Bioinformatics: an introduction ii

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Bioinformatics an introduction (ii)

• Nick Rhodes
• Room 323 (Lynch Laboratory)
• n.rhodes_at_sheffield.ac.uk

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Week 1

• Lecture
• I - definitions
• II - the central dogma - DNA, RNA, proteins
• III - data in biology molecular biology
• IV - (information management interoperation)
• Practical
• information retrieval
• display of macromolecules

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Week 2

• Lecture - Extracting meaning from sequences
• I comparing sequences
• II - alignment
• III content and features
• IV (content analysis in more detail) -
  subcellular location prediction
• Practical
• prediction servers
• working with multiple sequences

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I Comparisons

• The ability to compare sequences is fundamental
  to bioinformatics.
• by comparing an unknown protein or nucleic acid
  sequence against the databases, the unknown may
  be identified if a match is found
• if highly similar entries are identified,
  conclusions may be drawn about the identity and
  function of the unknown

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Definitions

• similarity
• sequences are in some sense similar, implies no
  evolutionary connotations
• homology
• evolutionarily related sequences stemming from a
  common ancestor

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Comparisons - two techniques

• sequence comparison
• merely detects common features
• sequence alignment
• places the residues or bases into an optimal
  one-to-one correspondence
• Implies selection of an appropriate scoring
  scheme which takes the form of a scoring matrix.

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Comparison criteria

• comparisons utilise certain criteria
• e.g.
• substitution of K for R is a conservative
  substitution (chemically similar)
• substitution of D for N not conservative in
  chemical terms but is in structural ones
• many sets (schemes) of criteria
• SCORE according to significance
• C v. C, W v. W score high
• C v. W scores low

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Comparison schemes

• chemical
• acidic (DE), aliphatic (AGILV), amide (NQ),
  aromatic (FWY), basic (RHK), hydroxyl (ST), imino
  (P), sulphur (CM)
• functional
• acidic, basic, hydrophobic, (AILMFPWV), polar
  (NCQGSTY)
• charge
• acidic, basic, neutral
• structural
• ambivalent (ACGPSTWY), external (RNDQEHK),
  internal (ILMFV)

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Willie Taylors classification
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The PAM 250 scoring matrix
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Similarity helps identify residues

• functional
• catalytic residues
• binding residues
• structural
• interactions may be essential for maintenance of
  3D structure
• formation of e.g. helixes sheets
• S-S bridges

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Similarity aids interpretation

• inferred relationships
• e.g. GPCRs to elucidate relationships between
  receptor families
• functional
• structure prediction
• (secondary and perhaps tertiary)
• interpretation of genome sequence data
  inferences concerning the proteome

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Similarity - phylogenetic analysis

• evolutionary inference
• illustrates relationships and divergence between
  sequences
• stages
• multiple alignment
• calculate similarity scores between each pair of
  sequences
• construct a dendrogram or evolutionary tree

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Looking for similarities

• Simplest method is DIAGON
• plot of every residues in one sequence
• against every residue in the other
• this gives a matrix
• plot a dot for an identity
• plotting two identical sequences should produce a
  diagonal line

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Diagon DotPlot
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Removing noise from DotPlots
Use a window, where for each diagonal the number
of hits must exceed a certain threshold (here 2,
2)
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Scoring identities
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II Aligning two sequences

• 2 sequences
• ABCDXEFGHI, ABCDEFGHI
• alignment gives
• ABCDXEFGHI
• 5 identities
• ABCDEFGHI
• but introduce a gap (and penalise...)
• ABCDXEFGHI
• 9 identities
• ABCD EFGHI

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Sample alignment

• mark identities
• indicate conservative substitutions
• gaps obvious
• remainder mismatches

EAGIGAVLKVLTTGLPALISWIKRKRQQG--GIGAVLKVLTTGLPALIS
WISRKKRQQ-
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Pairwise alignment

• Basic Local Alignment Search Tool
• BLAST
• finds high-scoring local alignments between
  target sequence and a database (both either NA or
  protein)
• used for searching databases for sequences
  similar to a query sequence

http//www.ncbi.nlm.nih.gov/BLAST
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BLAST comparisons

• blastp
• compares a protein query sequence against the
  protein sequence databases
• blastn
• compares a nucleotide query against the
  nucleotide sequence databases
• blastx
• compares the translation products of all six
  frames of a nucleotide query against the protein
  sequence databases

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TBLAST comparisons

• tblastn
• compares a protein query against a nucleotide
  sequence database dynamically translated in all
  six frames
• tblastx
• compares the six frame translations of a
  nucleotide query against a nucleotide sequence
  database dynamically translated in all six frames

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Multiple alignments

• CLUSTALW
• Higgins D., Thompson J., Gibson T., Nucleic Acids
  Res. 224673-4680 (1994).

