Psi-BLAST Position-specific iterative BLAST Combines BLAST searching with PSSMs! 1- Start with regular BLAST search- look at the results 2-Pick the ones you believe are really homologous 3- Now align these sequences to the query sequence and make up a pssm that tells how much to weigh each amino acid in each position in the alignment 4-Use this pssm to do another blast search 5. Add any new sequences that come up to the old ones if you believe they are really homologous 6-Repeat the alignment to make a new and improved PSSM that tells how much to weigh each amino acid in each position in the alignment
Psi-BLAST Position-specific iterative BLAST Combines BLAST searching with PSSMs! 1- Start with regular BLAST search – look at the results 2- Pick the ones you believe are really homologous 3- Now align these sequences to the query sequence and make up a PSSM that tells how much to weigh each amino acid in each position in the alignment 4- Use this PSSM to do another BLAST search 5- Add any new sequences that come up to the old ones if you believe they are really homologous 6- Repeat the alignment to make a new and improved PSSM that tells how much to weigh each amino acid in each position in the alignment
s1502225LsbAX2155,1 AB39211 (Ar339021) polo-like prete 61·129 以4274鱼AB(A043097)p6101 ike kinas1 Healer,,.1·1 giL7510512IreINP 12102.Al IN))Y?1F9B 7 p ICarnorhabdi..392.-10B sil0436451s.U (Ar057165) putative serine/threonine p.. 223.-l08 op 40324392lrs ElNE-491779411 (N0M-066369)Protein kinase ICaenerh..2 gL4s57B1RPALnR CAKL Hypothetical 41.8 kDa protein C14... 287 t-107 y gilzz22s79re NP_22412 (N_o70455) pole I Drosophila melano.. 271+-102 3 g8415Z1nplE222LBIQ-DBOM PRonIN KINASE PoLo >gi|7293666. 279+-101 区c1922MMAD线1(Ar740)p1-1ikk1nAs,,24> g silgp2u1olshlA3919L. [021392) putative serine/threonine kina M suazne91rafINP-_9940.1 INM_04073) eytokine-indueible kinas.. 1 2e-85 区a1225吨M,MHo1) Unknown Iprotein f.,2132, n B giLl2879428LmlQ9ROLAICNK_RAT Cytokine- inducible senne/threonin., al 7.g4 5205xfNP1tm00622)srm- inducible kinas1…,206-自2 B siL9424IretIXP 941712.1 101041712) serus-indueibl, knas...294 14-82 区叫umls皿cn- INDOCIBLE SERINE/TOUC.,20.1 M gilm92912lre NR 14999,A1 INM 031821) seruminducible kinase.,2914e-8o a gLzulmlP3351LsNx NDUSE Serine/threonine-protein kinase s 9皇 区叫1902ML11An1tAn5042)pl- like kinase,,24-7 区叫n4LA8I3HA421taF4421, educable kt,7 M gLat999le blcMZ439Lll (Y139601 polo-like protein kinase ITr..26464-70 3 glzilzlra INP 29119,ll INM068795) protein kinase ICaenorh., 2994e-6e a silAz99451l SCHR Serine/threonine-protein kinase.. 229 le.59 叫31rf1t001451cDC5;d甲 sable for…,12241 3 sil33557921sbIA28424,L (Ar0530921 polo-like kinase isoform IR..159 4e.38 区m160 M916 AIADSZALI IAF3701ple-1;k如in4s…,.12e-3 区1 4184661eAIMXP7154HIAF1A4B424)rm-1 duc ibl….12-34 M silBs91i4rflxP-92292121 I 0029931) similar to eytokine-ind..145 4s-34 区毗1lrfx03t0039btht; al protein..55e-1
Psi-BLAST 7- Use this pssm to do another blast search 8-Keep iterating until no new sequences are found Very good for finding weakly related sequences on to Molecular phylogenetics
Psi-BLAST 7-- Use this PSSM to do another BLAST search 8– Keep iterating until no new sequences are found Very good for finding weakly related sequences …on to Molecular Phylogenetics
Gene structure AUG 5 Flank 3 Flank 目exon I intron Poly (A)addition splIcing 3'UTR 5 UTR AUG UGA AAAAAAA
1 2 3 4 5 6 7 Gene Structure 5’ Flank AUG 3’ Flank exon intron Poly(A) addition splicing 3’ UTR 5’ UTR AUG UGA…AAAAAAA
Mutation rates deleterious Mutations: - neutral advantageous +-substantial minority Consider 2 sequences KE of substitutions since they shared a common ancestor T= divergence time R=mutation rate=K/(2T) KEY PREMISE OF PHYLOGENETICS: If R=constant for all species, then K will provide insight into evolutionary relatedness for which no other physical evidence is available
Mutation Rates Mutations: deleterious neutral advantageous ←substantial minority Consider 2 sequences K= # of substitutions since they shared a common ancestor T= divergence time R = mutation rate = K/(2T) KEY PREMISE OF PHYLOGENETICS: If R=constant for all species, then K will provide insight into evolutionary relatedness for which no other physical evidence is available