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Scenario 3 Anotation Process What follows is a simulation of the process of annotating, using the proposed graphical interface. The interface does not yet exist. As you go through the simulation please consider what capabilities you would want to serve your research and annotation interests. A narrative to help you go through the simulation appears in a red-bordered box, such as the one below. To begin: 1. Click on Slide Show, (on the upper toolbar) 2. Click View Show 3. Click Continue button Continue Scenario 3 Anotation Process You find yourself annotating a set of genes that includes all1290 from Anabaena PCC 7120. You want to assess the validity of the automated annotation, particularly the assertion of function and start codon. If there are any problems, you want to fix them. Continue Main Menu Options Annotate History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Evidently no human has touched this annotation (System is given as the only source). Is the functional annotation sound? Mouse over* to the blue motif bar above the graphical representation of all1290. *In real life, information will pop up when you mouse to an informative position. In this simulation you’ll also need to click. System Main Menu Options Annotate History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase N6,N4 DNA methyltransferase (PF01515): 2e-18 Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Pfam found a motif consistent with the annotation with a good expectation value. Curiously, the motif extends beyond the start of the gene. Mouse away from the bar.* *In real life, information will go away when you mouse away from an informative position. In this simulation you’ll also need to click anywhere.. System Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase System Cyanobacterial orthologs: none PvuIIM: DNA methyltransferase (NP_072082): 8e-64 Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Mouse over to the first aligned gene, from Proteus vulgaris. Again, good support, and again, the gene extends beyond the annotated start codon of the Anabaena orf. *Remember to click to view the popup and click to make it disappear. Main Menu Annotate Options Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis You begin to suspect the annotated start codon. To visualize alternatives, click on the OptionsOptions button, then click on Show alternate starts. History View: graphic Show all Show hits: <1e-6 Predicted Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features Predicted Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Mouse over to the annotated start site* and you see the sequence preceding it: a plausible ribosome binding site. Mouse over to the most distant start site. Not as good. *Click to show, click to disappear System 1533628 and upstream: 1533931 and upstream: CTTCCGGGAAATCCTGTG TAACTTAAATGTCGCTTG Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Nonetheless, the position of the distant site relative to the other sequences makes it a reasonable candidate. Click on it to provisionally change the start site. System Main Menu Annotate Options Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Length = 200 amino acids Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris History View:View graphic Show all Show hits: <1e-6 System Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features System Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis Much better! More similarity and more sequences that are similar (and Neisseria better meningitidis E-values, too, if you were to look). However, it is always a good practice to check the alignment before accepting the conclusions of a More and