Mutants isolation

Fraenkel et al. (17), who isolated mutant strains which had lost the ability to grow on glycerol, succinate, or acetate but grew normally on hexoses or pentoses. These organisms were shown to be deficient in a specific FDPase, which could be distinguished from the nonspecific acid hexosephosphatase present in both mutant or wild-type strains by the fact that the latter was present in the periplasmic space (86) and did not require a divalent metal cation. The properties of the specific FDPase were confirmed with a partially purified preparation (87) the E. coli enzyme was shown to be highly specific for FDP and to be active with very low concentrations of this substance. The requirement for a divalent cation was satisfied by Mg2+, which was far more effective than Mn2+ other divalent cations were either inactive or inhibitory. The partially purified enzyme showed optimum activity at pH 7.8, with very little activity below pH 7 or above pH 9. The enzyme resembled mammalian and Candida FDPases in its sensitivity to low concentrations of AMP it was approximately 50% inhibited at an AMP concentration of 2.5 X 10-° M. 

It is indispensable for any formal genetic analysis to be able to isolate mutants defective in genes coding for important cellular functions. In addition to the spontaneous mutations mentioned above, as caused by ISH elements, several protocols of induced mutagenesis were introduced. The most efficient method so far is the use of the alkylating agent ethyl methane sulfonate to cause point mutations... 

Characterization of isolated mutants or new round of mutagenesis and screening... 

It is not clear what specific functions the starch synthase isoforms may have in synthesis of the starch granule. There has been considerable effort to isolate mutant plants specifically deficient in one of the isoforms of the starch synthase in order to determine their individual functions. These have provided some insight into the possible... 

Replacement of amino acid residues in a polypeptide chain still is achieved most easily by isolating mutant proteins from bacteria, but Laskowski, Jr. and his colleagues at Purdue University have developed a method for enzymatically removing an essential arginine residue from the reactive site of soybean trypsin inhibitor (Kunitz) and replacing it with a lysine residue (84). 

Techniques for Isolating Mutants with Novel Capabilities... 

Although it is counterintuitive, chemical or UV mutagenesis appears to be no more effective than reliance on spontaneous mutations to produce the desired phenotypes. The key elements for success in isolating mutants with novel capabilities, especially those capabilities that require multiple mutations, appear to be (1) large populations, (2) intense selection, and (3) prolonged periods of selection that may last up to several weeks. 

Recently, Brill and co-workers (43, 44) have isolated mutant strains of Azotobacter vinelandii which produce an inactive nitrogenase component. This component can be reactivated by treatment with the neutralized acid-hydrolysis products of other nitrogenases (which themselves become inactive on such a treatment) but not apparently with any other molybdenum enzymes. This may either reflect a difference between the cofactor in nitrogenase and other molybdenum enzymes or may be caused by the reconstitution conditions used which may not have been sufficiently varied to allow for different molybdenum oxidation states to be attained. In any event, the chemical characterization and authentication of the molybdenum cofactor should reveal some of the intimate details of the molybdenum site. 

Sites within the carboxy-terminal domain core of phage T4 lysozyme were substituted singly and as a group with methionine to produce a simplified core sequence. The properties of such mutant lysozymes are briefly described. In addition we describe a method to isolate mutant protein from inclusion bodies and a sensitive enzymatic assay to detect small differences in mutant protein activities. 

During the process of isolating mutants, we noticed that one culture produced a very intense musty-green odor. Upon isolation and culturing of the strain responsible, it was found to have 2-methoxy-3-isopropyl- and 2-methoxy-3-secbutyl pyrazines at levels of 12,500 and 150 ng/mL, respectively, when grown in nutrient broth culture. The bacterium isolated was not found to have different growth requirements from the parent strain. 

If after characterization of isolated mutants it is determined that further affinity maturation is required, new libraries can be constructed, using plasmid DNA isolated from high-affinity mutants as a template (rrrNote 17). 

Improved Clones A selective pressure designed to isolated mutants with improved... 

Repeat the steps in Subheading 3.5.1 and 3.5.2 for 2-3 rounds of selection. Each successive round of selection should yield a population of cells with increased antigen-binding fluorescence (Fig. 4). Analyze individual clones once incremental improvements are no longer observed between rounds of selection and/or apply increased selective pressure to isolate clones with the highest degree of improvement. However, sequence diversity of isolated mutants will be decreased. 

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