Point mutation

Point mutations are smallest (invisible) microscopic modifications in the molecular construction of the DNA. Substances which react on a chemical basis with the DNA are basically able to provoke this. 

The most important test methods for the examination of point mutations are in vitro bacterium tests. A well known bacterium test is the so called Ames test developed by the American scientist Prof Bruce Ames. Here, the test substance is transferred onto a culture medium on which the bacterium used is no longer able to grow. The number of bacteria which grow under the influence of the test substance is proportional to the mutagenic potential of the substance (see Fig. 2.11). 

A large number of point mutations have been described. Several show marked ethnogeographic clustering related to the occurrence of a mutation in a founder whose descendants migrated and increased in frequency in different geographical regions in accordance with the history of human populations. Only the common mutations that illustrate some of the key genetic aspects of inherited prion diseases are described here. 

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How can we apply molecular dynamics simulations practically. This section gives a brief outline of a typical MD scenario. Imagine that you are interested in the response of a protein to changes in the amino add sequence, i.e., to point mutations. In this case, it is appropriate to divide the analysis into a static and a dynamic part. What we need first is a reference system, because it is advisable to base the interpretation of the calculated data on changes compared with other simulations. By taking this relative point of view, one hopes that possible errors introduced due to the assumptions and simplifications within the potential energy function may cancel out. All kinds of simulations, analyses, etc., should always be carried out for the reference and the model systems, applying the same simulation protocols. 

Binding and complexation data Folding processes Transition temperatures Free energies for point mutations Free energies of binding... 

Point Mutations. Since the advent of recombinant DNA technology, a number of researchers have used point mutation techniques either to delete one or more residues within the hGH molecule or systematically to change from one amino acid to another to probe hGH stmcture/function relationships (33). 

In Figure 7b, the data are plotted as AG yielding a linear function. Extrapolation to 2ero denaturant provides a quantitative estimate of the intrinsic stability of the protein, AG, which in principle is the free energy of unfolding for the protein in the absence of denaturant. Comparison of the AG values between mutant and wild-type proteins provides a quantitative means of assessing the effects of point mutations on the stability of a protein. 

Figure 2 Genetic operators used to create a population of children chromosomes from a population of parent chromosomes, (a) Single-point mutation. A gene to he mutated is selected at random, and its value is modified, (b) One-point crossover. The crossover point is selected randomly, and the genes are exchanged between the two parents. Two children are created, each having genes from both parents.

Equations (5)-(8) assume that the energy functions (7 and Ub operate on the same conformation space i.e., A and B must have the same number N of degrees of freedom. In practice, this almost always implies that A and B have the same number of atoms or particles. Most biochemical changes of interest (e.g., point mutations of a protein) do not obey this requirement, but they can often be made to do so artificially through the use of dummy atoms (see below). 

V De Filippis, C Sander, G Vriend. Pi edictmg local structural changes that result from point mutations. Protein Eng 7 1203-1208, 1994. 

A protein with the innocuous name p53 is one of the most frequently cited biological molecules in the Science Citation Index. The "p" in p53 stands for protein and "53" indicates a molecular mass of 53 kDa. The p53 protein plays a fundamental role in human cell growth and mutations in this protein are frequently associated with the formation of tumors. It is estimated that of the 6.5 million people diagnosed with one or another form of cancer each year about half have p53 mutations in their tumor cells and that the vast majority of these mutations are single point mutations. 

By examining some of the over one thousand tumor-causing point mutations of p53 in the light of its structure, we can identify features of p53 that are necessary for tumor suppression. The amino acids most frequently changed in cancer cells are at or near the protein-DNA interface residues that are infrequently mutated, if at all, are in general far from the DNA-binding site. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

Figure 17.14 Model of evolved mutant from cephalosphorinase shuffling. The sequence of the most active cephalosporinase mutant was modeled using the crystal structure of the class C cephalosporinase from Enterobacter cloacae. The mutant and wild-type proteins were 63% identical. This chimeric protein contained portions from three of the starting genes, including Enterobacter (blue), Klebsiella (yellow), and Citrobacter (green), as well as 33 point mutations (red). (Courtesy of A. Crameri.)...

Other examples include rifampin resistance due to mutations in the ipoB gene encoding the (3-subunit of RNA polymerase, or oxazolidinone resistance due to a G2576T mutation in the gene for the 23 S rRNA as central part of the 50S large ribosomal subunit. Macrolide resistance is based upon the alteration of nucleotide A2058 by a point mutation. 

In contrast to macrolides, the targets of (3-lactams, the penicillin binding proteins (PBPs) require several mutations in order to become resistant while simultaneously maintaining their viable function as cell wall transpeptidases/transglycosidases. Thus, in order to achieve clinically relevant resistance Streptococcus pneumoniae uses a unique strategy to rapidly accumulate several point mutations. Due to its natural competence for transformation during respiratory tract... 

Sorcin is associated with the development of multidrug resistances in leukemia and other cancels. Sorcin is also able to improve cardiac contractility independently of (3-adienergic stimulation and may prove beneficial in treatment of heart failure. A point mutation in sorcin causes familial hypertrophic cardiomyopathy. 

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