Molecular techniques used in nutrition research

Nota:

• Single nucleotide polymorphisms: Single base substitution in genes  --> AA subs in their encoded proteins --> Replacements do not necessarily result in inactivation of respective proteins    --> Basis for our individuality, the differences in our ability to metabolize nutrients; may be due to epigenetics    
• Differences in wobble position [3rd position] does not change protein BUT there is a limit of tRNAs in body, we may have a difference in proteins that the cell can make.     
• Alteration of protein specific activity may have major consequences for nutrient metabolism. If we can identify SNPs and link with function, we can see what defines nutrient metabolism   May bind ligand tighter, cant be released -> either be activated or inhibited ex: sub in LXR, protein may not be able to recognize binding site, less bound to DNA, indv may not be able to respond to stimulus    
• 1 gene could have many different variations that affect function in body (ex: lipid transport). Difference in nucleotide positions in an individual determines type of alleles [rare or common]    

1. Synonymous

   Nota:

   • Distinct SNPs at the same position lead to the same polypeptide sequence    
   • May require rarer tRNA to produce protein so less efficient -> affects gene expression    
2. Nonsynonymous

   Nota:

   • Distinct SNPs at the same position lead to different polypeptide sequences    
   • Substitution of AA at that position in the protein; protein might not work as well or work better    
3. Advantageous variations

   Nota:

   • Normally don't know ex: protect against obesity    
4. Deleterious variations

   Nota:

   • Identified a lot, looking/studying those genes    
   1. ApoE polymorphisms

      Nota:

      • [1] E7 (E244K,E245K): Glutamic acid in position 244 and at 245 becomes lysine = base instead = affects binding with protein/lipid --> Hypertriglyceridemia, diabetes (Japanese population, may only have gene variation in that population)    
      • [2] E4 (C112R):  Cysteine to arginine --> Alzheimer's disease    
      • [3] SNP -219G: In promoter region [glycine] --> Optic neuropathy    
5. DIFFERENCES?

   Nota:

   • ESTS:  - Know which genes are activated at any one time [nutrigenomics-> how food affects our genes] - Type of cDNA cloning and sequencing (also includes mRNA sequence) SNPs:  - Know which one varies between 2 people [nutrigenetics->how gene variations affect metabolism of nutrients] - Type of gene cloning and sequencing (also includes genome sequence)      

Nota:

• Expressed sequence tags:      Short sequences that are generated by sequencing either one or both ends of an expressed gene (i.e., sequences from cDNAs of mRNAs)     # ESTs > # SNPs
• Come from mRNA; sequence ends of gene to identify genes expressed in body    - Take known kind of cell - Compare ESTs to identify genes present in both or in only 1 type of cell **Genes would be very similar but will have some differences**   
• Some ESTs are identified (correspond to fully characterized genes e.g. LDL gene); many correspond to unidentified genes (like those involved in the acquisition and metabolism of nutrients)    

Nota:

• i.e. amount gene is expressed Both can be used to study which and how specific mRNA species are altered by changes in state of nutrition or metabolic status.    

1. Q-PCR
2. Microarray Analysis

   Nota:

   • Microarray: slide on which there are pcs of DNA that came from genome [or ESTs], covalently link sequence [DNA] to glass    
   • Procedure that can survey the expression of genes, by measuring and detecting mRNA from these genes; if gene not expressed, no mRNA. Can differentially label samples between other samples ex 3 colours: 1 for each cell type, and 1 for both Way for us to look at global gene profile (which ones are repressed, respond or no response)    
   1. Method

      Nota:

      • Ways to analyse data: - Image analysis - Cluster Analysis
      • How to interpret data? When looking at ratios of signals.  - Black = no diff between 2 tissue types so gene present in both [ex brain and gene] - Red = liver specific gene - Green = brain specific gene
      • Use?  Good way to look at global changes in gene expression of a cell, look at families of genes turned on/off/not affected   

Nota:

• Can be used to study which and how proteins/enzymes are affected when there is a change in nutrition or metabolic status 

1. 2D Gel/HPLC

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   • 2D: according to size and charge  HPLC: according to how substances in solvent interact with adsorbent material--> lead to separation in pressurized column
   1. Cleanup/Prep
      1. Mass Spectrometry

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         • ID composition of purified protein
         1. ID each protein and relate it to a gene that expresses it

            Nota:

            • Each protein has a characteristic nucleotide sequence

Nota:

• QUALITATIVE: Provides sequence information; including genomic sequences that have not yet been determined. Allows us to see everything going on in cell, no need for ESCs [so info already known]
• QUANTITATIVE: Provides information regarding the relative abundance of transcripts. 
• For this example [EST], no need to know info, everything in tube, getting all the sequences of the RNA present at a time. If gene is repressed: less prevalent /less sequences   
• Other example [MICROARRAY]: Put on microarray with target sequences, that tell us what's expressed, need to already know something about RNA we are looking for, need to know target.    

1. Reasons for use

   Nota:

   • a) short development cycle  b) easy to standardize across different labs  c) small size d) easy to house e) variety of inbred strains available: C57BL/6J common in nutrition; swiss albino, common in immunology studies f) mouse genome fully sequenced  g) corresponding human genes have been mapped.
2. Disadvantages

   Nota:

   • as a model for human physiology a) metabolic differences b) nutritional differences; nutrients non-essential for mice may be essential for us  c) genetic differences  

Nota:

• inject DNA into male nucleus make it easier to see  Natural or synthetic gene(s) introduced into another genome for permanent propagation DNA sol'n injected into nucleus of fertilized egg of host natural genes: obtained from another species Synthetic genes:contains components from other genes or species 

1. Process of testing

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   • 1) transgene construction: promotor + structural protein + 3' end 2) cloning 3) in vitro evaluation: to check if things are assembled properly before injection into cells to test for effects  4)Microinjection--> recovery period necessary; nutrient medium used to feed mice  is important to supply embryo with all that it needs 5) Embryo Transplant--> transfer into oviduct of pseudopregnant mice (induced by hormone therapy) 6) Genomic analysis
2. Over- expression of LDL receptors in transgenic mice

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   • -Synthetic transgene direct overexpression LDL receptors in  liver - LDLR transgenic mice fed low cholesterol diet-->low blood LDL concentration (lower than wild-type mice) - LDLR transgenic mice exhibit resistance to diet-induced hypercholesterolemia
3. Targeted Gene Disruption

   Nota:

   • -more complicated compared to transgenesis  -allows for specific mutations to be created--> can be global(all animal) or local (specific region) -mutations can also be introduced at any stage in development cycle  -allows for direct assessment of gene function
   1. Effect on mice

      Nota:

      • Wild-type(blastocyst host) crossed with agouti coat (es cell donor)--> modified genome ES inserted into blastocysts--> implanted into pseudopregnant mice  results in chimeric offspring (expresses both genomes) Test cross: albino(host) x chimeric mouse = original parents (albino and agouti) 
   2. e.g targeted LDLR knockout in mice

      Nota:

      • by knocking out LDLR in mice (-/-) this leads to characteristics similar to FH in humans  one difference is that while humans have premature atherosclerosis mice are only susceptible and will only develop this condition if fed a cholesterol diet while they lack LDL