>
<br>
</p>
<p>After a while you will se a new window with the aligned sequences. There are three proteins that have a large deletion in the region around 450 to 550. Which? <font color="red"><b>Vibcho, Vibsp and Vibalg</b></font><br>
</p>
<p>One sequence seems to have a deleted C-terminal. Who? <font color="red"><b>ProK</b></font><br>
<br>
<br>
<br>
</p>
<p>8. To see which sequence are most related, we will run the phylogenetic program PROTPARS (Protein Sequence Parsimony Method) (Accessory Application>Protpars protein parsimony method). Which sequences are SerX most related to?<br>
<br>
<br>
</p>
<p>Protein parsimony algorithm, version 3.573c<br>
<br>
<br>
One most parsimonious tree found:<br>
<br>
<br>
</p>
<p> +-----Vibcho <br>
</p>
<p> +--------5 <br>
</p>
<p> ! ! +--Vibsp <br>
</p>
<p> ! +--6 <br>
</p>
<p> +--4 +--Vibalg <br>
</p>
<p> ! ! <br>
</p>
<p>! ! +--<font color="red"><b>Altsp</b></font><br>
</p>
<p> ! ! +--3 <br>
</p>
<p>--1 +--------2 +--<font color="red"><b>AltspAS11</b></font><br>
</p>
<p> ! ! <br>
</p>
<p> ! +-----SerX <br>
</p>
<p> ! <br>
</p>
<p> +-----------------ProK <br>
<br>
</p>
<p> remember: this is an unrooted tree!<br>
<br>
</p>
<p></p>
</body>
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