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Gene expresion


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Gene expresion

  1. 1. What is Gene Expression?Gene expression is the process by which information froma gene is used in the synthesis of a functional geneproduct.These products are often proteins, but in non-proteincoding genes such as rRNA genes or tRNA genes, theproduct is a functional RNA.The process of gene expression is used by all known life -eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses - to generate themacromolecular machinery for life.
  2. 2. Gene expression process may be modulated at transcription, RNAsplicing, translation, and post-translational modification of a protein.It gives the cell control over structure and function,it is the basis for cellular differentiation, morphogenesis and theversatility and adaptability of any organism.It also serve as a substrate for evolutionary change,(since control of the timing, location, and amount of gene expressioncan have a profound effect on the functions (actions) of the gene in acell or in a multicellular organism).In genetics gene expression is the most fundamental level at whichgenotype gives rise to the phenotype.The genetic code is "interpreted" by gene expression, and theproperties of the expression products give rise to the organismsphenotype.
  3. 3. Gene Expression MechanismTranscription, RNA processing, non-coding RNA maturation, RNAexport, Translation,FoldingEnzymes called chaperones assist the newly formed protein to attain(fold into) the 3-dimensional structure it needs to function. Similarly,RNA chaperones help RNAs attain their functional shapes.Protein transportMany proteins are destined for other parts of the cell than the cytosolIn prokaryotes this is normally a simple process due to limitedcompartmentalisation of the cell.In eukaryotes the export pathway is well developedProteins is translocation to the endoplasmatic reticulum, followed bytransport via the Golgi apparatus.
  4. 4. Gene Expression MeasurementThe ability to quantify the level at which a particular gene isexpressed within a cell, tissue or organism can give a hugeamount of information.For example measuring gene expression can:•Identify viral infection of a cell (viral protein expression)•Find if a bacterium is resistant to penicillin (beta-lactamaseexpression)Ideally measurement of expression is done by detecting the finalgene product (is the protein)However it is often easier to detect one of the precursors, typicallymRNA, and infer gene expression level
  5. 5. mRNA quantificationLevels of mRNA can be quantitatively measured byNorthern blotting which gives size and sequence information about themRNA molecules.The main problems with Northern blotting stemUse of radioactive reagents (which make the procedure time consumingand potentially dangerous) andLower quality quantification than more modern methods (due to the factthat quantification is done by measuring band strength in an image of agel).Northern blotting is, however, still widely used as the additional mRNA sizeinformation allows the discrimination of alternately spliced transcripts.
  6. 6. A more modern low-throughput approachis reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR).RT-PCR first generates a DNA template from the mRNA by reversetranscription. The DNA template is then used for qPCR where thechange in fluorescence of a probe changes as the DNA amplificationprocess progresses.qPCR can produce an absolute measurement such as number ofcopies of mRNA, typically in units of copies per nanolitre ofhomogenized tissue or copies per cell.qPCR is very sensitive (detection of a single mRNA molecule ispossible), but can be expensive due to the fluorescent probesrequired.
  7. 7. Northern blots and RT-qPCR are good for detecting whether a singlegene is being expressed, But impractical if many genes within thesample are being studied.Using DNA microarrays transcript levels for many genes at once(expression profiling) can be measured. Recent advances in microarraytechnology allow for the quantification, on a single array, of transcriptlevels for every known gene in several organisms genomes, includinghumans.Alternatively "tag based" technologies like Serial analysis of geneexpression (SAGE), which can provide a relative measure of the cellularconcentration of different messenger RNAs, can be used. The greatadvantage of tag-based methods is the "open architecture", allowing forthe exact measurement of any transcript, with a known or unknownsequence.
  8. 8. Protein quantificationThe most commonly used method is to perform a Western blotagainst the protein of interest - this gives information on thesize of the protein in addition to its identity.A sample (often cellular lysate) is separated on apolyacrylamide gel, transferred to a membrane and thenprobed with an antibody to the protein of interest.The antibody can either be conjugated to a fluorophore or tohorseradish peroxidase for imaging and/or quantification.The gel-based nature of this assay makes quantification lessaccurate but it has the advantage of being able to identify latermodifications to the protein, for example proteolysis orubiquitination, from changes in size.
  9. 9. By replacing the gene with a new version fused a greenfluorescent protein (or similar) marker expression may bedirectly quantified in live cells.This is done by imaging using a fluorescence microscope. It isvery difficult to clone a GFP-fused protein into its nativelocation in the genome without affecting expression levels sothis method often cannot be used to measure endogenous geneexpression.It is, however, widely used to measure the expression of a geneartificially introduced into the cell, for example via anexpression vector.It is important to note that by fusing a target protein to afluorescent reporter the proteins behavior, including its cellularlocalization and expression level, can be significantly changed.
  10. 10. The enzyme-linked immunosorbent assay (ELISA)works by usingantibodies immobilised on a microtiter plate to capture proteins of interestfrom samples added to the well.Using a detection antibody conjugated to an enzyme or fluorophore thequantity of bound protein can be accurately measured by fluorometric orcolourimetric detection.The detection process is very similar to that of a Western blot, but byavoiding the gel steps more accurate quantification can be achieved.LocalisationAnalysis of expression is not limited to only quantification; localisation canalso be determined.mRNA can be detected with a suitably labelled complementary mRNAstrand and protein can be detected via labelled antibodies. The probedsample is then observed by microscopy to identify where the mRNA orprotein is.
