Molecular Characterization of the Canine HMGB1

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Molecular Characterization of the Canine HMGB1

• Presented by Melissa Metcalfe and Shauna Maguire

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Canine Genome

• An overview

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• First Sequenced by Celera with the DNA from
  Shadow, a male Standard Poodle, in 2002. (WGS 80
  coverage rough draft)
• Next major sequencing, done by the Broad
  Institute, was of Tasha, a 7 year old female
  boxer. (95 coverage)

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Genome Information

• Genome Size 2.8x109 bp
• Gene sequences ESTs 900
• BAC end sequences 668
• Sequence Tag sites (STS) 106
• 2n Chromosome number 78
• 38 autosomes sex chromosomes

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Why sequence the canine genome?

• Human genome and dog genome are evolutionarily
  and physiologically similar.
• The canine genome could help us understand
  analogous human hereditary diseases. Half of dog
  hereditary diseases have a human analog.
• Canines have been selectively bred for many years
  and their pedigrees have been kept. They are
  also have high levels of inbreeding. These
  factors make them ideal for genetic studies.
• Improvement of canine therapeutic treatments and
  purebred bloodline maintenance.

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HMGB1

• High mobility group box protein 1
• Originally identified as a transcriptional
  regulatory molecule that can modify chromatin
  structure by bending DNA.
• Recently, it has been attracting considerable
  interest by oncologists because it has been said
  to have a double life.
• What does it mean a double life?

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HMGB1

• Besides its function as a transcriptional
  activator, HMGB1 has been found outside of the
  nucleus by secretion from macrophages.
• As a extracellular protein, HMGB1 participates in
  developmental and differentiation processes,
  triggers and modulates many of the inflammatory
  cascades in the body, and may be involved in the
  metastic invasion programme of cancer cells.
• Specifically, HMGB1 is a ligand for the receptor
  for advanced glycation end products (RAGE) thus
  activating p38mapk ,JNK, and p42/p44mapk which
  are key signaling pathways.
• This signal via a receptor induces inflammatory
  responses.

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Sepsis

• Is the bodys systemic inflammatory response to
  infection or trauma.
• Can also be a caused by an infecting agent (such
  as bacteria).
• The body can mount a widespread inflammatory
  response to an infection that can quickly become
  out of control and cause even more harm.
• Can cause shock, organ damage, permanent
  disability, or death.

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Figure 1. Structure of the genomic elements and
cDNA of the canine HMGB1.
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Table 1. Detailed analysis of the canine HMGB1
cDNA and genomic elements.
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Fig. 3 Comparison of Canine, Human, Mouse, and
Bovine HMGB1 Protein
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Northern Blot Analysis
Performed to define a basic expression pattern of
the protein in canine heart, lung, muscle,
kidney, and spleen tissue.
Lane 1 Kidney Lane 2 Spleen Lane 3
Spleen Lane 4 Heart Lane 5 Heart Lane 6
lung Lane 7 Muscle Lane 8 - Fibroblasts
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Fluorescent In Situ Hybridization

• Makes it possible to map any cloned locus to its
  position on the metaphase chromosome.
• Good for quickly obtaining the chromosomal
  position of a newly cloned locus. Does not
  require mapping to be in relation to another.

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FISH protocol

• Drop cells onto glass slide
• Gently denature DNA with DNase
• Hybridize with fluorescent probe and wash away
  unhybridized probe
• Expose to UV light and take a picture of the
  fluorescent chromosome with a Fluorescence
  microscope.

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Metaphase spread after FISH with signals on both
chromosomes (a) and after GTG-banding (b).
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Conclusion

• The molecular characterization of the canine
  HMGB1 gene and protein showed that humans and
  dogs share an identical HMGB1 protein.
• Molecular targeting of HMGB1 in dogs is very
  significant for therapeutic approaches in humans
  due to the similarity of genesis and development
  of diseases in both species.
• Thus, the canine shows great potential as a model
  organism in the study of human diseases.