Ch. 21 (19) - Analysis of Gene Expression

Question 1

Functional genomics

Answer 1

The study of the whole genome and its expression. Includes transcriptome analysis, proteomics, and metabolomics.

Transcriptome:
The total sum of all the RNA transcripts found in a cell, under any particular set of conditions.

Question 2

Monitoring gene expression

Answer 2

Measurements of gene expression means estimating the level of gene product synthesized, or monitoring the RNA or protein products that are made. Since most genes vary in expression during different conditions, it is necessary to measure the level of gene expression under a variety of conditions. Gene expression can be assayed by analyzing a single gene, or the transcriptome.

Proteins may be assayed by running cell extracts on PAGE or antibody-based assays. If the protein is an enzyme, its activity can be measured. Direct detection and assay of proteins is gene specific.

The first method used to measure mRNA expression was Northern blotting. However this method is not used a lot anymore. “Semi”-quantitative PCR is used instead.

Question 3

Reporter genes

Answer 3

Genes that are used in genetic analysis because their products are convenient to assay or easy to detect. These reporter genes are often artificial regions. Reporter genes are often used to report on gene expression, or the presence of a particular segment of DNA, or to detect the location of a protein. Examples of reporter genes are antibiotic resistance genes used to verify uptake of plasmids in bacteria, and the lac-z gene encoding beta-galactosidase.

Question 4

Reporter gene constructs

Answer 4

Study expression pattern from a specific promoter, how active is the promoter at a given time: promoter of interest and reporter gene.

Study expression pattern and subcellular localization: strong promoter (often viral), reporter gene, and gene of interest.

Study expression pattern, and cellular and subcellular localization, measure the activity of the promoter and gene of interest: promoter of interest, reporter gene, and gene of interest.

Question 5

Reporter gene lacZ and AP

Answer 5

The lacZ gene is a very common reporter gene that codes for beta-galactosidase, which cleaves galactosides. Cleavage of some artificial galactosides such as X-gal (blue) and OPNG (yellow) generated colored products that can be measured.

The phoA gene is also a common reporter gene that codes for alkaline phosphate, which cleaves phosphate groups from a broad range of substrates. Cleavage of o-Nitrophenyl phosphate gives yellow o-nitrophenol, and cleavage of X-phos gives a color change from purple to blue.

Question 6

Reporter gene luciferase

Answer 6

The luciferase enzyme emits light when provided with/it alters the structure of the substrate luciferin. The emitted light can be measured by a photodetector, which makes it useful for quantification.

Question 7

Green Fluorescent Protein (GFP)

Answer 7

GFP is a protien isolated from a jellyfish, and is independent of any substrate as it is not an enzyme. It can be observed directly in living tissue cells if excited by light with the right wavelength. Genetically modified GFP have enhanced fluorescence and emit light at different wavelengths, yielding different colored light. GFP is one of the most used reporter genes in eukaryotes.

Question 8

GFP for protein localization

Answer 8

The GFP gene can be fused in frame with the structural gene that encodes the protein of interest. The fused gene is then expressed in the host cell, yielding fused proteins. GFP can be located at the C- or N-terminal of the protein. The fused proteins are distributed to the localization of the protein of interest. The cell is excited with long-waved UV-light (or the wavelength required), and visualized under a microscope. The localization of the protein of interest can be observed.

Question 9

Gene fusion

Answer 9

Gene fusions can be used to study the expression of a gene. Reporter genes are fused to the regulatory region of the target gene. The regulatory sequences now control the expression of the easily detected reporter gene in the same manner that the original gene is controlled. Assaying the level of reporter genes reveals how the target gene would be expressed under given conditions.

Question 10

Deletion analysis of upstream regulatory regions using reporter genes

Answer 10

Reporter genes can be used to study the regulatory regions of genes. A series of constructs of the upstream regulatory region of a gene is produced by deleting segments of the DNA, yielding sequences with different lengths. Each of them are fused with reporter genes, which makes it possible to identify regulatory regions important/necessary to activate expression of the gene.

Question 11

Gel retardation assay

Answer 11

Binding of regulatory proteins to a regulatory region retards the mobility of the fragment, and will slow down its migration through a gel. This can be used to determine whether a regulatory protein binds to the regulatory region.

Question 12

DNA footprinting

Answer 12

Method for testing binding of a protein to DNA by its protection of DNA from chemical degradation. When proteins are bound to DNA they protect the region on the DNA that they cover from chemical attack. Separation on a denaturing PAGE/sequencing gel, yields a footprint where the DNA fragments bound to DNA binding proteins would be, as these are missing.

Question 13

Primer extension

Answer 13

Method to locate the 5’ start site of transcription by using reverse transcriptase to extend a primer bound to mRNA so locating the 5’ end of the transcript. Primer extension allows precise location of the start of transcription to the exact nucleotide.

mRNA is isolated from the cells or tissue that expresses the gene. A sequence specific antisense-primer, which binds near the suspected transcription start site is used to make cDNA by reverse transcriptase. cDNA is made from the primer to the 5’ end of the mRNA, which is where the transcription started. The size of the extended primer is compared to a sequencing ladder of the same region of DNA.

Question 14

Chromatin Immunoprecipitation (ChIP)

Answer 14

Technique that identifies the DNA binding site for a particular TF by crosslinking the DNA to the TF, and then immunoprecipitating the TF.

Proteins are crosslinked to DNA, and the segments are cut into smaller fragments. A primary antibody of the protein/TF of interest is added to the DNA/protein complex. Agarose or magnetic beads with a secondary antibody that binds to the primary antibody, is used to isolate the DNA/protein/antibody complex by immunoprecipitation. The DNA is recovered (removing crosslinks) and can be further analyzed/sequenced.

