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Several steps in the gene expression process may be modulated, including the transcription step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.
The expression of many genes is regulated after transcription (i.e., by microRNAs or ubiquitin ligases), so an increase in mRNA concentration need not always increase expression. Nevertheless, mRNA levels can be quantitatively measured by Northern blotting, a process in which a sample of RNA is separated on an agarose gel and hybridized to a radio-labeled RNA probe that is complementary to the target sequence. Northern blotting requires the use of radioactive reagents and can have lower data quality than more modern methods (due to the fact that quantification is done by measuring band strength in an image of a gel), but it is still often used. It does, for example, offer the benefit of allowing the discrimination of alternately spliced transcripts.
A more modern low-throughput approach for measuring mRNA abundance is real-time polymerase chain reaction or RT-PCR. With a carefully constructed standard curve RT-PCR can produce an absolute measurement such as number of copies of mRNA per nanolitre of homogenized tissue. The lower level of noise in data obtained via RT-PCR often makes this the method of choice, but the price of the required equipment and reagents can be prohibitive.
In addition to low-throughput methods, transcript levels for many genes at once (expression profiling) can be measured with DNA microarray technology or "tag based" technologies like Serial analysis of gene expression (SAGE) or the more advanced version SuperSAGE, which can provide a relative measure of the cellular concentration of different messenger RNAs. Recent advances in microarray technology allow for the quantification, on a single array, of transcript levels for every known gene in the human genome. The great advantage of tag-base methods is the "open architecture", allowing for the exact measurement of any transcript, known or unknown. Especially SuperSAGE recommends itself therefore also for studying organisms with unknown genomes.
Protein levels themselves can be estimated by a number of means. The most commonly used method is to perform a Western blot against the protein of interest, whereby cellular lysate is separated on a polyacrylamide gel and then probed with an antibody to the protein of interest. The antibody can either be conjugated to a fluorophore or to horseradish peroxidase for imaging or quantification. Another commonly used method for assaying the amount of a particular protein in a cell is to fuse a copy of the protein to a reporter gene such as Green fluorescent protein, which can be directly imaged using a fluorescent microscope. Because it is very difficult to clone a GFP-fused protein into its native location in the genome, however, this method often cannot be used to measure endogenous regulatory mechanisms (GFP-fusions are therefore most often expressed on extra-genomic DNA such as an expression vector). Fusing a target protein to a reporter can also change the protein's behavior, including its cellular localization and expression level.
The pattern of detection of a gene or gene product may be described using terms such as facultative, constitutive, circadian, cyclic, housekeeping, or inducible.
Regulation of gene expression
Regulation of gene expression is the cellular control of the amount and timing of appearance of the functional product of a gene. Any step of gene expression may be modulated, from the DNA-RNA transcription step to post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism.
An expression system consists, minimally, of a source of DNA and the molecular machinery required to transcribe the DNA into mRNA and translate the mRNA into protein using the nutrients and fuel provided. In the broadest sense, this includes every living cell capable of producing protein from DNA. However, an expression system more specifically refers to a laboratory tool, often artificial in some manner, used for assembling the product of a specific gene or genes. It is defined as the "combination of an expression vector, its cloned DNA, and the host for the vector that provide a context to allow foreign gene function in a host cell, that is, produce proteins at a high level".
In addition to these biological tools, certain naturally observed configurations of DNA (genes, promoters, enhancers, repressors) and the associated machinery itself are referred to as an expression system, as in the simple repressor 'switch' expression system in Lambda phage. It is these natural expression systems that inspire artificial expression systems, (such as the Tet-on and Tet-off expression systems).
Each expression system has distinct advantages and liabilities, and may be named after the host, the DNA source or the delivery mechanism for the genetic material. For example, common expression systems include bacteria (such as E.coli), yeast (such as S.cerevisiae), plasmid, artificial chromosomes, phage (such as lambda), cell lines, or virus (such as baculovirus, retrovirus, adenovirus).
In the laboratory, the protein encoded by a gene is sometimes expressed in increased quantity. This can come about by increasing the number of copies of the gene or increasing the binding strength of the promoter region.
Often, the DNA sequence for a protein of interest will be cloned or subcloned into a plasmid containing the lac promoter, which is then transformed into the bacterium Escherichia coli. Addition of IPTG (a lactose analog) causes the bacteria to express the protein of interest. However, this strategy does not always yield functional protein, in which case, other organisms or tissue cultures may be more effective. As for example the yeast, Saccharomyces cerevisiae, is often preferred to bacteria for proteins that undergo extensive Posttranslational modification. Nonetheless, bacterial expression has the advantage of easily producing large amounts of protein, which is required for X-ray crystallography or nuclear magnetic resonance experiments for structure determination.
Gene networks and expression
Genes have sometimes been regarded as nodes in a network, with inputs being proteins such as transcription factors, and outputs being the level of gene expression. The node itself performs a function, and the operation of these functions have been interpreted as performing a kind of information processing within cell and determine cellular behaviour.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "Gene_expression". A list of authors is available in Wikipedia.|