If DNA were altered irreversibly during development, the chromosomes of a differentiated cell would be incapable of guiding the development of the whole organism. To test this idea, a nucleus from a skin cell of an adult frog was injected into a frog egg developed normally into a tadpole, indicating that the transplanted skin cell nucleus cannot have any critical DNA sequences figure 8-2. Such nuclear transplantation experiments have also been carried out successfully using differentiated cells taken from adult mammals, including sheep,cows, pigs, goats, and mice. And in plants, individual cells removed from a carrot, for expample, can be shown to regenerate an entire adult carrot plant. These experiments all show that the DNA in specialized cell types still contain the entire set of instruction needed to form a whole organism. The cells of an organism therefore differ not because they contain different genes, but they express them differently. The tryptophan repressor, is a repressor protein: in its active form, it switches genes off, or represses them. Some bacterial transcription regulators do the opposite: they switch genes on, or activate them. These activator proteins work on promoters that-in contrast to the promoter for the trptophan operator-are, on their own, only marginally able to bind and position RNA polymerase; they may, for example, be recognized only poorly by the polymerase. However, these poorly functioning promoters can be made fully functional by activator proteins that bind to a nearby site on the DNA and contact the RNA polymerase to help it initiate transcription figure 8-8.In some cases, a bacterial transcription regulator can repress transcription at one promoter and activate transcription at another; whether the regulatory protein acts as an activator or repressor depends, in large part, on exactly where the regulatory sequence to which it bind are located with respect to the promoter.
---Like the tryptophan repressor, activator proteins often have to interact with a second molecule to able to bind DNA. For example, the bacterial activator protein CAP has to bind cyclic AMP (cAMP) before it can bind to DNA. Genes activated by CAP are switched on in response to an increase in intracellular cAMP concentration, which signals to the bacterium that glucose, its preferred carbon source, is no longer available; as a result, CAP drives the production of enzymes capable of degrading other sugars.
Initiation of transcription in eucaryotic cells must also take into account the packaging of DNA into chromosomes. The genetic material in eucaryotic cells is packed into nucleosomes, which, in turn, folded into higher-order structures.
---How do transcription regulators, genreal transcription factors, and RNA polymerase gain access to such DNA?
---Nucleosomes can inhibit the initiation of transcription if they are positioned over a promoter, probably because they physically block the assembly of the general transcription factors or RNA polymers on the promoter. In fact, such chromatin packaging may have evolved in part to prevent leaky gene expression-initiation of transcription in the absence of the proper activator proteins.
---In eucaryotic cells, activator and repressor proteins exploit chromatin structure to help turn genes on and off.
Eucaryotic gene activator proteins can direct local alterations in chromatin structure: Activator proteins can recruit histone-modiying enzymes and chromatin-remoldeling complexes to the promoter region of a gene. The action of these proteins renders the DNA packaged in chromatin more accessible to other proteins in the cell, including those required for transcription initiation. In addition, the covalent histone modifications can serve as binding sites for proteins that stimulate transcription initiation.
In addition to being able to switch individual genes on and off, all organisms-wheter procaryotes or eucaryote-need to coordinate the expression of different genes. When a eucaryotic cell receives a signal to divide, for example, a number of hitherto unexpressed genes are turned on together to set in motion the events that lead eventually to cell division. One way in which bacteria coordinate the expression of a set of genes is by having them clustered together in an operon under control of a single promoter. This is not the case in eucaryotes, in which each gene is transcribed and regulated individually. So how do eucaryotes coordinate gene expression?
---In particular, given that an eucaryotic cell uses a committee of transcription regulators to control each of its genes, how can it rapidly and decisively switch whole groups of genes on or off?
---Even though control of gene expression in combinatorial, the effect of a single transcription regulator can still be decisive in switching any particular gene on or off, simply by completing the combination needed to activate or repress the gene. This like dialing in the final number of combination lock: the lock will spring open if the other numbers have been previously entered. Just as the same number can complete the combination for several different genes. As long as different genes contain DNA sequences recognized by the same transcription regulator, they can switched on or off together,as a unit.
A striking example of the effect of a single transcription regulator on differentiation comes from studying the development of muscle cells. A mammalian skeletal muscle cell is a highly distinctive cell type. It is typically an extremely large cell that is formed by the fusion of many muscle precursor cells called myoblasts. The mature muslce cell is distinguished from other cells by the production of a large number of characteristic proteins, such as the actin and myosin that make up the contractile apparatus as well as the receptor proteins and ion channel proteins in the cell membrane that make the muscle cell sensitive to nerve stimulation. Genes encoding these muscle-specific proteins are switched on coordinately as the my blasts begin to fuse. Studies of muscle cells differentiating in the culture have identified key transcription regulators, expressed only in potential muscle cells, that coordinate gene expression and thus are crucial for muscle cells, that coordinate gene expression and thus are crucial for muscle-cell differentiation. These regulators activate the transcription of the genes that code for the muscle-specific proteins by binding to specific DNA sequences present in their regulatory regions.
---These key transcription regulators can convert non muscle cells to my blasts by activating the changes in gene expression typical of differentiating muscle cells. For example, when one of these regulators, MyoD, is artificially expressed in fibroblasts cultured from skin connective tissue, the fibroblasts start to behave like myoblasts and fuse to form muscle like cells. The dramatic effect of expressing the MyoD gene in firbroblasts is shown in figure 8-18. it appears that the fibroblasts, which are derived from the same broad class of embryonic cells as muscle cells, have already accumulated many of the other necessary transcription regulators required for the combinatorial control of the muscle-specific genes, and that addition of MyoD completes the unique combination that directs the cells to become muscle. Some other cell types fail to be converted to muscle by the addition of MyoD; these cells presumably have not accumulated the other required transcription regulators during their developmental history.
