Chapter 1: Levels Of Gene Control Flashcards
(32 cards)
[Protein content]
Methods used to study the expression of individual proteins in tissues and cells?
- Western blotting
- PAGE
- Mass spectrometry
[Protein composition]
Methods to study overall protein composition of tissues and cells
-2D PAGE
Principle of Western Blotting and the expected results
■Proteins are isolated from various tissue cells
■They are separated based on size by using gel electrophoresis (SDS-PAGE)
■ They are then transferred to a membrane
■ A protein-specific antibody is used to detect the protein of interest
■Results are observed by flouscerance
♤ Expected results
♡A band will be observed in tissue types where the antibody has bound.
How to read western blotting and what the results mean
■If a band is shown in all tissues— it indicates that a particular gene is expressed in various tissue types
■If a band is shown on a particular tissue type— it means the gene is differentially expressed
Gel electrophoresis (PAGE)
A technique whereby proteins are separated according to their size
Principle of 2D PAGE Analysis & the expected results
■isolate proteins from tissue cells
■Separate proteins by their charge (isometric focusing)
■ then separate them based on size by using gel electrophoresis (SDS PAGE)
■has better resolution than SDS PAGE
Expected results
♤ protein spots observers with different intensities
How to interpret 2D PAGE Analysis and What it means
■You check the size and it’s intensity
■ big spot/ high intensity —means the there is high level of the protein
■small/low intensity— means there is a low level of the protein
[mRna content]
Methods to study the expression of individual mRNAs
■Northern Blot
■RT(q)- PCR
[mRNA content]
Method to study mRNA population (many genes)
Microarray
[Expression of mRNA/transcriptional level]
Principle of Northern Blot
■Isolated proteins 4rm tissues are separated by size
■transfered to a membrane and hybridized to a radioactive probe from gene encoding the mRNA of interest
○mRNA will anneal to single
Stranded DNA probe with
Complementary sequence
○Hybridize/anneal to agene specific probe
■visualize by xray
[mRNA expression]” northern blot
Expected results and how to interpret the blot
■when a band is observed the mRNa of the gene is transcribed
■ no band= mRNA of gene is not transcribed
Interpretation
□when a band with similar intensity is observed in various tissue types= the gene is not differentially expressed
□when a band with different intensities or absent 4rm other tissue types= differentially expressed
□absence of band= gene not transcribed or transcribed at low levels
□ A band shows the gene is transcribed in the cell
Method to detect low levels of mRNA
RT-PCR
Why and when to use RT-(q)PCR
When we want 2 compare transcripts between samples with high accuracy n high sensitivity
■method used 2 detect low concentration of cDNA
[mRNA content at low level]
Principle of RT-qPCR and expected results
■isolate RNA and change it to double stranded cDNA
■DNA is amplified using gene specific primers
■primers are Labelled with flouscerant markers
■The quantity of amplified DNA is related to the level of mRNA in the cell
□amplified DNA observed when mRNA is transcribed
□amplified DNA not observed when mrna is inactive
[mRNA at low level]
How 2 interpret RT-qPCR
■high quantities of amplified gene —shows that more mRna are present in that tissue
■comparing results 4rm diff tissue types allows me to know in which cell type is a gene most abandantly transcribed
2 Principles that:
1- measure how well a gene transcription is initiated (whether it is regulated at the level of transcription initiation or at a downstream level, such as RNA stability)
2- can compare the rate of transcription of different genes in different tissues
●Pulse-labelling (in vivo- in cells)
●Nuclear run-oon assay(In vitro-test tube(
Pulse labeling principle
■The transcription rate of a gene in cells is monitored during RNA synthesis by adding a radioactive labelled UTP for Short period of time
■ Radioactive UTP is incorporated into newly synthesized mRNA during transcription by RNA Pol
■specific transcript of interest can be analyzed by hybridization using gene-specific probe
■visualize by auto radiography
How 2 interpret pulse labelling[rate]
■high intensity means more mRNA is present–meaning there is a higher transcription rate happening= transcription activation
■no signal means no transcription rate and no transcriptional activation
Principle of Nuclear run-on Assay[vitro-glass]
■Transcription rate is monitored by measuring the amount of radioactively labelled 3H-UTP
■labeling occurs for short period of time
■genes that are being transcribed in the labeling period will be radiolabelled
■specific transcripts of interest detected by hybridization to unlabeled genes
Exceptional cases where changes to DNA occurs
- DNA loss
- DNA Amplification
- DNA Rearrangement
DNA Loss example of mammalian red blood cell
During differentiation of red blood cells the nucleus is removed and degraded in the erythroblast cells, resulting in a anucleated cell(no nuclear & DNA)
It sysnthesiz3s small amounts of other proteins by translation of mRNA produced in red blood cells before loss of nucleus
DNA Amplification example
Case wherw high level of mRNA is required in short period of time such that a gene could not produce enough mrna/Protein
Chorion genes are amplified in DNA, allowing synthesis in these cells of the large amounts of chorion mRNA at short period of timr
How to know if gene is regulated at Transcriptional or post-transcriptipmal control
- Nuclear run on assay
- pulse labeling
When to know if it’s regulated at post-transcriptional level
■when genes are transcribed/expressed in all tissue types
