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Janice Drew Molecular Nutrition Nutrition and Cell Signalling Laboratory

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Dietary modulation of genes regulating cell signalling ... Influence of diet:gene interactions on the aetiology of colorectal cancer ... – PowerPoint PPT presentation

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Title: Janice Drew Molecular Nutrition Nutrition and Cell Signalling Laboratory


1
Janice DrewMolecular Nutrition - Nutrition and
Cell Signalling Laboratory
  • Dietary modulation of genes regulating cell
    signalling
  • Investigation of dietgene interactions in vitro
    and in vivo
  • Influence of dietgene interactions on the
    aetiology of colorectal cancer

(http//www.rowett.ac.uk/divisions/ghp/j_drew.html
)
2
Molecular Approaches to Nutrition
  • Molecular Biology 1
  • DNA Structure and Organisation Dr. Janice Drew

(http//www.rowett.ac.uk/divisions/ghp/j_drew.html
)
3
DNA/RNA Structure and Organisation
  • Relevance to nutrition ?
  • DNA/RNA structure and organisation
  • Genetic code
  • DNA replication
  • Genetic mutations
  • DNA Damage and Repair
  • Gene expression and regulation

4
Relevance to nutrition ?
  • Fundamental to all biology!
  • Power of molecular biology procedures as research
    tools
  • Action of mutagens/carcinogens in food
  • Nutrient excess/deficiency
  • Individual variation
  • Development

Genetic modification of food plants/bacteria
5
DNA / RNA structure and organisation
DNA
( Blueprint ) ( Plans ) ( Engines )
RNA
PROTEIN
6
DNA / RNA /Protein
  • Gene, cistron, allele, locus
  • 2 copies per cell
  • Encode proteins via RNA intermediate
  • Encoded proteins control cellular functions and
    maintenance including gene regulation

7
DNA / RNA /Protein
GENOME
TRANSCRIPTOME
PROTEOME
8
DNA is a double helix
gene x
gene y
chromosome
DNA helix
9
DNA strands are base-paired together
Sugar-phosphate backbone
Sugar-phosphate backbone
Base pairing (bp)
10
DNA contains deoxyribonucleotides
Adenine Thymine Cytosine Guanine
1
4
2
11
RNA contains ribonucleotides
Adenine Uracil Cytosine Guanine
12
Types of RNA
Messenger RNA (mRNA) synthesized from a gene
segment of DNA protein coding. The mRNA carries
the code into the cytoplasm where protein
synthesis occurs
Ribosomal RNA (rRNA) combines with protein to
form a nucleoprotein called a ribosome. The
ribosome serves as the site and carries the
enzymes necessary for protein synthesis.
Transfer RNA (tRNA) short RNA (75 nucleotides).
The tRNA reads the code and carries the amino
acid to be incorporated into the developing
protein.
13
Types of RNA
Small interfering RNAs (siRNAs) target the
degradation of specific messenger RNAs (mRNAs),
resulting in gene knockdown at both the mRNA and
the protein level.
Micro RNAs (miRNAs) related to small interfering
RNAs (siRNAs), non-coding, small RNAs (21-25 nt),
may play a role in tissue specific regulation of
mRNA transcripts.
14
Genetic code
15
Genetic code
  • 64 triplets of nucleotides called codons
  • each codon encodes for one of the 20 amino acids
    used in the synthesis of proteins.
  • redundancy in the code most of the amino acids
    being encoded by more than one codon.

16
Exceptions to the code
Three exceptions to codon rule.
  • AUG (ATG) serves two related functions
  • it signals the start of translation
  • it codes for the incorporation of the amino acid
    methionine (Met) into the growing polypeptide
    chain
  • UAA (TAA), UAG (TAG), UGA (TGA) do not encode
    amino acids
  • signal the end of translation
  • Mitochondrial DNA
  • UGA (TGA) encodes tryptophan
  • AUA (ATA) for methionine not isoleucine

17
Amino Acids
Alanine ala a CH3-CH(NH2)-COOH Argin
ine arg r HNC(NH2)-NH-(CH2)3-CH(NH2)-CO
OH Asparagine asn n
H2N-CO-CH2-CH(NH2)-COOH Aspartic acid asp d
HOOC-CH2-CH(NH2)-COOH Cysteine cys c
HS-CH2-CH(NH2)-COOH Glutamine gln q
H2N-CO-(CH2)2-CH(NH2)-COOH Glutamic acid glu
e HOOC-(CH2)2-CH(NH2)-COOH Glycine
gly g NH2-CH2-COOH Histidine his h
NH-CHN-CHC-CH2-CH(NH2)-COOH Isoleucine ile
i CH3-CH2-CH(CH3)-CH(NH2)-COOH Leucine
leu l (CH3)2-CH-CH2-CH(NH2)-COOH Lysine
lys k H2N-(CH2)4-CH(NH2)-COOH Methionin
e met m CH3-S-(CH2)2-CH(NH2)-COOH Pheny
lalanine phe f Ph-CH2-CH(NH2)-COOH Proline
pro p NH-(CH2)3-CH-COOH Serine
ser s HO-CH2-CH(NH2)-COOH Threonine thr
t CH3-CH(OH)-CH(NH2)-COOH Tryptophan
trp w Ph-NH-CHC-CH2-CH(NH2)-COOH Tyrosine
tyr y HO-p-Ph-CH2-CH(NH2)-COOH Valin
e val v (CH3)2-CH-CH(NH2)-COOH
18
Nonstandard Amino Acid
  • selenocysteine. This amino acid is encoded by
    UGA. UGA is still used as a chain terminator, but
    the translation machinery is able to discriminate
    when a UGA codon should be used for
    selenocysteine rather than STOP.

