7. Understanding a Genome Sequence

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7. Understanding a Genome Sequence
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Outline

• 7.1. Locating the Genes in a Genome
  Sequence
• 7.2. Determining the Functions of Individual
  Genes
• 7.3. Global Studies of Genome Activity
• 7.4. Comparative Genomics

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7.1. Locating the Genes in a Genome Sequence
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Methods of locating the genes

• Simply inspecting the sequence by eye.
• Inspecting by computer
• Bioinformatics

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Gene location by sequence inspection

• ORF scanning
• Initial codon ATG (usually)
• Termination codon TAA, TAG, TGA

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ORF scanning (1/3)

• A double-stranded DNA molecule has six reading
  frames

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ORF scanning (2/3)

• Length of gene
• E. Coli 317 codons
• S. cerevisiae 483 codons
• Human approximately 450 codons
• Simple ORF scanning is an effective way with
  bacterial

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ORF scanning (3/3)

• Real gene-red line, spurious ORF-blue line

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Simple ORF scanning

• Effective with bacterial (in most case)
• Real gene do not overlap
• No genes-within-genes
• LESS effective with higher eukaryotic DNA
• More space between the real genes
• Genes are often split by introns
• Many exon are shorter than 100 codons

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Higher eukaryotic DNA
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Three modifications to the basic procedure for
ORF scanning

• Codon bias
• Exon-intron boundaries
• Upstream regulatory sequences

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Codon bias

• All organisms have a bias
• Bias is different in different species
• In human genes, leucine is most frequently coded
  by CTG

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The genetic code
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Exon-intron boundaries

• Sequence of the upstream
• Sequence of the downstream

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Upstream regulatory sequences

• Locate the regions where genes begin
• Have distinctive sequences feature
• Variable
• Not all genes have the same collection of
  regulatory sequence

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Other strategy

• CpG island
• Vertebrate genomes contain CpG island upstream of
  many genes
• Some 40-50 of human genes are associated with
  an upstream CpG island

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Homology search

• Search the DNA database
• If the sequence is similar to any known genes
• Assign functions to newly discovery

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Experimental techniques of gene location

• Hybridization tests
• cDNA sequencing
• Exon-intron boundaries

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Northern hybridization
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Zoo-blotting
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cDNA capture

• Preparing cDNA

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RACE rapid amplification of cDNA ends
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Exon-intron boundaries

• Exon trapping

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7.2. Determining the Functions of Individual Genes
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We know rather less than we thought

• E. Coli
• 4288 protein-coding genes
• 1853 (43) previously identified
• S. cerevisiae
• 30 previously identified

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Homology reflects evolutionary relationships

• Orthologous
• Homologous genes located in the genomes of
  different organisms
• Paralogous
• Two or more homologous genes located in the same
  genome

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Amino acids or nucleotides
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Homologous domain

• Tudor domain (120-amino-acid motif)

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Homology analysis in the yeast genome project

• Yeast genome 6000 genes
• Identified by conventional genetic analysis 30
• Homology analysis 70

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Assign gene function by experimental analysis

• Gene inactivation
• Ultraviolet radiation
• Mutagenic chemical
• Mutants are present in a natural population

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Homologous recombination

• Inactivate individual gene by homologous
  recombination

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Example Yeast deletion cassette

• Disruption has occurred are identified
• Antibiotic-resistance gene is expressed

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Example Gene inactivation with mice

• Identifying the function of unknown human genes
• Use embryonic stem to make knockout mice
• Some gene inactivations are lethal

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Gene inactivation without homologous
recombination (1/2)

• Transposon tagging
• Most genomes contain transposable elements are
  inactive, but still few that retain their ability
  to transpose
• Difficult to target individual genes

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Gene inactivation without homologous
recombination (2/2)

• RNA interference
• Not disrupting gene itself, but its mRNA
• Effectively in the worm Caenorhabditis elegans
• Difficult applying to mammalian

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RNA interference (cont.)