Question 15

Transcriptome analysis

Answer 15

Transcriptome: The total sum of all the RNA transcripts found in a cell, under any particular set of conditions. This includes mRNA, rRNA, tRNA and various non-coding RNA. The transcriptome varies under different conditions, and the transcriptome analysis aims to measure the level of transcripts at a given condition/state. Two main techniques are RNA-seq (high throughput cDNA sequencing) and microarrays.

RT-PCR can determine the amount of mRNA for a particular gene in two different conditions. Differential Display PCR isolates mRNA in two different conditions and converts it to cDNA by oligo(dT) primers.

Question 16

RNA-seq(uencing) analysis

Answer 16

The use of high throughput cDNA sequencing to characterize an RNA sample. The entire transcriptome at a given condition can be identified by sequencing a cDNA library in its entirety. Next-generation sequencing makes this process possible, resulting in the identification of each and every mRNA that was expressed.

mRNA is isolated by binding to poly-T beads through their poly-A tails. The RNA is then transformed to cDNA as DNA is more stable than RNA. rRNA with poly-A tails can be removed by biotin-labeled anti-rRNA (complementary to rRNA). Streptavidin coated beads are added and bind to the biotin. The beads are then be removed by centrifuge.

Provide more data and can also identify SNPs, alternative splicing of genes and/or RNA editing.

Question 17

Isolation of mRNA and removal of rRNA

Answer 17

mRNA can be isolated by hybridizing to sequences with repeating T, as mRNA have poly-A tails. In addition to eukaryotic mRNA, some rRNAs have poly-A tail.

Removing rRNA from RNA sample

• Biotinylated ss probes, complementary to rRNA, bind to rRNA
• Streptovidin covered beads bind to biotin, used to remove rRNA

Distribution of RNA in cells:
80 % rRNA
15 % tRNA
5 % mRNA

Question 18

Single cell RNA-seq

Answer 18

Cells from a complex mixture are isolated. Single cells are mixed with barcoded primers, and the cells are lysed in their microenvironment. Reverse transcription yields cDNA containing the cell-specific, unique barcode. Samples from hundred or thousands of cells are pooled and the barcoded cDNA are amplified and sequenced.

Question 19

Microarray analysis

Answer 19

Global scale method, in contrast to qRT-PCR and reporter genes. RNA hybridizes with the cDNA in the array. Determines which RNA is present in different conditions. Determine amount of each transcripts in different conditions.

For total transcriptome analysis, the solid support (chip) has DNA sequences complementary to all possible mRNA molecules that a cell might express. mRNA extracted from cells are labeled with fluorescent dye. The labeled mRNA is placed on the array in conditions that favor hybridizing of complementary sequences. The intensity of the label at each spot correlates to the amount of that particular mRNA.

Most gene expression studies compare two different conditions at the same time, direct comparison. Both mRNA samples can be hybridized to the chip at the same time if two different fluorescent dyes (Cy3: green and Cy5: red) are used for each mRNA set. The two colors indicate the expression of each of the mRNA sets, while a yellow spot indicates expression of both of them.

Labeled cDNA can also be used. These samples are hybridized with DNA probes attached and immobilized on a glass slide. The slides are washed and scanned, and images are analyzed to determine gene expression.

Question 20

DNA microarray production

Answer 20

In practice, two types of DNA microarray are used for binding mRNA: arrays of cDNA or arrays of oligonucleotides.

cDNA is generated by PCR of each gene in the organism. Each amplified cDNA is attached to the chip/glass slide for use.

The immobilized DNA molecules in oligonucleotide arrays are synthetic segments of single-stranded DNA. Oligonucleotides are synthesized for each gene in a genome. Often more than one probe for each gene. The lengths of the oligonucleotide probes depend on the slide type. GeneChip uses 25 nt, and the oligonucleotides are synthesized directly on the chip:
A glass slide is covered with a reactive/coupling group, and then a photosensitive blocking group that can be removed by light. In each synthetic cycle, those sites where a nucleotide is not added are covered with a mask to protect the blocking group from light. The other sites are treated with light, which removes the blocking group. When nucleotides are added to these open spots, they attach to the reactive group on the glass slide. The nucleotides are also attached to a reactive group and a blocking group for the next cycle. Masks and light are added, and a new nucleotide can be added to the open spots.

Question 21

Visualizing microarray data: Heat map

Answer 21

Computer analysis determines the intensity of the fluorescence in microarrays, and can be presented as a heat map grid. The genes for the control set of data is listed on one axis, and the experimental conditions are listed on the other axis. Each square of grid is colored and illustrates the comparison of gene expression between the control and the test sample in the given condition. Red indicates an increase in gene expression, whereas green/blue indicates a decrease in gene expression compared to the control at the given condition.

Microarray data can also be presented as gene cluster in an interaction map. Correlation values between gene pairs can be calculated and visualized as clusters with connections between them. Interaction map:

• Shows co-regulation at a given condition
• Genes expressed in the same pattern may be close to each other on the map
• Following the same pattern, probably involved in the same processes

Question 22

Serial Analysis of Gene Expression (SAGE)

Answer 22

A DNA sequencing procedure that can be used to measure the expression level of many genes simultaneously. The mRNAs are converted to cDNA, which are cut into smaller fragments (tags) by a restriction enzyme. These are joined together by ligase, producing long concatemers, which is sequenced. The tags are mapped against known mRNAs and counted. Each segment in the concatemer represents a single mRNA, therefore the number of repeats of each segment correlates with the level of expression of the corresponding gene in the cell.

All the mRNA in a cell is converted to cDNA, cut in smaller fragments, and joined end-to-end. This concatemer is sequenced, which reveals how many copies of each mRNA were present - hence the level of expression.