21 - Analysis of Gene Expression Flashcards
functional genomics
study of the whole genome and its expression
transcriptome
total sum of the RNA transcripts found in a cell under any particular set of conditions
Easily assayable enzymes as reporters
beta-galactosidase/ /lacZ/ gene.
can also split other compounds than lactose, such as ONPG and X-gal.
phoA reporter gene encodes alkaline phosphatase, an enzyme that cleaves phosphate groups from a broad range of substrates. can also use a variety of artificial substrates (o-nitrophenyl phosphate, X-phos 4-methylumbelliferyl phosphate)
Light emmision as reporter system
luciferase emits light if provided with the substrate luciferin.
bacterial luciferase (from lux gene) uses a reduced form of the co-factor FMN as luciferin (also needs oxygen and long chain aldehyde)
whereas the one from fireflies (luc genes) uses ATP as well as oxygen and its luciferin.
If DNA carrying a gene for luciferase is incorporated into a target cell, it will emit a light only when the appropriate luciferin is added.
Green fluorescent protein as reporter
GFP is not an enzyme, does not need a nonprotein coffactor for fluorescence. stable and nontoxic. can be observed without adding reagents.
enhanced GFP (EGFP), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescence protein (ECFP) can be detected simultaneously using an argon-ion laser plus an appropriate detector.
GFP is used for monitoring gene expression and for locating proteins within the cell. Usually the GFP is added to the N- or C-terminal end of the POI.
Gene fusions
Many gene products are complicated or tedious to assay by directly measuring them. The original gene product can be replaced by fusing its regulatory region to the structural region of a reporter gene. To create this fusion, the target gene is cut between its regulatory region and coding region. the same is done with the reporter gene. The regulatory region of the gene under investigation is joined to the coding region of the reporter gene, in a hybrid structure called gene fusion.
The regulatory region will controll the expression of the reporter gene in the same manner as the original gene. This can be utilized to study the regulation of the gene under varying conditions.
deletion analysis of the upstream region
the upstream regulatory region of a gene often contains several sites where regulatory proteins such as TFs bind, in addition to the promoter region where RNA polymerase binds. these regulatory sites enhance or suppress the expression of the gene under varying conditions.
In order to determine the function of the regulatory elements, it is often useful to construct a series of altered upstream regions in which presumed binding sites have been eliminated, like by removing successive segments from the 5’ end of the upstrema region. PCR is used rather than REs, as it has higher specificity.
Locating protein-binding sites in the upstream region
regulatory sites are often found upstream. regulatory proteins often bind here. computer-predicted binding sites will often not function in vivo, and deletion analysis does not tell us of a protein binds, even if they tell us whether the region is part of gene regulation.
The electrophoretic mobility shift, bandshift, or gel redardation assay tests whether e suspected protein binds to DNA from the upstream region.
First, the DNA carrying the gene and its upstream region is labeled with digoxygenin or radioactivity, and then cut with an RE to get a series of fragments. After the cutting, the DNA is separated into two tubes (one control sample). The DNA-binding POI is added to the main sample, and the two are run side by side on a nondenaturing agarose gel. If the protein binds to one of the DNA fragments, the complex will be larger and run slower than the original DNA.
Gel redardation reveals binding. To locate the binding site, a footprint analysis is performed. The fragment of DNA that binds the protein is labaled at one end with radioactivity or fluorescence. As before, the sample is split into two portions and the protein is mixed with one batch. Both portions are then treated with a small amount of a reagent that breaks the DNA strands (DNase I is often used, as it is nonspecific and cuts between any two nts). The DNA is attacked everywhere except for the place the protein has bound, as it is covered. the labeled fragment is the only one visible in the gel, even if there are other fragments that “survive” the treatment.
DNA-protein complexes can be isolated by chromatin immunoprecipitation
chromatin immunoprecipitation = ChIP = technique that identifies the DNA-binding site for a particular TF by crosslinking the DNA to the TF, and then immunoprecipitation the TF.
the protein-freezing is done by creating covalent bonds between different AA side chains. The crosslinking reagents also fuse proteins to DNA, so they will be attached to their DNA binding sites. The DNA is then broken into smaller pieces, sometimes by sonication (sound waves).
next step of ChIP (isolating the POI) uses antibody isolation from chap 15.
The isolated complexes are studied in a variety of ways:
DNA seq can be determined by seq. this info can identify the exact genomic location to which the POI binds. normal seq demands cloning. To avoid cloning, the DNA fragment can be subjected to ChIP-Seq, where one of the second-generation DNA seq methods like Illumina is used. Additionally, the DNA frags can be used as a target for a DNA microarray (ChIP-CHIP), which reeals if the DBD of the POI is found in any other regions in the genome.
ChIA-PET (chromatin interaction analysis-paired end-tag sequencing) is another method for studying the POI after ChIP. the POI is corsslinked to DNA and isolated from the cellular components with immunoprecipitation. Esp useful to analyze protein complexes that create loops in DNA (like enhancers). multiple DNA segs are attached to this type of complex. After sonication and immunoprecipitation, the isolated complexes are mixed with two different oligonucleotide half-linkers (A and B), which anneal to the end of the DNA segs and have complementary seq to pair with each other. Upon binding of DNA from differnet complexes, the linker is a chimera that is easily identified.