• Fusion with liposomes can be used to deliver
  double-stranded RNA into a human cell

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Using gene overexpression to assess function

• Test gene is much more active than normal (gain
  of function)
• Vector multicopy (40-200 copies per cell)
• The vector must contain a highly active promoter

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Function analysis by gene overexpression
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Directed mutagenesis

• Probe gene function in detail
• Delete or alter the relevant part of the gene
  sequence
• Applications lie in the area of protein
  engineering

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Directed mutagenesis

• Knowing which cells have undergone homologous
  recombination
• Placing a marker gene

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Reporter genes

• Function of a gene can often be obtained by where
  and when gene is active
• What the reporter genes can do?

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Immunocytochemistry

• Searching for where the protein is located
• Labeled antibody
• Fluorescent labeling and colloidal gold

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7.3. Global Studies of Genome Activity
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Global Studies of Genome Activity

• Understanding how the genomes as a whole operates
  within the cell
• From genome itself, transcriptome and proteome

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Studying the transcriptome

• mRNAs that are present in a cell at a particular
  time
• Identify the mRNAs that is contains and determine
  their relative abundances

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Assay the composition of a transcriptome

• Convert its mRNA into cDNA, and then to sequence
  every clone in the resulting cDNA library
• Feasible but laborious
• SAGE (Serial analysis of gene expression)
• Study short sequence (12bp in length)
• Short but sufficient to enable the gene to be
  identified

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SAGE why 12-bp tags is enough?

• 412 16,777,216 bp
• Average size of eukaryotic mRNA is about 1500 bp
• 150011000 16,500,000

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Using chip and microarray technology

• Converting target transcriptomes mRNA into cDNA
• Chip - Immobilized oligonucleotides
• Microarray - cDNA

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Studying the proteome

• Proteome plays as the link between the genome and
  the biochemical capability of the cell
• Between transcriptome and proteome
• Not all mRNAs are actively translated at any
  particular time
• The protein content is variable

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Proteomics - methodology

• Protein electrophoresis
• Mass spectrometry

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Identifying proteins that interact with one
another

• An interaction with a second well-characterized
  protein can indicate something
• Two most useful method
• Phage display
• Yeast two-hybrid system

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Phage display

• Insert particular DNA for protein of phage coat
• More powerful strategy
• Prepare a phage display library

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Phage display
M13???????????phage display????,M13????????DNA ???
???,??DNA???????????????????
M13 filamentous phage ??????
pIII?pVIII phage display?????, ?????full?hybrid??,
??hybrid?????
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Phage display???????
1.?????????????phage????????????????phage???
2.??2???????phage?????????????????
3.?????????????ligand???,??low pH????,???eluted???

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Yeast two-hybrid system

• Activator
• Bind to a DNA sequence upstream
• Polymerase activation

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(No Transcript)
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Using homology analysis to deduce protein-protein
interactions

• 5 region of the yeast HIS2
• E. coli his2 and 3 region of the E. coli his10

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7.4. Comparative Genomics
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Comparative genomics as an aid to gene
mapping (1/3)

• Genomes of related organisms are similar.
• The closer two organisms are on the evolutionary
  scale, the more related their genomes will be.

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Comparative genomics as an aid to gene
mapping (2/3)

• The pufferfish genome is just 400Mb, but
  containing approximately the same number of gene
  with human.
• It should be possible to use the pufferfish map
  to find human homologs of pufferfish genes.

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Comparative genomics as an aid to gene
mapping (3/3)

• For example
• Wheat genome 16000Mb
• Rice genome 430Mb

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Comparative genomes in the study of human disease
genes

• Gain access to the sequences of genes involved in
  human disease.
• Discovery of a homolog of a human disease in a
  second organism.
• Find the biochemical role of the human gene from
  the homolog that have already been characterized.

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Example of human disease genes that have homologs
in Saccharomyces cerevisiae