CLUSTAL W (1.5) multiple sequence alignment
Bovine_Ins MALWTRLRPLLALLALWPPPPARAFVNQHLCGSHLVEAL
YLVCGERGFFYTPKARREVEG Human_Insu MALWMRLLPLLALLALW
GPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAED

. Bovine_Ins PQVGALELAGGPGAG-----GLE
GPPQKRGIVEQCCASVCSLYQLENYCN Human_Insu LQVGQVELGGG
PGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN
. ..

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III Sequence content proteins

• endoprotease and modification sites
• hydrophobicity
• secondary structure prediction
• e.g. Chou and Fasman, Garnier and Robson
• http//bmbsgi11.leeds.ac.uk/bmb5dp/gor.html
• transmembrane helix prediction server
• http//www.cbs.dtu.dk/services/TMHMM-1.0/
• secretory and transport signal peptides
• http//psort.nibb.ac.jp/
• phosphorylation, glycosylation etc.

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Structural predictions

• secondary structure (helices, sheets)
• simple reasonably well understood
• association of secondary structures
• hydrophobic/hydrophilic faces
• transmembrane domains
• tertiary structure
• given a very similar molecule

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Predicting hydrophobicity
Hydrophobic regions of the molecule tend to
associate with other hydrophobic regions.
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Prediction of helices (GOR)
helices in blue, sheet in yellow taking the last
helix... (next slide)
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Helical wheel plots
Can provide information about the association of
helices.
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Transmembrane helices
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Sequence content DNA RNA

• cognate sites within NA sequences
• endonuclease and modification sites
• coding regions
• promoter sequences
• terminator sequences
• local deviations in structure due to variations
  in sequence

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Restriction mapping
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Searching for ORFs

• in any given DNA sequence
• 3 reading frames for each strand
• identify start and stop codons
• identify promoter/terminator sequences?
• look for the longest?
• some genomes have overlapping genes
• exploit codon preferences

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IV Subcellular location

• closely correlated with protein function
• predict location, predict function?
• all proteins synthesised in cytoplasm
• Blobel, Milstein and Sabatini
• proposed "signal hypothesis
• secretory proteins marked by a signal peptide

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Sorting signals

• generally, the signals for any such "sorting"
  reside in the primary sequence and take two
  forms
• short and contiguous sequences of amino acid
  residues
• specific 3D structures formed by two or more
  non-contiguous sequences of amino acid residues

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Mammalian signal system

• well characterised
• proteins synthesised with N-terminal signal
  peptides 13-36 residues long
• consist of
• 7 to 13 residue hydrophobic core
• several relatively hydrophilic residues
• ? 1 basic residues close to the N-terminus

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Rule-based prediction methods

• "expert system" servers
• EMail access
• require knowledge of processing system
• rely on existence of leader sequences
• unreliability of automatically assigned 5-regions

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Statistical prediction methods (i)
based on amino acid (AA) composition

• Kou-Chen Chou and David W. Elrod, Protein
  Engineering 12 2, 107-118, 1999.
• different organelles have different
  physio-chemical environments
• protein surface is highly likely to correlate
  with AA composition
• structural class correlates with AA composition
• ? total AA composition may carry a signal that
  identifies the subcellular location

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Statistical prediction methods (ii)

• three stage refinement of the technique
• predict
• intracellular / extracellular
• extracellular / integral membrane / anchored
  membrane / intracellular / nuclear
• chloroplast / cytoplasm / cytoskeleton /
  endoplasmic reticulum / extracell / Golgi
  apparatus / lysosome / mitochondrion / nucleus /
  peroxisome / plasma membrane / vacuole