then on View. similarity search, so click on Options button. Main Menu Annotate Options Anabaena PCC 7120: Graphicall1290 Genome Alignment with homologs Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Alignment with motif Replicon: Chromosome Length = 200 amino acids Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis Click on Alignment with motif to see Neisseria meningitidis how All1120 compares to other protein that make up the Pfam motif represented by the motif bar. More History View Show all Show hits: <1e-6 System Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features System Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Conserved Domain Database PubMed Nucleotide Protein CD: Pfam01555.5, N6_N4_Mtase Structure CDD Taxonomy Help Source: Pfam[US], Pfam[UK] PSSM-Id: 8054 Description: DNA methylase. Members of this family are DNA methylases. The family contains both N-4 cytosine-specific DNA methylases and N-6 Adenine-specific DNA methylases. Taxa: root Status: Alignment from source Created: 13-Jun-2002 Aligned: 52 rows PSSM: 199 columns Representative: Consensus Proteins: [Click here for CDART summary of Proteins containing pfam01555] View 3D Structure Subset Rows with Cn3D up to 10 All1290 consensus Pvuii Dna Methyltransfer Modification methylase S BglI modification methyl adenine specific DNA met adenine specific DNA met Modification methylase M Modification methylase M Modification methylase B gi_3257411 78 1 77 119 92 174 131 133 178 134 123 using Virtual Bonds (To display structure, download Cn3D) of the most diverse members 10 20 30 40 50 60 ....*....|....*....|....*....|....*....|....*....|....*....| KRVLTENGSFILDLGgaylpgnp----VRSIYQYELLVKLCKEVGFFL----------- 122 ARRVLKPDGSIFIFGG------------NKSQKNIFRFIDILIEKGFKF----------- 37 DGSFVVDFGGAYMKGVp-----------ARSIYNFRVLIRMIDEVGFFL----------114 This is output from NCBI showing the PRTGWKRNKQLSLVP--------------------FRVALALQDDGWWI----------- 147 alignment that went into making Pfam FQAVNLKKHRVSVTREd----------iLNALKLEPELTKYQLAKKFNCs---------e 132 ARDLLKEDGVIFISIDdnecan-lkilcDEIFGEDNFVGDFIRKTKSTTndakiglnyqh 232 motif 01555... but it was intercepted by ILRTLLPIYARALKDLmrt------iknVFKQKGQAQAQLVLKEQIKELs---------q 175 the interface in order to incorporate IIDPFMGSGTTLVQCMem--------giNSIGIDISPFNCLIAEVKLQKydi------qk 178 LSRCNLKDGDYILDPFagtgttgavvkkMKYQLYPKDLNVILIEKGKKF----------226 the sequence of All1290 for SHIAFEPKSFIFEYNLeeldraykeiyeISPTKIEQKFNHLSITEGAFPeete----ndl 189 comparison. Click on ANTYAMPSELILKGFP-------------KVKDVVLLFVDDKVLVGETVr---------159 More to get to the best part of the alignment. More Back to Annotation Conserved Domain Database PubMed Nucleotide All1290 consensus Pvuii Dna Methyltransfer Modification methylase S BglI modification methyl adenine specific DNA met adenine specific DNA met Modification methylase M Modification methylase M Modification methylase B gi_3257411 All1290 consensus Pvuii Dna Methyltransfer Modification methylase S BglI modification methyl adenine specific DNA met adenine specific DNA met Modification methylase M Modification methylase M Modification methylase B gi_3257411 Protein Structure CDD Taxonomy Help 228 131 223 339 238 367 279 329 464 319 255 310 320 330 340 350 360 ....*....|....*....|....*....|....*....|....*....|....*....| rrcKAAGVKPHPARFPQGFAEFFIKFLTD-------------------EGDLVLDPFAGS ---NLKGKKKHPTQKPEALLERIILESSN-------------------EGDLVLDPFAGS ancKLMGIKAHPARFPAKLPEFFIRMLTE-------------------PDDLVVDIFGGS sipTAPSGANHFAVMPRKLAHFALKATLP-------------------MNGSCLDPFMGS ---SVRKNDDHEAKFPLLLPQRLIKLLTQ-------------------KGDTVLDCFMGS ellNLGMGEYFTYPKGVEFMKKIILHSTTp-----------------nEGDIILDFFAGS ----LGLKGLFKTPKPVALIKYLLLCSTP-------------------KDSIILDFFAGS vfdPYYCYKHKKICRPVQTILRHLEEYTNdvisri-------eefskiRKDAYYLIINGD icyNCQKEIYGKNSLPILKESKEIIESLEsvengeyqvgenikpqyqkRCKESKSKFAGM tspPYCNRYDYTRTYALELAYLGVDEKKIrelrqa-------qlsctvENKSKLKQLKDY ---KVCDVRRLRKCFPRTRFDAIITEPYM-------------------GKPLKYKPSRGE 269 169 264 380 276 410 316 382 524 372 293 370 380 390 400 410 ....