  11. 11. Regulation of Gene ExpressionRegulation of gene expression refers to the control of the amount andtiming of appearance of the functional product of a gene. Control ofexpression is vital to allow a cell to produce the gene products it needswhen it needs them; in turn this gives cells the flexibility to adapt to avariable environment, external signals, damage to the cell, etc. Moregenerally gene regulation gives the cell control over all structure andfunction, and is the basis for cellular differentiation, morphogenesis andthe versatility and adaptability of any organism.Any step of gene expression may be modulated, from the DNA-RNAtranscription step to post-translational modification of a protein.The stability of the final gene product, whether it is RNA or protein, alsocontributes to the expression level of the gene - an unstable productresults in a low expression level.In general gene expression is regulated through changes in the numberand type of interactions between molecules that collectively influencetranscription of DNA and translation of RNA.
  12. 12. Transcriptional regulationRegulation of transcription : Three main routes of influence1.Genetic (direct interaction of a control factor with the gene),2.Modulation (interaction of a control factor with the transcriptionmachinery) and3. Epigenetic (non-sequence changes in DNA structure whichinfluence transcription).Direct interaction with DNA is the simplest and most direct method aprotein can change transcription levels and genes often have severalprotein binding sites around the coding region with the specificfunction of regulating transcription.There are many classes of regulatory DNA binding sites known asenhancers, insulators, repressors and silencers.
  13. 13. The mechanisms for regulating transcription are veryvaried, from blocking key binding sites on the DNA for RNApolymerase to acting as an activator and promotingtranscription by assisting RNA polymerase binding.The activity of transcription factors is further modulated byintracellular signals causing protein post-translationalmodification including phosphorylated, acetylated, orglycosylated.These changes influence a transcription factors ability tobind, directly or indirectly, to promoter DNA, to recruit RNApolymerase, or to favor elongation of a newly synthetizedRNA molecule.
  14. 14. The nuclear membrane in eukaryotes allows further regulation oftranscription factors by the duration of their presence in the nucleuswhich is regulated by reversible changes in their structure and bybinding of other proteins.Environmental stimuli or endocrine signals may cause modification ofregulatory proteins eliciting cascades of intracellular signals, whichresult in regulation of gene expression.More recently it has become apparent that there is a huge influence ofnon-DNA-sequence specific effects on translation. These effects arereferred to as epigenetic and involve the higher order structure of DNA,non-sequence specific DNA binding proteins and chemical modificationof DNA.In general epigenetic effects alter the accessibility of DNA to proteinsand so modulate transcription.
  15. 15. DNA methylation is a widespread mechanism for epigenetic influenceon gene expression and is seen in bacteria and eukaryotes and hasroles in heritable transcription silencing and transcription regulation.In eukaryotes the structure of chromatin, controlled by the histonecode, regulates access to DNA with significant impacts on theexpression of genes in euchromatin and heterochromatin areas.Post-Transcriptional regulationIn eukaryotes, where export of RNA is required beforetranslation is possible, nuclear export is thought to provideadditional control over gene expression.All transport in and out of the nucleus is via the nuclear poreand transport is controlled by a wide range of importin andexportin proteins.
  16. 16. Expression of a gene coding for a protein is only possible if themessenger RNA carrying the code survives long enough to betranslated.In a typical cell an RNA molecule is only stable if specificallyprotected from degradation. RNA degradation has particularimportance in regulation of expression in eukaryotic cellswhere mRNA has to travel significant distances before beingtranslated.In eukaryotes RNA is stabilised by certain post-transcriptionalmodifications, particularly the 5 cap and poly-adenylated tail.Intentional degradation of mRNA is used not just as a defencemechanism from foreign RNA (normally from viruses) but alsoas an route of mRNA destabilisation. If an mRNA moleculehas a complementary sequence to a small interfering RNA thenit is targeted for destruction via the RNA interference pathway.
  17. 17. Translational regulationDirect regulation of translation is less prevalent than control oftranscription or mRNA stability but is occasionally used.Inhibition of protein translation is a major target for toxins and antibioticsin order to kill a cell by overriding its normal gene expression control.Protein synthesis inhibitors include the antibiotic neomycin and the toxinricin.Protein degradationOnce protein synthesis is complete the level of expression of that proteincan be reduced by protein degradation. There are major proteindegradation pathways in all prokaryotes and eukaryotes of which theproteasome is a common component.An unneeded or damaged protein is often labelled for degradation byaddition of ubiquitin
  18. 18. Gene Expression SystemAn expression system is a system specifically designed for the productionof a gene product of choice.This is normally a protein although may also be RNA, such as tRNA or aribozyme.An expression system consists of a gene, normally encoded by DNA, andthe molecular machinery required to transcribe the DNA into mRNA andtranslate the mRNA into protein using the reagents provided.In the broadest sense this includes every living cell but the term is morenormally used to refer to expression as a laboratory tool.An expression system is therefore often artificial in some manner.Expression systems are, however, a fundamentally natural process.Viruses are an excellent example where they replicate by using the hostcell as an expression system for the viral proteins and genome.
  19. 19. In natureIn addition to these biological tools, certain naturally observedconfigurations of DNA (genes, promoters, enhancers, repressors) andthe associated machinery itself are referred to as an expressionsystem.This term is normally used in the case where a gene or set of genes isswitched on under well defined conditions. For example the simplerepressor switch expression system in Lambda phage and the lacoperator system in bacteria.Several natural expression systems are directly used or modified andused for artificial expression systems such as the Tet-on and Tet-offexpression system.
  20. 20. The following experimental techniques are used to measure geneexpression and are listed in roughly chronological order, starting withthe older, more established technologies.They are divided into two groups based on their degree of multiplexity.•Low-to-mid-plex techniques: •Reporter gene •Northern blot •Western blot •Fluorescent in situ hybridization •Reverse transcription PCR•Higher-plex techniques: •SAGE •DNA microarray •Tiling array •RNA-Seq
  21. 21. tutorial/northern.html#prot-troub /