Location of the start of transcription by primer extension
primer extension = method to locate the 5’ start site of trc by using reverse transcriptase to extend a primer bound to mRNA so locating the 5’ end of the transcript.
The primer that is ysnthetizes if complementary to a seq close to the suspected start, and does not bind to anything other than the desired mRNA. reverse transcriptase synthetizes cDNA with labeled nts (radioactive, fluorescence). the resulting DNA/RNA hybrid is denatures and run on the same type of denaturing gel used in DNA seq. to determine the excact nt where mRNA starts, a sample of DNA is also seq using the same primer. they are run side by side with the primer extension fragment. the primer extension product migrates to the same location aas the seq fragment that represent the precice start of trc.
Location of the start of trc by S1 nuclease
S1 nuclease cleaves ssRNA or DNA, but not ds nucleic acids. the DNA carrying the suspected start of trc must first be cloned onto a vector. the DNA is digested win RE that yields a fragment containing the presumed start site. Fragment can also be generated using PCR.
Either way, DNA must be denatured to give a ssDNA, which is then labeled on the 5’ end, then denatured and hybridized to the corresponding mRNA. the 5’ end of the mRNA corresponds with the start site. DNA beyond this point remains single stranded. the sample is split into two portions, and S1 nuclease is added to one, degrading the ss overhangs. The samples are run on a denaturing gel. the different in length (the location of the start site) can then be located.
Modification: S1 nuclease mapping can be used for locating the 3æ end of the transcript. the DNA probe will then contain the sustepcted stop site.
not as accurate as primer extension, as S1 nuclease may degrade the ends of the RNA/DNA hybrid slightly
Transcriptome analysis - assessing the purity of RNA
mRNAs with poly(A)-tails can be isolated by binding to a poly(T)-linker attached to magnetic beads.
in proks, a poly(A)-tail can be artificially added.
The binding of complementary oligomer probes linked with a purification tag can isolate the transcripts.
rRNA can be isolated by hybridizing an rRNA probe labeled with a biotin tag
DIFFERENTIAL DISPLAY PCR PROVIDES RELATIVE EXPRESSION PROFILES
See chap 6. This method isolated mRNA in two different conditions and then converts the sample to cDNA. In theory, if the correct primers are used, this method can isdentify all the transcripts in the sample. not used anymore, replaced by RNA-Seq and microarrays
RNA-Seq
simultaneously quantifies expression and maps the transcriptome.
uses high-throughput cDNA sequencing to charecterize an RNA sample.
1) create cDNA library from total or fractioned RNA samples.
2) sequence cDNA, the seq may only occur from one end (single-end seq) or both ends (pair-end seq). With computer assistance, the seqs are aligned with a known genome or other transcripts (or assembled without a reference!) The relative copy number of each cDNA seq is an indication of gene expression levels.
SIngle-Cell RNA-Seq captures trc profiles of a single cell
Single-cell RNA sequencing (scRNA-seq) = the use of RNA-Seq to charecterize the transcriptome of a single cell within a population of cells.
p 676
microarrays for gene expression
See chap 8
For total transcriptome analysis, the solid support (“chip”) has DNA seqs complementary to all possible mRNA molecules that a cell might express.
1) the DNA is printed ontp a nylon membrane or a glass slide.
2) mRNA is extracted from cells and labeled (fluorescently)
3) labeled mRNA is placed on the DNA array in coditions that favor binding of the complementary seqs.
4) after binding, the intensity of the label in each spor correlates to the amount og that perticular mRNA.
Usually a control sample and one experimental one under the conditions we want. Can be hybridized to the chip at the same time if labeled with different colors. (red, green and yellow if both bind)
in practise, two types of DNA microarray are used for binding mRNA; cDNA- or oligio-nt arrays.
cDNA is generated by PCR ampl of each gene in the genome. One problem is the existance of gene families, which increases the risk of cross-hybridization. To avoid this, seqs from the 3’ end of the cDNA (often incl 3’UTR) is used.
oligo-nt arrays are synthetic segments of ssDNA synthetized for each gene in a genome. determining the seq of an oligo-nt requires some investigtion. At least 16 bp long fragments must be made to ensure that it does not bind due to random complementary stuff. The seq does not create any stem-loop structure, and it must not hybridie with stable mRNA secondary structures. to avoid this, multiple different oligo-nts for one gene are included at different locations on the chip.
Synthetizing the oligo-nts:
directly on the chip!
1) glass slide is covered with a reactive group
2) this is then coveren by a photosensitive blocking group that can be removed by light
3) in each synthetic cycle, those sites where a nt will not be attached are coevred with a mask. Those sites where a aprticulat nt (say, A) is to be attached are illuminated to remove the blocking group. Only one type of nt can be added at a time. New blocking, then repeat.