---How the accumulation of different transcription regulators can lead to the generation of different cell types is illustrated schematically in figure 8-19. This figure also illustrates how, thanks to the possibilities of combinatorial control and shared regulatory DNA sequence, a limited set transcription regulators can control the expression of a much larger number of genes.
---The conversion of one cell type (fibroblast) to another (muscle) by single transcription regulator emphasizes one of the most important principles... The dramatic difference between cell types such as size, shape, and function - are produced by differences in gene expression.
Once a cell in a multicellular organism has become differentiated into a particular cell type, it will generally remain differentiated, and all its progeny cells will remain that same cell type. Some highly specialized cells never divide again once they have differentiated; for example, skeletal muscle cells and neurons. But many other differentiated cells, such as fibroblasts, smooth muscle cell,s and liver cells (hepatocytes), will divide many times in the life of an individual. All of these cell types give rise only to cells like themselves when they divide: smooth muscle does not give rise to liver cells, nor liver cells to fibroblasts.
---This preservation of cellular identity means that the changes in gene expression that give rise to a differentiated cell must be remembered and passed on to its daughter cells through all subsequent cell divisions. For example, in the cells illustrated in figure 8-19, the production of each transcription regulator, once begun, has to be perpetuated in the daughter cells of each cell division. How might this be accomplished?
---Cells have several ways of ensuring that their daughter "remember" what kind of cells they are supposed to be. One of the simplest is through a positive feedback loop, where a key transcription regulator activates transcription of its own gene in addition to that of other cell-types specific genes figure 8-20. The MyoD protein discussed earlier functions in such feedback loop. Another way of maintaining cell type is through the faithful propagation of a condensed chromatin structure from a parent to daughter cell.
---A third way in cell can transmit information about gene expression to their progeny is through DNA methylation. In vertebrate cells, DNA methylation occurs exclusively on cytosine bases(figure8-21). This covalent modification occurs exclusively cytosines generally turns off genes by attracting proteins that black gene expression. DNA methylatoin patterns are passed on to progeny cells by the actio of an enzyme that copies the methylation pattern on the parent DNA strand of an enzyme that copies the methyaltion pattern on the parent DNA strand to the daughter DNA strand immediately after replication figure 8-22. Because each of these mechanisms- positive feedback loops, certain forms of condensed chromatin,and DNA methylation- transmits information from parent to daughter cell without altering the actual nucleotide sequence of the DNA, they are considered forms of epigenetic inheritance.
2) Ribosqitcjes are parts of an mRNA that can undergo changes in conformation in response to signals , thereby altering transcription or translation.
---Transcription regulators control gene expression by switching on or off transcription initiation. The vase majority of genes in all organisms are regulated in this way. But additional points of control can come into play later in the pathway form DNA to protein, giving cells a further opportunity to manage the amount of gene product that is made. These post-transcriptioal controls, which operate after RNA polymerase has bound to a gene's promoter and started to synthesize RNA, are crucial for the regulation of many genes.
---One type of post-transcriptional control:alternative splicing, which allows different forms of a protein to be made in different tissues.
----Riboswitches Provide An Economical Solution to Gene Regulation:
----The mechanisms for controlling gene expression we have described thus far all involve the participation of a regulatory protein. But scientist have recently discovered a number of mRNAs that can regulate their own transcription and translation. These self-regulating mRNAs contain riboswitches:short sequences of RNA that change their conformation when bound to small molecules such as metabolites. Many riboswitches hanse been discovered, and each recognizes a specific small molecule.
The conformational change that is driven by the binding of that molecule can regulate gene expression(figure 8-24). This mode of gene regulation is particularly common in bacteria, where riboswitches sense key small metabolites in the cell and adjust gene expression accordingly.
---Riboswitches are perhaps the most economical examples of gene control devices, because they bypass the need for regulatory proteins altogether. The fact that short sequences of RNA can form such highly efficient gene control devices offers further evidence that, before modern cells arose, world run by RNAs may have reached a high level of sophistication.
---Some of the proteins that process and package miRNAs also serve as a cell defense mechanism: they orchestrate the destruction of 'foreign' RNA molecules, specifically those that are double-stranded. Many viruses-and transposable genetic elements-produce double-stranded RNA some time in their life cycles. This targeted RNA degradation mechanism, called RNA interference (RANi), helps to keep these potentially dangerous invaders in check.
---The presence of foreign, double stranded RNA in the cell tigers RNAi by first attracting protein complex containing nuclease called dicer. Dicer cleaves the double stranded RNA into short fragments (approx 23 nucleotide pairs in length) called small interfering RNAs (siRNAs). These short, double stranded RNAs are then incorporated into RISCs, the same complexes that can carry miRNAs. The RISC discards one strand of the si RNA duplex and uses the remaining single stranded RNA to locate a complementary foreign RNA molecule (figure 8-27). This target RNA molecule is then rapidly degraded, leaving the RISC free to search out more of the same foreign RNA molecules.
---RANi is found in a wide variety of organisms, including single celled fungi, plants, and worms,indicating that it is evolutionarily ancient. In some organisms, including plants, the RNAi activity can spread from tissue to tissue by movement of RNA between cells. This RNA transfer allows the entire plant to become resistant to a virus after only a few of its cells have been infected. In a broad sense, the RNAi response resembles certain aspects of the human immune system. In both cases, an infectious organism elicits the production of 'attack' molecules (either siRANs or antibodies ) that are custom designed to inactivate the invader and thereby protect the host.
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