Mutation Operator 0 mutants

In any hypothesis concerning the nature of the operator, its specific complementary configuration must be genetically determined therefore the operator could be affected by mutations which would alter or abolish its specific affinity for the repressor. Such mutations might result in constitutive synthesis of the protein or proteins if no other functions were impaired. These mutations should define an operator locus the most distinctive predictable property of operator mutants is that the constitutive allele should be dominant to the wild type, since the repressor-sensitive operators should not prevent the functioning of repressor-insensitive operators. 

The organization of the linked genes is of interest. From the tentative identification of one operator mutant (Sherratt and Collins, in preparation) and the location of a frameshift mutation near to the C-... 

Within Section 2, we describe the basic concept of our novel model-based mutation debugging approach. Section 3 illustrates a model of a car alarm system, which is used as a running example during this paper. In Section 4 we define our deterministic Input/Output Timed Automata. In Section 5, we explain model mutation and a set of mutation operators for TA models and describe the linkage between model mutants and corresponding implementation faults. We explain our notion of the timed input/output conformance relation tioco and show the equivalence between language inclusion and tioco conformance in Section 6. 

Mutant Generation First, we create a set of all model mutants our framework supports (Mutantl, Mutant2, Mutants, Mutantd in Figure 1). Details on the different supported mutation operators can be found in Section 5. 

Mutation of a specification is done by altering the model in a small way, mimicking common implementation errors. In our setting, this is done via 9 different predefined mutation operators, that were already introduced in a previous publication [3]. Each of them represents an individual type of implementation fault, that can occur at multiple parts of the model. We only create first order mutants, which means we only apply one operator at a time to one part of the model at a time. Each operator creates a set of mutants ... 

Mutations in the regulatory genes usually produce dramatic results. A mutant repressor gene may produce a defective protein which cannot bind to the operator and would result in continued protein synthesis. Enzymes which, in normal cells, are inducible thus become constitutive. Constitutive operator mutants also occur where an altered base sequence in the DNA of the operator region results in loss of the ability to bind the normal repressor protein. 

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

In 1991, several factors were demonstrated to affect the efficiency of relE controlled killing of E. colt when under lac promoter control (Knudsen Karlstrom, 1991)- Cells escaped suicide primarily because of the mutation rate and the leakiness of repression during normal growth. When relF was under lac JV5 promoter/operator control, the inactivation of suicide function through spontaneous mutation occurred at a frequency of <5 x 10 9. Knudsen has further theorized that if the number of suicide minus (mutant) bacteria in a culture can be kept at zero before induction, all cells will die. This can be achieved in two ways provide the suicide function in duplicate, and control the suicide gene expression so stringently, with a chromosomally located laclq gene, that a basal level of the suicide function to which cells can adapt will not be present (Knudsen et al., 1995). 

The laser photolysis results on the ET behavior of these mutants have been confirmed by steady-state kinetic measurements [58, 59]. Interestingly, the latter experiments have shown that nonconservative mutations at F65 and E94 not only severely inhibit reactivity with FNR, but also with two other ferredoxin-dependent enzymes, nitrite reductase and nitrate reductase [58]. Apparently, similar structural constraints in their interactions with Fd are also operative in these other enzymes. 

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