Chemical genetics development

Figure 1.24 Orthogonal chemical genetics development of analog-sensitive kinase alleles (ASKAs).

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (elF2 GTP Met-tRNAjMet) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target. 

In reverse chemical genetics, it is crucial to synthesize proteins of interest using appropriate foreign gene expression systems and cDNA resources. Since cell-free protein synthesis systems have the potential to synthesize any desired proteins, including both native proteins and those that are toxic to cells (1), with high throughput, they can be powerful tools for this objective. We developed a cell-free protein synthesis system from Spodop-tera fm iperda 21 (S 21) insect cells, which are widely used as the host for baculovirus expression systems, and commercialized it as the Transdirect insect cell. 

The progress of reverse chemical genetics research is influenced by the efficiency of generation of active recombinant proteins. In recent years, numerous systems for the expression of the recombinant proteins have been developed. Of these, the baculovirus system is considered to be the most efficient. Typically in the baculovirus system, an insect cell line (for example, Sf9) is used as a host for the expression of recombinant proteins. In the present report, we describe the novel application of Kaiko as a host in the baculovirus system for the expression of recombinant... 

To date, complex proteins with biological activity (6), for use in X-ray crystal structure analysis (7) and ELISA systems (8), and for the development of animal drugs (9) have successfully been produced by using this system. Therefore, the Kaiko-baculovirus protein production system has broad applicability across the field of reverse chemical genetics for the analysis of protein function on the basis of interactions with chemical compounds. 

Another case, much more serious and also more pertinent to the chemical process development area, occurred in 1989 when over 1600 people became ill with eosinophilia-myalgia syndrome (EMS) and 38 died, worldwide, after taking l-tryptophan (Trp) manufactured by one producer in Japan. Prior to the outbreak, this producer whose Trp met the >98.5% purity specification had decided to employ a new genetically modified strain of the established Bacillus amyloliquefaciens and also to halve the amount of activated charcoal used in the purification step. These changes cause the Trp product to become contaminated with several new impurities, principally I to III, all associated to some extent (using a crude animal model) with EMS.5... 

Individual susceptibility is one of the most important variables in the development of chronic benzene toxicity. Aksoy et al. (1976) suggested familial susceptibility as one of the main factors in the frequency of chronic benzene toxicity. Two patients with pancytopenia associated with chronic exposure to benzene were cousins. One of these two patients died of aplastic anemia the son of this patient presented with leukopenia a short time after exposure to this chemical. Genetic polymorphism with regard to the P-450 enzymes that metabolize benzene to its reactive metabolites may render some individuals at higher risk to the toxic effects of benzene (Kato et al. 1995). Although there are possible indications of genetic... 

Recently, new comprehensive approaches have been developed that use compound libraries for screens in whole living cells using specific phenotype readouts. Some of the applications of this type of approach have been called chemical genetics or chemogenomics [9-27]. In many instances it is probably more accurate to call... 

A higher throughput version of this approach takes advantage of combinatorial technologies and has been recently developed and exploited with the name of chemical genetics (16-18). More specifically, two complementary approaches have been defined and will be exemplified ... 

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