*....|....*....|....*....|....*....|....*....|....*. NTTGFVAETW-----------QRRWIAVEINQDYVLGS--------------RYRF 299 GTTAAAAKEL-----------GRKFIGIEINEEYVKLA--------------KKRL 199 Note that All1290 matches the NTTGLVAERE-----------SRKWISFEMKPEYVAAS--------------AFRF 294 GTTGRVVREL-----------GGRFVGVDVNEHYMTDY--------------LVES 410most alignment in almost all of the GTTAVAALSE-----------SRNFIGIEKEPKYIQLS--------------NKNV 306 informative positions (in 452 red), GTTVHAVMELnae-----dkgNREFILVQIDEEIKEDEsay--------dfcKKEL GTTAQAVIEV-----------NRDYCLNWSFYLCQKEE--------------KIKN 346 those presumably related to SRTVDIEEELkkhpnfyelykNKKIDGIFTSPPYLGQIdyheqhayayelfdIPRL 437 common function. Click554 on ERMNWGASPG-----------ARKTIIGDSFSKMRLYR--------------LDQP YHSLFLESRYae--------iERLVTGNEVLNEINYAL--------------RKRW 405 Back to Annotation AIKIAKGLDR------------LYYQAFESFADVLKRGs-------------IIVF 323 More Back to Annotation 268 168 263 379 275 409 315 381 523 371 292 Main Menu Annotate Options Anabaena PCC 7120: Graphicall1290 Genome Alignment with homologs Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Alignment with motif Replicon: Chromosome Length = 200 amino acids Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis Since the motif does not extend to the beginning of the Neisseria protein, meningitidis it might be a good idea to check alignment with the full length of protein identified by the similarity search. Click on button then on View,More and finally Options on Alignment with homologs. History View:View graphic Show all Show hits: <1e-6 System Show neighbors: +/- 1 Show motifs Show alternate starts Show annotated features System Show predicted features Show transposon ends Show tandem repeats Show inverted repeats Show dispersed repeats Show private set Main Menu Annotate Options History Anabaena PCC 7120: all1290 NmeSIM (Neisseria) ScaIM (Streptomyces) All1290 (Anabaena) PvuIIM (Proteus) SptAIM (Salmonella) SpcM (Streptomyces) SpcG (Streptomyces) BglIIM (Bacillus) MJ1498 (Methanococcus) CfrBIM (Citrobacter) BamHIM (Bacillus) 3 6 1 6 1 1 1 5 1 66 68 ---TISSIMSLEN---------------FQLIKTQLGVPSYEND--RVAIYQGDCIQLMD FGYVIQSSAALWNRLSTFSQRGKALDTRLADIKKALGKPYYETS--DVLLYHGDSLELLK LNSQQTTTIKLTN-----------------------FIPSYTQQ--HGAVYLGDCLEIIK LQTMSSNDMLNFG-----------------------KKPAYTTS--NGSMYIGDSLELLE --------MMFDD-----------------------KKPAYKTD--LGAMYIADSLEMLE ----MTESTTAAG-----------------------DRRALGAE--HGSYHWGDSLALLE ----MTELT-PTE-----------------------DRCPLEAE--HGSYHWGNSLELMK QYKQIKLHLGMED---D-----------NEDLPNHIPSSFPKQH--LNKIYNGDTMNMLL ------------------------------------------ME--INKIYCMDCLEGMK VINSKTSTIDYKKSSKSPFLIKKAMEVVHTPKKVKKEKKIISKSPLNNMLLQGNCAETLK KILENKNSVKLNEFN-----------------SIQEIKPYFYTD--LGKLYNGDCLELFK NmeSIM (Neisseria) 43 KISKGSFQMTITSPPYNIGKEYES----ILDLEHYLEWCERWMQSIHHLTEDSGCFWLNL ScaIM (Streptomyces) 64 SMPQQIFDLTVTSPPYNIGKEYEG----VLSIEEYISWCETWMSRVHRATSAGGAFWLNV All1290 (Anabaena) 36 SIPDNSVNLILTSPPFALTRKKEYG---NESAEKYIEWFLPFADEFKRVLTENGSFILDL PvuIIM (Proteus) 41 SFPDESISLVMTSPPFALQRKKEYG---NLEQHEYVDWFLSFAKVVNKKLKPDGSFVVDF SptAIM (Salmonella) 28 SMPDNSLNLVMTSPPFALQRKKEYG---NHDQEQYIDWFLKFGELVFKKLKDDGSFVVDF SpcM (Streptomyces) 32 GVPDGSVRAVVCSPPFEGPQLIADE---DRAGACSSDWLMPFFGQFERVLRPDGCVAFEL SpcG (Streptomyces) 32 GIPDASVDAVVCSPPFEGDALISDE---GRRGDAFVAWIEPFFRQFQRVLRPAGCVAFEL BglIIM (Bacillus) 49 DIPDNSVDLVVTSPPYNINKFKND----RRPLEEYLKWQTEIIEQCHRVLKPSGSIFWQV