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PATH2211 L13 Gene expression Technologies
Terms in this set (33)
What is gene expression
Gene expression is the process by which the heritable information in a gene, the sequence of DNA base pairs, is made into a functional gene product, such as protein or RNA.
The basic idea is that DNA is transcribed into RNA, which is then translated into proteins.
Gene expression plays a central role controlling cell phenotype - i.e. the behaviour and function of a cell
Gene expression regulation
Eukaryotic gene expression is regulated during transcription and RNA processing, which take place in the nucleus, and during protein translation, which takes place in the cytoplasm. Further regulation may occur through posttranslational modifications of proteins.
purpose of control of gene expression
The regulation of gene expression conserves energy and space. It would require a significant amount of energy for an organism to express every gene at all times, so it is more energy efficient to turn on the genes only when they are required.
In addition, only expressing a subset of genes in each cell saves space because DNA must be unwound from its tightly coiled structure to transcribe and translate the DNA. Cells would have to be enormous if every protein were expressed in every cell all the time.
The control of gene expression is extremely complex. Malfunctions in this process are detrimental to the cell and can lead to the development of many diseases, including cancer
In multi-cellular organisms different cell-types will express different genes, for example:
1. Between tissues: muscle versus brain cells
2. Within tissues: Multiple distinct types of cells constitute tissues
3. Embryonic development and adult regeneration: Pluripotent ("stem") cells drive embryonic growth and adult tissue maintenance
4. Transient responses to external stimuli (e.g. stress, nutrition)
5. Associated with disease
typically expressed in most cells at high levels and are required to maintain cell function. e.g. BETA-ACTIN (structural protein), GAPDH (involved in glycolysis).
Cell-specific or inducible genes
show a restricted pattern of expression which may depend on cell-type, stage of development or exposure to external stimuli. e.g. BETA-GLOBIN (HBB), INSULIN (INS), MYOSIN (MYH2
gene expression between/ within tissues
Gene-expression between tissues
Gene expression within tissues
Transient responses to external stimuli
In response to environmental signals. Examples:
Nutrition Immune activation Circadian rhythm
1. gene-expression technologies
1. RNA profiling technologies - quantify RNA molecules
2. Gene-expression reporter systems - indirect assays of transcriptional activity that label positive-cells expressing gene of-interest to facilitate observation of gene-expression in vivo
RT-qPCR 2 Reverse transcription quantitative polymerase chain reaction
involves the synthesis of cDNA from mRNA in the sample before amplification of the template
cDNA is synthesised by reverse transcriptase which is an RNA-dependent DNA polymerase found in retroviruses
One-Step or Two-Step RT-qPCR
1) The addition of a fluorescent probe that binds to DNA
2) Measurement of fluorescence signal after each PCR cycle
is a common fluorescent probe used to quantify double stranded DNA. SYBR-green is a dye that emits low florescence in solution When bound to double stranded DNA SYBR-green absorbs best 497 nanometer blue light (λmax = 497 nm) and emits green light (λmax = 520 nm). Optical measurements are taken at the end of the extension step of PCR cycle (72oC)
RT-qPCR data must be normalized to house-keeping gene(s) expression
are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence corresponding to a gene.
Gene expression micro-arrays are tools that are used to measure the expression of thousands of known genes
Hubridization of labeled RNA to micro-array
Summary of micro-array design and use
RNA-seq and CAGE-seq
. utilise next-gen sequencing to identify & measure abundance of expressed genomic regions
CAGE-seq: Capped Analysis of Gene Expression -seq
. methods that are used to measure expression of thousands unknwon & known genes
. no. genes that can be assayed dependent on how deeply samples have been sequenced
- shallow sequencing - detect ~4,000 genes deep sequencing - can detect a large fraction of the genome
Are non-coding RNA transcripts functional molecules or byproducts of other processes?
"Annotated exons of protein-coding genes cover 2.94% of the genome or 1.22% for protein-coding exons" (ENCODE Project Consortium, 2012)
BUT: 75-80% of the human genome is capable of transcription
Categories of non-coding RNA transcripts: 1. Antisense RNAs
Locus that has at least one transcripts that intersect any exon of a protein-coding locus on the opposite strand, or published evidence of antisense regulation of a coding gene.
Categories of non-coding RNA transcripts: 2
Locus is intergenic non-coding RNA loci.
Categories of non-coding RNA transcripts: 3 Sense overlapping
Locus contains a coding gene within an intron on the same strand
Categories of non-coding RNA transcripts: 4 Sense intronic
Locus resides within intron of a coding gene, but does not intersect any exons on the same strand.
Categories of non-coding RNA transcripts: 5 proceed transcript
Locus where none of its transcripts contain an open reading frame (ORF) and cannot be placed
CAGE-seq: genome-scale 5′ rapid amplification of cDNA ends
RNA-seq - diagram
Genome browser screen shots of RNAseq and CAGE-seq mapped reads
RNA-seq sequences broad regions of expressed genes CAGE-seq sequences narrow sites of activity with greater depth
Advantages of different RNA-seq platforms
Single cell RNA-seq
Single-cell RNA sequencing analyzes gene expression at single-cell resolution for heterogeneous samples
• Isolation of single cells
• Capturing their transcripts and barcoding
• Generating sequencing libraries in which the transcripts are mapped to individual cells using the barcodes
There are different ways to barcode single cells including Chromium 10X and SPLIT-seq methods
Single cell RNA-seq
Can potentially reveal rare cells associated with disease
This set is often in folders with...
PATH2211 L1 DNA chemistry
L2 DNA & chromosome strctuure
PATH2211 L3 Genes and Genomes
PATH2211 L4 RNA
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