Humans synthesize 25 different proteins
containing selenium. Se from dietary sources is
essential to supply the demand for selenocysteine
for protein synthesis.
19
Nucleic acid strand growth occurs in one
direction only 5' ? 3'
P
P
P
BASE
CH2
5
O
3
H
O
20
Polymerase enzymes copy DNA and RNA
DNA
DNA polymerase (Reverse Transcriptase)
DNA polymerase
DNA
RNA
RNA polymerase
21
Polymerases require a primer to make new
nucleic acids
A primer is a piece of single stranded starter
template DNA/RNA
A primer provides a free 3-OH group onto which
a new strand (DNA or RNA) is synthesised.
22
Polymerases require a template to work
A template consists of single stranded DNA or
RNA
A template serves as the master copy onto
which a new strand (DNA or RNA) is copied.
23
DNA-dependant DNA polymerase
ssDNA
molecular biology uses PCR DNA
Sequencing
24
RNA-dependant DNA polymerase
RNA
Reverse Transcription (RNA viruses) e.g. Reverse
Transcriptase
DNA
molecular biology uses making cDNA
libraries cDNA for quantitative gene
expression
25
DNA polymerases make mistakes
POINT MUTATION / SINGLE NUCLEOTIDE POLYMORPHISM
(SNP)
A G T T C A G
A G T T G A G
serine
Stop codon
e.g. Obesity
Different individuals have different SNPs and
these can be detected allowing identification of
an individual and to explain individual responses
to nutrition.
26
Single-nucleotide polymorphisms (SNPs)
  • 93 of all known genes (most genes evaluated so
    far 2 coding-region SNPs, Sachidanandam et al.,
    2001 Marsh et al., 2002)
  • initial SNP map from Human Genome Project
    discovered 1.42 million variants
  • public databases, which are growing rapidly,
    over 5 million human SNPs
  • research on the impact of common genetic
    variants on nutrient responses will constitute a
    considerable research effort over the next years

27
DNA Damage
  • Chemical/dietary mutagens
  • Food additives
  • Pesticides
  • Environmental chemicals
  • Micronutrient depletion
  • Excess micro/macronutrients

28
DNA Damage
  • Deamination (spontaneous or induced by
    chemical/dietary mutagens)
  • The 6-amino group of C is deaminated to yield U
  • Changes deoxycytosine to deoxyuracil (a base not
    normally found in DNA)
  • If not corrected, a C/G base pair is changed to a
    T/A (from a (U/A) base pair

29
Alkylation
  • Addition of methyl group results in distortion of
    helix
  • May result in improper base pairing (nucleotide
    substitution) if not corrected

30
Oxidative Damage
Mutagenic transversions
TA activation of oncogenes eg
k-ras inactivation of tumour suppressors eg p53
oh8GC oxidative damage
COLORECTAL CANCER
31
DNA Repair
  • Direct Reversal
  • Base Excision Repair
  • Nucleotide Excision Repair
  • Mismatch Repair
  • Recombination Repair

APOPTOSIS cell suicide/programmed cell death
32
Gene expression
  • Bacteria
  • Eukaryotes

33
Human Genome Sequence
3,000 million bp of DNA Only 20,000 -
25,000 genes Only 1.1 (33 million bp) is
coding sequence
(Venter et al, Science, 2001, 291 1304-1351)
34
Human Genome Sequence
Protein Coding DNA (1.1) (Exons)
Non-Coding DNA (98.9)
- Intragenic DNA (24) Introns -
Intergenic DNA (75)
35
Eukaryotic Genes Contain Introns intragenic DNA
Gene
CHROMOSOMAL DNA
Exon 1
Exon 2
Exon 3
Intron 1
Intron 2
Transcription
Primary RNA Transcript
Exon 1
Exon 2
Exon 3
Intron 1
Intron 2
Intron 1
RNA splicing
Intron 2
messenger RNA (mRNA)
36
Intragenic DNA regions
Gene
DNA
intron
intron
intron
RNA polymerase Transcription/splicing
mRNA
37
Promoter regions promote transcription, but not
all of the time
Promoters can be 1) Cell-specific 2)
Tissue-specific 3) Developmental-specific
4) Species-specific
38
  • Specific promoters
  • switch genes on/off, increase/decrease
  • limit gene expression to specific cell types or
    tissues
  • regulate expression in response to specific
    stimuli e.g. macro/micronutrients/dietary
    metabolites

39
Transcription and Translation
Transcription factors
40
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