The instructions for assembling every organism

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• The instructions for assembling every organism
• on the planet--slugs and sequoias, peacocks and
• parasites, whales and wasps--are all specified in
  DNA
• sequences that can be translated into digital
• information and stored in a computer for
  analysis. As
• a consequence of this revolution, biology in the
  21st
• century is rapidly becoming an information
  science...

...hypotheses will arise as often in silico as in
vitro.
Eric Lander, Science 287 (5459), 1777-1782
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Midterm IIIQuestion and Answer Sessions

• May 19th Q/A, 12 - 1, ES100,
• May 21st Q/A during class,
• the assigned paper for 5/21 is postponed,
• see WEB site for revised syllabus and sreading
  list,
• Midterm (May 5th - May 19th materials).

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(No Transcript)
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The Problem(s)
Big Problem Cardiovascular Disease 40.1 of
deaths in 1999 (USA)
Genomic Challenges

• recognizing genes in the genome,
• Exons/Introns,
• Alternative Splicing/Termination,
• Alternate transcription start/stop sites,
• Tandem Repeats, Psuedogenes, etc.
• We dont really understand all there is to know
  about gene structure,
• etc.

• determining the function of the gene,
• Transgenic mice,
• Knock-out mice.

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Cardiovascular Disease
Nature 407, pp. 234
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Lipidsmajor players in heart disease.

• Triglycerides,
• most common fat in the body, energy storage
  molecule,
• found in fat cells, blood plasma, and cell
  membranes,
• derived from eaten fats or made from other energy
  sources.

• Cholesterol,
• a steroid alcohol (sterol) used to insulate
  nerves, produce human sex hormones and as a
  component in cell membranes,
• produced by the body and supplied by diet.

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Apolipoproteinsand classes.
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Chromosome 11 (H.s.)

• Several apoproteins map to a region of chromosome
  11, including two APOA genes,
• There is a polymorphism that is linked with high
  serum triglyceride levels that maps to the same
  region.

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Hypertriglyceridemia Why bad?

• high levels have been linked with coronary
  artery disease,

• American Health Association Recommendation
• Changes in lifestyle habits are the main therapy
  for hypertriglyceridemia
• If you're overweight, cut down on calories to
  reach your ideal body weight.
• Reduce the saturated fat and cholesterol content
  of your diet. 
• Reduce your intake of alcohol considerably.
• Be physically active for at least 30 minutes on
  most days each week. 
• Substitute monounsaturated and polyunsaturated
  fats  
• Substitute fish high in omega-3 fatty acids
  instead of meats that are high in saturated fat.

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A Solutionto finding genes.

• Mouse Genome
• 2.75 Gb,
• 19 autosomes X and Y chromosomes,
• 99 per cent of the mouse genes have a sequence
  match in the human genome,
• 96 lie within 'syntenic' regions of mouse and
  human chromosomes.

Nature 420, 515 - 516
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The SolutionComparative Genomics
High homology, no annotation.
How did they know the region was expressed? - not
including the Northerns.
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New Gene Structure?
Mouse 1107 Open Reading Frame (ORF), 368
AA, Human similar structure, 366 AA,

• Most similar to each other (71 identity, 78
  similarity),
• Next most similar to APOAIV (27, 48)

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The Solutionto determining function (I, II).
movie

• I. Transgenic Mice
• - DNA is integrated in a random fashion
• by injecting it into the pronucleus of a
• fertilized ovum,

• II. Knock-Out Mice
• - DNA is introduced first into embryonic
• stem (ES) cells. ES cells that have
• undergone homologous recombination are
• identified and injected into a 4 day old mouse
• embryo - a blastocyst.

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Pronucleus either of the 2 haploid gamete nuclei
just prior to their fusion in the fertilized
ovum. Transgenic lines are often generated by
microinjection of the transgene into the
pronuclear region of these haploid gametes.
Transgenic Mice Pronuclear Injection
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Fig. 2
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APOAV Transgenic Miceoverexpressors

• n48 (transgenics), n46 (controls), P lt 0.0001

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Typical KO vectorhomologous recombination.
tkthymidine kinase gancyclovir lethal
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KO Mice w/ Embryonic Stem Cells
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Transformed ES cells are injected into blastocysts
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Chimeric mouse
Black/White Chimeric Example
Cross and look for offspring with germ-line
transfection.
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Ref. 25
Fig. 2
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APOAV KO Miceunderexpressors
n13 (wt), n22 (hets), n 10 (homozygous mut.)
P lt 0.001
Fig. 3
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Murine Models are Goodbut what about us?

• Identified 4 SNPs in and near the human APOAV
  locus (each occurs at gt8),

• Surveyed 500 Caucasians for numerous lipid
  parameters before and after high- and low-fat
  diets,

Fig. 1A
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1,1 homozygous for major allele
1,2 heterozygous for major allele

• Individuals heterozygous for the minor alleles at
  SNPs 1-3 had significantly higher triglyceride
  levels and VLDL mass.
• SNP4 does not show a significant difference in
  triglyceride levels or VLDL mass.

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Linkage disequilibrium When the observed
frequencies of haplotypes in a population does
not agree with haplotype frequencies predicted by
multiplying together the frequency of individual
genetic markers in each haplotype, English
Translation The co-occurrence of genetic markers
more often than would be expected.
SNP1, SNP2, SNP3 have significant
disequilibrium with each other while SNP4 is not
in linkage disequilibrium with any other
SNP, English Translation this genetic region
containing SNP1-3 segregates as a unit.
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Independent Association Study

• Data from an independent set of individuals
  demonstrating that subjects with minor alleles at
  SNP3 are significantly more likely to have high
  triglyceride levels than low.

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Summary

• SNP data shows that APOAV plays a significant
  role in plasma triglyceride and VLDL homeostasis.
• Polymorphisms of APOAV could be used as
  prognostic indicators for hypertriglyceridemia.
• Modulation of APOAV is a potential strategy for
  reducing risk of cardiovascular disease.

• Comparative Genomics uncovered a new gene in the
  human genome.
• Mouse transgenic and KO experiments suggest that
  the protein contributes to triglyceride and VLDL
  levels.

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APOAV Update
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Wednesdayone for Genomicists.

• Genome scale KO experiment.