Lecture 10 Flashcards
Once LDL is internalized, it is hydrolyzed to form…
Endosomes and lysosomes.
Genes involved in the metabolism of lipids and lipoproteins.
ABCA-1, Apo E, and Cyp 7a1.
Heterodimer nuclear receptors.
They can make heterodimers with the RXR nuclear receptors.
Releasing co-activators from the DNA sequence results in…
Loss of transcription because the help is gone.
Do polymorphisms have to occur in the exons of genes?
No, they can be in the introns as well.
cDNA method.
Obtain the mRNA sequence. Convert DNA to mRNA to cDNA. See EST: expressed sequence tags.
mRNA in the cDNA method.
Because it is mRNA, you know the genes in question are being actively transcribed, and are therefore expressed.
2 methods of studying changes in gene expression.
Quantitative PCR and microarray analysis.
Quantitative PCR.
Treat RNA with DNase; the DNA is destroyed, but not the mRNA. Reverse transcription: take RNA sequence and transcribe it into DNA using primers made from known ESTs. Place the samples in a thermal cycler which causes synthesis of mRNA copies. You can see how gene expression is altered by changes in nutrient status.
Microarray analysis.
A larger scope that Q-PCR. Reverse transcription of RNA to DNA. You then use cDNA to make RNA that will be fluorescently labelled cRNA. It is fragmented and hybridized into a microarray. The microarray contains both ESTs and DNA. If a gene is not expressed, it cannot hybridize and will therefore not show,
What is meant by hybridization in the microarray?
RNA binding to DNA.
RNA-sequencing.
Provides information regarding abundance of the transcripts. Make cDNA or label RNA fragments. Convert RNA into an EST or cDNA, thereby making a cDNA library. Chop it into pieces and add adaptors that convert it into a cDNA library. Place a tag on each fragment to give the quantity.
Open reading frame (ORAF) in RNA sequencing.
Tells you the repeats in DNA by combining with a nucleotide position in a chromosome.
Proteomic analysis.
Purpose: separating proteins according to unique properties. Uses the charge of the protein and its mass. When placed into a mass spectrometer, it measures the mass of the protein. It is 2-dimensional.
Why are mice good models?
Purebred strains are available, short developmental cycles, easily modified through transgenics, and easy to house.