MJ1498 (Methanococcus) 17 QLKDKTVDVVVTSPPYNIGIKYNKYSD-NLSREDYLNWIEEVVKEIKRVLKDDGSFFINV CfrBIM (Citrobacter) 126 KLPDESVNLVFTSPPYYNAKPEYS--E-YHTYDEYLSLLRSVIKECHRVLSEGRFFVINV BamHIM (Bacillus) 68 QVPDENVDTIFADPPFNLDKEYDEGVTDKNSFSGYLDWYYKWIDECIRVLKPGGSLFIYN To one knowledgeable on DNA methyltransferases, the identity of All1290 at positions 48-51 (SPPF) is particularly striking, as that is part of the AdoMet binding site and one of the signatures of N4-cytosine methyltransferases. Press Continue . Continue Main Menu Annotate Options History Anabaena PCC 7120: all1290 NmeSIM (Neisseria) ScaIM (Streptomyces) All1290 (Anabaena) PvuIIM (Proteus) SptAIM (Salmonella) SpcM (Streptomyces) SpcG (Streptomyces) BglIIM (Bacillus) MJ1498 (Methanococcus) CfrBIM (Citrobacter) BamHIM (Bacillus) 3 6 1 6 1 1 1 5 1 66 68 ---TISSIMSLEN---------------FQLIKTQLGVPSYEND--RVAIYQGDCIQLMD FGYVIQSSAALWNRLSTFSQRGKALDTRLADIKKALGKPYYETS--DVLLYHGDSLELLK LNSQQTTTIKLTN-----------------------FIPSYTQQ--HGAVYLGDCLEIIK LQTMSSNDMLNFG-----------------------KKPAYTTS--NGSMYIGDSLELLE --------MMFDD-----------------------KKPAYKTD--LGAMYIADSLEMLE ----MTESTTAAG-----------------------DRRALGAE--HGSYHWGDSLALLE ----MTELT-PTE-----------------------DRCPLEAE--HGSYHWGNSLELMK QYKQIKLHLGMED---D-----------NEDLPNHIPSSFPKQH--LNKIYNGDTMNMLL ------------------------------------------ME--INKIYCMDCLEGMK VINSKTSTIDYKKSSKSPFLIKKAMEVVHTPKKVKKEKKIISKSPLNNMLLQGNCAETLK KILENKNSVKLNEFN-----------------SIQEIKPYFYTD--LGKLYNGDCLELFK NmeSIM (Neisseria) 43 KISKGSFQMTITSPPYNIGKEYES----ILDLEHYLEWCERWMQSIHHLTEDSGCFWLNL ScaIM (Streptomyces) 64 SMPQQIFDLTVTSPPYNIGKEYEG----VLSIEEYISWCETWMSRVHRATSAGGAFWLNV All1290 (Anabaena) 36 SIPDNSVNLILTSPPFALTRKKEYG---NESAEKYIEWFLPFADEFKRVLTENGSFILDL PvuIIM (Proteus) 41 SFPDESISLVMTSPPFALQRKKEYG---NLEQHEYVDWFLSFAKVVNKKLKPDGSFVVDF SptAIM (Salmonella) 28 SMPDNSLNLVMTSPPFALQRKKEYG---NHDQEQYIDWFLKFGELVFKKLKDDGSFVVDF SpcM (Streptomyces) 32 GVPDGSVRAVVCSPPFEGPQLIADE---DRAGACSSDWLMPFFGQFERVLRPDGCVAFEL SpcG (Streptomyces) 32 GIPDASVDAVVCSPPFEGDALISDE---GRRGDAFVAWIEPFFRQFQRVLRPAGCVAFEL BglIIM (Bacillus) 49 DIPDNSVDLVVTSPPYNINKFKND----RRPLEEYLKWQTEIIEQCHRVLKPSGSIFWQV MJ1498 (Methanococcus) 17 QLKDKTVDVVVTSPPYNIGIKYNKYSD-NLSREDYLNWIEEVVKEIKRVLKDDGSFFINV CfrBIM (Citrobacter) 126 KLPDESVNLVFTSPPYYNAKPEYS--E-YHTYDEYLSLLRSVIKECHRVLSEGRFFVINV BamHIM (Bacillus) 68 QVPDENVDTIFADPPFNLDKEYDEGVTDKNSFSGYLDWYYKWIDECIRVLKPGGSLFIYN The substantial agreement between All1290, PvuIIM, and SptAIM turns out to extend through the DNA recognition domain. That’s good enough for me! Let’s start making this public by clicking Annotate . Main Menu Annotate Options History Anabaena PCC 7120: all1290 Coordinates Start site Replicon: Chromosome Gene name Coordinates: 1533026 (stop) <- 1533628 (start-GTG) Function Length = 200 amino acids Comment Internal note Strand: Complementary Remove orf Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis That click gets you back to the annotation Neisseria meningitidis page. Let’s begin by changing the start site. Click Start site.. More System System Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533628 1533628 (start-GTG) Length = 200 amino acids System Strand: Complementary Function: PvuII DNA methyltransferase System Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis You could type in the new Neisseria meningitidis coordinate, but it’s easier just to click on the new start site. More Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533931 1533628 (start-TTG) Length = 301 amino acids IC: Inferred by curator Use this when your assertion is based on your own judgement Function: PvuII DNA methyltransferaseof sequence similarity or other information, when there is no direct experimental