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

• Complexities of food components
• Mode of nutrient gene interactions
• Molecular Tools and Techniques

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Diet essentials

• Amino acids,
• Fatty acids
• Water
• Vitamins
• Minerals
• ?

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Examples of known nutrient effects on gene
expression
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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?

Can molecular approaches answer these questions?
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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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Dietary Polyphenols

• Fruit, vegetables, beverages and grains are rich
  in polyphenolic compounds

• Some epidemiological studies show an inverse
  relationship to dietary intake of polyphenols and
  the incidence of chronic diseases

• Powerful antioxidants and scavenge reactive
  oxygen and nitrogen in vitro

• Their antioxidant ability in vitro may
  contribute to health effects and this is
  currently being investigated

Humans may ingest up to 280 540mg of these
compounds per day
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Metabolites formed by
Tissue and cellular metabolism Microbial action
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The modulation of gene expression by either the
parent compound or the metabolites may be

• Tissue/cell specific

• Dependent on gene polymorphisms

• Dependent on other dietary components

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Modulation of gene activity may be regulated by
nutrients at several levels

• Transcription promoters, RNA processing,
  transport and stability control

• Translation translation of RNA into protein

• Post-translational control

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Post translational modifications proteolytic
cleavage, glycosylation phosphorylation
oligomerisation, sulfation, methylation, acylation
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Genomics and proteomics can be defined as the
qualitative and quantitative comparison of
genomes and proteomes under different conditions
to further unravel biological processes.
Molecular Tools and Techniques
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Proteomics study of proteins translated from
messenger RNA and modification of these proteins
within cells and tissues.  
Proteomics techniques used by nutritionists to

• determine outcome of nutrientgene interactions
  by analysing the modulation of protein profiles
  in a cell or tissue

• identify modulation of protein profiles and
  protein modifications in response to nutrients

• predict influence of nutrient on cell metabolism
  or gene regulation

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Genomics - studies at the gene level
Genomics techniques used by nutrionists 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  

• Proteomics
•  
• 2-D gel electrophoresis
• Mass spectrometry

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Molecular Tools and Techniques
Functional Genomics

•   RNA expression profiling
• Northern blots
• RT-PCR (Reverse Transcription-Polymerase Chain
  Reaction)
• Microarray analysis
• Gene Trapping
• SAGE (Serial Analysis of Gene Expression)

•   Microscopy In-situ Hybridisation (ISH)
• Fluorescence In-situ
  Hybridisation (FISH)

•   Cell culture Transfection studies, reporter
  assays

• Transgenic animals

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

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PROTEOMICS

• 2-D protein gel electrophoresis
• 1st dimension separation according to charges
• 2nd dimension separation according to mass

• Use of proteomics software to identify
  differences in protein levels between groups
• Cutting out of protein spots from the gel
• Determination of the molecular mass of the
  individual peptides by MALDI-TOF mass
  spectrometry after tryptic digestion of the
  eluted protein
• Protein identification can be obtained by
  searching the specific peptide profile of each
  protein against protein databases. The
  corresponding gene can be obtained from public
  DNA databases

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Protein spot detection
Isoelectric point (pI)
Mr
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Matching of the protein spots
Treatment A (reference gel)
Treatment B
Matched protein spots
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Quantitative analysis
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Advantages Separation of a large number of
proteins Quantitative of protein expression
levels Examination of global protein changes
under specific conditions
Disadvantages High (gt100 kDa) and low (lt10 - 15
kDa) molecular weight proteins Proteins with
extremes of pI Low abundance or
membrane-associated/bound proteins
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GENOMICS TECHNIQUES
Northern Blotting

• Facilitates measurement of differential gene
  expression
• Procedure
• Isolate RNA from cells/tissues
• Electrophorese
• Transfer to nylon membranes
• Hybridise with labelled cDNA (probe)
• to the gene of interest
• Detection/quantitation of labelled probe
•  

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RT-PCR (Reverse Transcription-Polymerase Chain
Reaction)

• Facilitates measurement of differential gene
  expression
• More sensitve than Northern blots
• 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 cDNA
• PCR performed on the cDNA with gene specific
  primers
• Products can be analysed using Southern
  blotting/fluorescent techniques
•  

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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
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Up-regulated genes in vitamin E depleted gut
mucosa gt2 fold
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Down-regulated genes in vitamin E depleted gut
mucosa gt2 fold
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Serial Analysis of Gene Expression (SAGE)

• Analysis of gene expression patterns
• Differential levels of gene expression
•  

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Serial Analysis of Gene Expression (SAGE)

•   A short sequence tag (10-14bp) contains
  sufficient information to uniquely identify a
  transcript provided that that the tag is obtained
  from a unique position within each transcript 

• Sequence tags can be linked together to from
  long serial molecules that can be cloned and
  sequenced and
•  

• Quantitation of the number of times a particular
  tag is observed provides the expression level of
  the corresponding transcript.
•  

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Short sequence tags identify gene
Ligate tags to form concatamer
Sequence tags
Software used to identify gene and quantify
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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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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
Frizzled
Cell membrane

-
Proteosome
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Transgenic animals

• What is transgenics? 
• Animals with genetic alterations in their DNA
  that is heritable

• Why transgenics?
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• Study gene function in vivo

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Transgenic animals

• Isolate embryonic stem cells from blastocyst
  donor.
• Genetically transform embryonic stem cells by
  insertion/rearrangment of chromosomal DNA
• Select genetically transformed embryonic stem
  cells
• Inject the transformed stem cells into blastocyst
• Transfer to uterus of surrogate dam
• Chimeric pups carry ES genome in some of their
  cells
• Mate chimeric mice
• Some of these pups carry a haploid genome, half
  of these are transgenic

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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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