Mutation study techniques

It has been possible to cultivate in vitro normal diploid human cell strains from a variety of tissues, including all the soft organs, skin, membranes, bone marrow, and peripheral leukoc)rtes this is also possible for many primates, rodents, and others. The techniques for initiation and maintenance of continuous cell cultures will not be described here. The reader is referred to standard texts and research papers on tissue and cell culture. We must, however, consider several aspects of the biology of cultured mammalian cells that have direct bearing on the genetic, especially mutational, studies. 

A number of techniques have been devised for mutation studies in the mouse, but undoubtedly the most productive has been the specific-locus method, which so far has found its greatest use for the assessment of the genetical hazards of ionizing radiations to man. The method has the potential for fulfilling the same role with respect to environmental and chemical mutagens, and, as the first results already demonstrate, it is likely to provide information which will elucidate the nature of the mutation process in the mammalian germ cell and indicate some of the variables which affect it. 

In our laboratory we first isolated the major lethal protein (termed Cobrotoxin) of non-enzymatic nature from the venom of Taiwan cobra Naja naja atra) in 1964 and subsequently purified and crystallized the protein. The primary structure and the disulfide linkages with various efforts by chemical modification and immunological methods in elucidation of the structure-function relationship of this important venom neurotoxin have since been accomplished. Structure-activity correlations have been drawn from chemical modification carried out on both pre- and post-synaptic neurotoxins. With recent advances in DNA recombination and protein engineering, we feel that the time is now ripe to apply these techniques to the isolation and characterization of the genes encoding these toxins. Detailed structural and site-specific mutational studies on the cDNA clones of neurotoxins of both types may complement our previous chemical modifications of the functional role of some amino acid residues in neurotoxins and lead to insight into the modes of action for these biologically active molecules. 

Genetic Control. Manipulation of the mechanisms of inheritance of the insect pest populations has occurred most successhiUy through the mass release of steri1i2ed males, but a variety of other techniques have been studied, including the environmental use of chemostetilants and the mass introduction of deleterious mutations, eg, conditional lethals and chromosomal translocations (58 ndash 60) (see Genetic engineering). 

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

The specific role of each amino acid residue for the function of the protein can be tested by making specific mutations of the residue in question and examining the properties of the mutant protein. By combining in this way functional studies in solution, site-directed mutagenesis by recombinant DNA techniques, and three-dimensional structure determination, we are now in a position to gain fresh insights into the way protein molecules work. 

Progress in molecular biology has provided a new perspective. Techniques such as the polymerase chain reaction and single-strand conformation polymorphism analysis have greatly facilitated the molecular analysis of erythroenzymopathies. These studies have clarified the correlation between the functional and structural abnormalities of the variant enzymes. In general, the mutations that induce an alteration of substrate binding site and/or enzyme instability might result in markedly altered enzyme properties and severe clinical symptoms. 

In approaching the study of the molecular mechanisms of heredity, this chapter first discusses the structural and functional roles of the genetic material, DNA. This includes an analysis of its replication and susceptibility to mutation. The health-related aspects of the use of recombinant DNA techniques are considered, and examples of then-use in the analysis of several human genetic diseases are used to illustrate the biochemical side of genetics. 

Spontaneous mutations in experimental animals provide insights about the structure and assembly of myelin. The myelin mutants often have names relating to their characteristic tremor due to the myelin deficit, e.g. shiverer, jimpy, quaking and trembler mice (Table 4-2). Although some of the mutants have been studied for many years [1], it is only recently that recombinant DNA techniques have led to identification of the primary genetic defects in most of them. Some of them are good... 

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

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