data. Cyanobacterial orthologs: none Strand: Complementary Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis Every assertion must be accompanied by a Neisseria meningitidis justification. The codes are standard from the Gene Ontology Consortium and facilitate searching. More Mouse over to IC to find out what it’s used for. Justification Predicted IC: Inferred by curator IDA: Inferred by direct assay IEP: Inferred from expression pattern IGI: Inferred from genetic interaction Predicted IMP: Inferred from mutant phenotype IPI: Inferred from physical interaction TAS: Traceable author statement NAS: Nontraceable author statement ND: No biological data available Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533931 1533628 (start-TTG) Length = 301 amino acids Strand: Complementary Function: PvuII DNA methyltransferase Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis Inferred by curator seems to fit Neisseria the meningitidis situation. Click on it. More Justification Predicted IC: Inferred by curator IDA: Inferred by direct assay IEP: Inferred from expression pattern IGI: Inferred from genetic interaction Predicted IMP: Inferred from mutant phenotype IPI: Inferred from physical interaction TAS: Traceable author statement NAS: Nontraceable author statement ND: No biological data available Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533931 1533628 (start-TTG) Length = 301 amino acids Comment Human Strand: Complementary Function: PvuII DNA methyltransferase Predicted Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis You would now fill in the reasons Neisseria meningitidis leading you to your assertion regarding the start codon. For now, just press the Enter key. More Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533931 (start-TTG) Length = 301 amino acids Human Strand: Complementary Function: PvuII DNA methyltransferase System Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi Streptomyces spectabilis Streptomyces netropsis The start codon has been changed. In a like fashion, a name is Neisseria given tomeningitidis the orf, and functional annotation added. Now that the activity seems real, I’m inspired to look through the literature, More and... it turns out that Bancroft et al (1989) described in vivo protection of Anabaena DNA against PvuII! Press Continue . Continue A Main Menu Annotate Options History Anabaena PCC 7120: all1290 Replicon: Chromosome Coordinates: 1533026 (stop) <- 1533931 (start-TTG) Length = 301 amino acids Human A Human Human A Experiment Experiment A Strand: Complementary Gene name(s): avaXM Function: Putative type II DNA cytosine methyltransferase (CAGCTG-specific) Classification: Type II beta (N4) Activity: Protects against: PvuII In vivo activity: exists Cyanobacterial orthologs: none Anab7120 Proteus vulgaris Salmonella paratyphi That’s how the annotation ends up. Now anyone clicking on Human or Experiment will find the annotations you made, and Streptomyces spectabilis anyone mousing over one of the annotator icons ( ) willA get More to your name, and your e-mail address. End A A Scenario 3 Anotation Process Summary • Each assertion is accompanied by a justification linked to the annotator • Interface provides tools for trying out different start codons and assessing their validity • Interface provides different alignments for the assessment of orf function • In the end, human expertise is required to recognize the critical information There are no new tools here, just a comprehensive interface to put these tools at the service of human annotators who prefer to do science rather than click keys. The proposal seeks resources to support the development of this interface. To quit, right click, then click End Show.