Gene expression machineries

Some of the challenges in performing efficient transfection are as described in detail in the chapter, to reduce toxicity, achieve higher efficiency without deleterious manipulation of gene expression machinery, reduce lipid-induced off target effects, and interferon responses. The procedure of transfection is still relatively poorly understood and the challenges posed to the researcher are still daunting at times. The objective of this chapter is to equip the user with vital information to enhance the success of transfection experiments. 

White RJ, Sharrocks AD (2010) Coordinated control of the gene expression machinery. Trends Genet 26 214—220... 

Keaveney M, Struhl K Activator-mediated recruitment of the RNA polymerase machinery is the predominant mechanism for transcriptional activation in yeast. Mol Cell 1998 1 917. Lemon B, Tjian R Orchestrated response a symphony of transcription factors for gene control. Genes Dev 2000 l4 255l. Maniatis T, Reed R An extensive network of coupling among gene expression machines. Nature 2002 416 499. 

Cortistatin was discovered as a result of the effort to characterize cortex-specific gene expression modulated by synaptic activity. It was named after its cortical expression and sequence homology to somatostatin (de Lecea et a ., 1996). The characterization of this peptide is yet another example of the use of reverse genetics to study the molecular components of the sleep machinery. 

The participation of the nuclear receptors in the machinery of gene transcription takes place by means of specific domains of the molecule known as transactivators (abbreviation for transcription activators). These are made up of sequences of amino acids that interact by means of protein-protein contacts with other transcription factors. The artificial alteration of these sequences has as a consequence the inability of the hormone to induce gene expression (Beato et al. 1996 Klug et al. 1987 Lones et al. 1995). 

Li FN, Johnston M (1997) Grrl of Saccharomyces cerevisiae is connected to the ubiquitin proteolysis machinery through Skpl coupling glucose sensing to gene expression and the cell cycle. EMBO J 16 5629-5638 Li SJ, Hochstrasser M (1999) A new protease required for cell-cycle progression in yeast. Nature 398 246-251... 

Whereas it is conceivable that a CRH/vasopressin hyperdrive contributes to behavioral, emotional, and hormonal symptoms of mood disorders, the question remains which mechanisms mediate this hyperactivity of CRH and vasopressin-secreting neurons. The gene expression of both neuropeptides is suppressed by ligand-activated glucocorticoid receptors. The efficiency of this negative feedback action of circulating corticosteroids depends on the number of corticosteroid receptors, their affinity, and the degree of interaction with other factors (e.g., heat shock protein) involved in the transcription machinery. 

Because the export machinery selectively binds transport-competent mRNPs, i.e., mRNAs associated with the appropriate nuclear proteins, the diffusion-based model for intranuclear RNA movement provides an additional checkpoint for gene expression at the level of mRNA export. This type of control has been demonstrated for tRNA export only mature tRNAs are transported to the cytoplasm because incompletely processed tRNAs do not efficiently bind to exportin-t, the tRNA-specific export receptor. 

For eukaryotic genes, expression in insect cells (Baculovirus system) or mammalian cells (Chinese hamster ovary cells, cos7 monkey cells) is possible and is often more likely to lead to active protein than in bacteria or yeast owing to a more efficient posttranslational machinery. However, expression rates are very low and not feasible for production of biocatalysts. 

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