Molecular Approaches to Nutrition

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Molecular Approaches to Nutrition

• Nutrient Gene Interactions
• Molecular Tools and Techniques
• Dr. Janice Drew

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Nutrient Gene Interactions

• Why do nutritionists need to study nutrient gene
  interactions?

• How can molecular approaches be used to
  investigate gene modulation by dietary components?

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Nutrient Gene Interactions

• Necessity for molecular approaches
• Mode of nutrient gene interactions
• Molecular Tools and Techniques

Hot topic in nutrition research
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Diet/lifestyle related diseases
Obesity Cardiovascular disease High blood
pressure Metabolic syndrome Diabetes Inflammatory
bowel syndromes Cancer
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30 to 40 of all cancers are directly linked to
the foods we eat, the exercise we get and how
well we watch our weight. (source World
Cancer Research Fund)
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Epidemiological studies and dietary surveys may
be helpful in highlighting nutrition problems but
we are left with some basic unanswered questions.
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Unanswered basic questions

• What compounds in diet increase risk or confer
  health protection?
• What are the mechanisms by which they increase
  risk or confer protection?
• Inter-individual variation?

Can molecular approaches answer these questions?
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Examples of known nutrient effects on gene
expression
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Diet and gene interactions??

• Dietary complexity

• Inter-individual variation

• Human studies vs in vitro/vivo model systems

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Diet and gene interactions

• Calorie consumption over consumption vs
  calorie restriction

• Glucose metabolism - refined sugar and
  wheatflour products

• Fibre

• Red meat

• Omega36 ratio

• Fruit and vegetable consumption

• Micronutrients selenium, vitamins, folate

• Antioxidants

• Dietary phenolic compounds

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Metabolites formed by
Tissue and cellular metabolism Microbial action
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Modulation of gene expression

• Parent compound vs Metabolites

• Tissue/cell specific

• Dependent on gene polymorphisms

• Dependent on other dietary components

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Mode of nutrient regulation of gene activity?

• Transcription promoters, RNA processing,
  transport and stability control

• Translation translation of RNA into protein

• Post-translational control

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Molecular biological approaches to investigate
dietary modulation of gene activity?Nutrigenomic
s- new era- multidisciplinary- a systems
approach- inter-individual variation
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Genomics, proteomics and metabolomics can be
defined as the qualitative and quantitative
comparison of genomes, proteomes and metabolomes
under different conditions to further unravel
biological processes.
Molecular Tools and Techniques
Bioinformatics to assist interrogation of complex
data sets generated.
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Genomics - studies at the gene level
Genomics techniques used by nutritionists to

• identify nutrientgene interaction

• identify gene function

• predict influence of nutrient on gene regulation
  and cellular metabolism

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Molecular Tools and Techniques
Functional Genomics

•   RNA expression profiling
• RT-PCR (Reverse Transcription-Polymerase Chain
  Reaction)
• Real-time PCR/QPCR
• Microarray analysis

•   Microscopy In-situ Hybridisation (ISH)

•   Cell culture Transfection studies, reporter
  assays

• Transgenic animals

• Genotyping - Restriction Fragment Length
  Polymorphisms RFLP
• Single Nucleotide Polymorphisms SNP
  analysis

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RT-PCR (Reverse Transcription-Polymerase Chain
Reaction)

• Facilitates measurement of differential gene
  expression
• Measurement of low abundance mRNA transcripts
• Measurement of mRNA in very small tissue samples
• Can be used for accurate quantitation of gene
  expression (QPCR, Real-time PCR)

• Procedure 
• Isolate RNA from cells/tissues
• RNA used as template for synthesis of
  complementary DNA (cDNA)
• PCR performed on the cDNA with gene specific
  primers
• Products can be analysed using Southern
  blotting/fluorescent techniques to assess
  amplification levels. Sequencing.
•  

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Real-Time PCR
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Microarray
Microarray is a technique used to monitor and
compare expression levels of multiple genes
simultaneously (100s 10,000s)  

• Procedure 
• Messenger RNA is isolated from cells/tissue
  samples being investigated
• The messenger RNA is copied into complementary
  cDNA that is radio/fluor labelled
• The labelled cDNA is hybridised to nylon/plastic
  membranes or glass slides printed with cDNAs or
  short oligo sequences representing specific genes
• The level of signal hybridising to the printed
  cDNA is compared between reference and treated
  samples to determine up/down regulation of gene
  expression
•  

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Vitamin E deficient rat model
plus E
minus E
minus E
plus E
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Genes regulated by vitamin E depletion in rat gut
mucosa gt2 fold
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In situ hybridisation (ISH)
In situ hybridisation (ISH) is the detection of a
target DNA or RNA sequence in a tissue section
using a labelled nucleic acid probe. It allows
cellular and subcellular localisation of the
target.
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In situ hybridisation rat colon
Proximal
Transverse
Distal
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In vitro - Reporter systems

• Reporter systems allow study of eukaryotic gene
  expression and regulation.

• A reporter gene is joined to a promotor sequence
  in an expression vector that is transfected into
  cells.

• Following transfection the cells are assayed for
  the presence of the reporter.

Reporter genes   Chloramphenicol
acetyltransferase (CAT) ß-galactosidase, (ß
-gal) firelfly luciferase (LUC) growth hormone
(GH), ß -glucuronidase (GUS) alkaline
phosphatase (AP) green fluorescent protein
(GFP) aequorin
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Luciferase reporter assays
Dietary component
Frizzled
Cell membrane

-
Proteosome
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In vivo - Transgenic animals

• What is transgenics? 
• Animals with genetically altered DNA that is
  heritable

• Why transgenics?
•  
• Study gene function in vivo

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In vivo imaging of NF-kB activity(Carlsen et al
2002)

• Combined transgenic/ reporter gene strategy
• NF-kB responsive gene transcription
• Inflammatory responses
• Human disorders

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GenotypingGenotyping includes a variety of
techniques that are used to identify the primary
localization and mapping of genes implicated in
human diseases.

• Polymorphisms (different forms of a gene) may be
  present in coding and non-coding regions of a
  gene.

• Polymorphisms may influence gene regulation in
  response to nutrients

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Primer Extension Theory
SNP Analysis - primer extension theory
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SNP Analysis
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