Lecture #4 : Comparing genes

1. Lecture #4 : Comparing genes 9/14/09

2. This week • Homework #2 due on Wed • Email with questions • Email me answers or hand in in class • Wed - I will be at Dept of Biology retreat • Lecture will be given by Kelly O’Quin - expert in phylogenetics • He will go over homework so it must be done before class

3. Questions for today 0. More BLAST • Where do we get high quality gene sequences? • How do genes evolve? • How do we compare genes?

4. How to find genes • Start with genes which are known from model organisms • Use these to pull out genes from genomes • Compare genes to learn about sensory evolution

5. Blast - Genbank • What database do you want to search? • What do you want to compare? • What program do you want to do the searching?

6. Types of blast queries

8. Defaults Database Program Confirm

9. Nucleotide BLAST = DNA nucleotide query vs nucleotide database

10. Choices for programs • Megablast Highly similar sequences >95% • Word length 28 • Discontiguous megablast • Pretty similar seqs • Word length 11 • Blastn Dissimilar seqs • Word length 11

11. Translated blast = protein query vs translated database

12. BLAST a genome Request ID AWJ4D4B7012

17. BLASTing is fun • This is meant to be enjoyable • Be a genome explorer • Find out what kind of data is out there • Find out what kind of data isn’t there • QUESTIONS?????

18. Q1. • There is so much data in Genbank. How do you find GOOD data? • Example • Bovine rhodopsin - 1st G protein coupled receptor to be sequenced • Search Genbank with text • 49 entries

19. Bovine opsin

20. Bovine rhodopsin

21. Searching for genes • Searching by text is fraught with peril • Genbank has too many links • Pull up many things that are not what you want • BLAST is better approach • NCBI has also made records which combine all similar sequences into one

24. NCBI has done some of the work • They have hand-curated data for some species to make a set of reference sequences • Nucleotide sequences - NMxxxxxxx • Protein sequences - NPxxxxxx • For human rhodopsin • NM000539 • NP000530 • These are the gold standard for sequences

25. Homologene

26. Homologs • Two genes which arise in the common ancestor of two organisms and are passed down • Implies genes perform same function in two organisms • Therefore they can be compared to learn about evolution

27. These 4 primates have many genes which are homologs and have been passed down from primate ancestor Human Chimp Macaque Bushbaby

28. Homologene search for rhodopsin

29. Homologene

30. Three primary sequence portals: 1. NCBI

31. 3. DNA database of Japan

32. 2. Ensembl - European Bioinformatics Institute (EBI)

33. Select just genes

34. Scroll down to find the gene you want

35. Location Orthologues are predicted and linked Links to transcript and protein

37. OMIM - Online mendelian inheritance in man

38. Good places to find genes • Model organisms: NCBI homologene • Genes from models and other organisms: Sanger Ensembl gene families • NOTE: These are often predicted from genome sequences • If there is a sequence in NCBI homologene, it may be different (and more accurate) than Sanger predictions • OMIM is a good reference

39. Q2. How do genes change through time? • Change in actual sequence • Mutation • Recombination • Change in frequency of a sequence • Selection - “survive” better • Drift - get passed on by chance • Migration - move between populations

40. Mutation vs selection • Mutation = sequence change • ATGCCGTGACGT • ATGCCTTGACGT • Selection/drift/migration = sequence frequency changes across a number of individuals • ATGTG ATGTG ATGTG ATGTG ATGTG ATGTG • ATGTG ATGTG ATGTG ATGTG ATGTG ATGTT • ATGTG ATGTG ATGTG  ATGTT ATGTT ATGTT • ATGTG ATGTG ATGTG ATGTT ATGTT ATGTT • ATGTT ATGTG ATGTG ATGTT ATGTT ATGTT

41. Evolution as tinkerer • Changes are typically small • Mutation is source of new sequence • Not all mutations are created equal • Some occur more often than others • Other forces shift frequency of particular sequence

42. Triplet amino acid code

43. Mutation causes nucleotide change • What about AA sequence? • Synonymous change • Syn = same • AA stays same • Nonsynonymous change • Not same • AA changes

44. Amino acid code

45. Amino acid (AA) types • Non-polar A, F, G, I, L, M, P, V, W • Polar N, Q, S, T, Y • Charged, + H, K, R • Charged, - D, E • Other C Often changing AA within a group does not affect protein function

46. Selection • Stabilizing selection - Acts to keep protein function the same • Synonymous change more frequent than nonsynonymous • Amino acid changes occur within group much more common than between • Non polar  nonpolar • Polar  polar

47. Similarity matrix A = alanine C = cysteine D = aspartic acid E = glutamic acid F = phenylalanine G = glycine H = histidine

48. Comparing sequences • Can do at either nucleotide or AA level • Gather sequences from a bunch of different organisms • Need to align them so that sites which perform the same function can be compared

49. Aligning sequences • Sequences may differ in length • Often have differences at amino- or carboxy- terminus of the protein • Need a way to align parts of protein that are performing the same function

50. Example - RH2 opsin in fishes Goldfish MNGTEGNNFYVPLSNR Medaka MENGTEGKNFYIPMNNR Zebrafish MNGTEGSNFYIPMSNR Killifish MGYGPNGTEGNNFYIPMSNK Trout MQNGTEGSNFYIPMSNR Halibut MVWDGGIEPNGTEGKNFYIPMSNR Cod MRMEANGTEGKNFYIPMSNR Tetraodon MVWDGGIEPNGTEGKNFYIPMSNR