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The orexin story

• Sakurai, T. et al. Cell 92, 573-585, 1998.
• Background
• ESTs - Expressed Sequence Tags
• active sequences
• tissue specific
• low quality sequence

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Some ESTs were GPCRs

• GPCRs are
• largest class of targets of pharmaceutical
  intervention
• superfamily - structural and functional
  similarities but no sequence similarity
• Of the GPCRs identified
• some had known ligands
• unknown ligands - orphan GPCRs

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Supporting research

• molecular biology, tissue culture and analytical
  biochemistry
• Cell lines expressing range of orphan GPCRs
• Challenged with tissue extract fractions.
• protein chemistry
• identification of active component
• Orexin-A - 33 residues, N-terminal pyroglutamate,
  C-terminally amidated. Bovine is identical to
  rat.
• Orexin-B - 28 residues, C-terminally amidated

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Isolation of precursor polypeptide

• PCR based methods using highly degenerate primers
  yielded cDNA fragment encoding part of orexin-A
• extension by RACE methods to obtain the full
  length cDNA
• found to encode both orexin-A and orexin-B

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The role of orexins?

• 46 identity with each other
• BLAST search revealed
• no similarity with other peptides
• not contained in any other existing GenBank entry

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Characterisation of orexins (i)

• 5-most ATG preceded by an in-frame stop codon
• ORF 130 residues long
• residues 1-33 secretory signal sequence
• (hydrophobic core followed by small polar
  residues)
• SignalP server predicted cleavage site to be
  Ala32/Gln33
• Last residue of mature peptide is followed Gly66,
  serving as NH2 donor for C-terminal amidation
• Lys67-Arg68 - recognition site for prohormone
  convertases

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Characterisation of orexins (ii)

• Arg69-Met96 Orexin-B
• Gly-Gly-Arg, a C-terminal amidation signal
• orexin-A
• rat, mouse and human are identical
• orexin-B
• rat and mouse identical
• human has two substitutions
• mouse rat human prepro-orexins show 95
  identity
• human rat show 83 identity

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Characterisation of receptors (i)

• OX1R most similar to
• neuropeptide Y receptor
• TRH receptor
• cholesystokinin type-A receptor
• NK2 neurokinin receptor
• consistent with orexins being small regulatory
  peptides.
• Radioimmunoassays indicated that orexin-A is a
  specific, high-affinity agonist for OX1R
• Orexin-B had much lower affinity - another higher
  affinity receptor?

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Characterisation of receptors (ii)

• tBLASTn against dbEST revealed
• two highly similar entries which were later
  shown to be both derived from the same transcript
  coding for a GPCR designated OX2R
• AA identity between human OX1R and OX2R
• 64
• OX2R binds both orexins with high affinity
• Both receptors were mapped and the subjects of
  extensive immunohistochemical and physiological
  studies.

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GenBank accession numbers

• prepro-orexin cDNA
• human AF041240
• rat AF041241
• mouse AF041242
• OX1R cDNA
• human AF041243
• rat AF041244
• OX2R cDNA
• human AF041245
• rat AF041246

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References - books (i)

• Richard Durbin and Sean Eddy
• "Biological sequence analysis - probabilistic
  models of proteins and nucleic acids", Cambridge
  University Press, 1998.
• Teresa K. Attwood and David J. Parry-Smith
• Introduction to Bioinformatics, Addison Wesley
  Longman Higher Education, 1999.

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References - books (ii)

• Andreas Baxevanis and B. F. Francis Ouellette
• Bioinformatics A Practical Guide to the
  Analysis of Genes and Proteins, John Wiley
  Sons, 1998.
• Gunnar von Heijne
• Sequence analysis in molecular biology
  treasure trove or trivial pursuit, Academic
  Press, 1987.

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Conference proceedings

• H.A. Lim and C.R. Cantor
• Bioinformatics and Genome Research, World
  Scientific Publishing, 1995.
• R. Hofestadt, T. Lengauer, M. Loffler and D.
  Schomburg
• Bioinformatics, Springer-Verlag, 1997.

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Week (ii) exercises

• Use a browser to access the file below at the
  CISRG web site. The exercises are all web-based.

http//cisrg.shef.ac.uk/inf6140/week2.htm