Introduction Of Random Mutations Research Articles

Efficient introgression of allelic variants by embryo-mediated editing of the bovine genome.

The recent development of designer nucleases allows for the efficient and precise introduction of genetic change into livestock genomes. Most studies so far have focused on the introduction of random mutations in cultured cells and the use of nuclear transfer to generate animals with edited genotypes. To circumvent the intrinsic uncertainties of random mutations and the inefficiencies of nuclear transfer we directed our efforts to the introduction of specific genetic changes by homology-driven repair directly in in vitro produced embryos. Initially, we injected zinc finger nuclease (ZFN)-encoding mRNA or DNA into bovine zygotes to verify cleavage activity at their target site within the gene for beta-lactoglobulin (LGB) and detected ZFN-induced random mutations in 30% to 80% of embryos. Next, to precisely change the LGB sequence, we co-injected ZFNs or transcription activator-like effector nucleases (TALENs) with DNA oligonucleotides (ODNs). Analysis of co-injected embryos showed targeted changes in up to 33% (ZFNs) and 46% (TALENs) of blastocysts. Deep sequence analysis of selected embryos revealed contributions of the targeted LGB allele can reach 100% which implies that genome editing by zygote injections can facilitate the one-step generation of non-mosaic livestock animals with pre-designed biallelic modifications.

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

Regulation of gene expression in prostate cancer cells with an artificially constructed promoter responsive to radiation

A promoter library was developed that is composed of DNA fragments constructed by randomly elongating the cis-acting elements of transcription factors presumably activated in prostate cancer by radiation, and linking to the TATA-box sequence. One promoter with the strongest reactivity to X-ray in the LNCap cells of the library was chosen and improved by the introduction of random mutations. The resultant promoter was designated clone 880-8, showing the highest dose-dependent activity enhancement with X-ray irradiation (X-irradiation). A recombinant retrovirus expressing the luciferase gene under the control of clone 880-8 was infected into LNCap cells that showed 9.12±0.36-fold enhancement of luciferase activity 12 h after X-irradiation at 10 Gy. When the infected cells were inoculated onto nude mice, enhancement of luciferase expression was 4.27±1.36-fold 12 h after X-irradiation at 10 Gy. When LNCap was infected with another recombinant carrying the fcy::fur gene downstream from clone 880-8, fcy::fur expression was enhanced by X-irradiation. It was also shown to increase the dose-dependent cell killing ratio with 5-FC as compared with a counterpart without X-irradiation. These results suggest that the method used in this study is effective to construct a promoter responsive to stimulation. Such promoters can be used for stimulation-controlled gene therapies.

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

We have simultaneously improved the activity, reaction specificity, and thermal stability of p-hydroxybenzoate hydroxylase by means of systematic and comprehensive combinatorial mutagenesis starting from available single mutations. Introduction of random mutations at the positions of four cysteine and eight methionine residues provided 216 single mutants as stably expressed forms in Escherichia coli host cells. Four characteristics, hydroxylase activity toward p-hydroxybenzoate (main activity), protocatechuate-dependent NADPH oxidase activity (sub-activity), ratio of sub-activity to main activity (reaction specificity), and thermal stability, of the purified mutants were determined. To improve the above characteristics for diagnostic use of the enzyme, 11 single mutations (C152V, C211I, C332A, M52V, M52Q, M110L, M110I, M213G, M213L, M276Q, and M349A) were selected for further combinatorial mutagenesis. All possible combinations of the mutations provided 18 variants with double mutations and further combinatorial mutagenesis provided 6 variants with triple mutations and 9 variants with quadruple mutations with the simultaneously improved four properties.

Unlocking hidden genomic sequence.

Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.

Unraveling the effect of changes in conformation and compactness at the antibody V(L)-V(H) interface upon antigen binding.

We have analyzed conformational changes that occur at the interface between the light (V(L)) and heavy (V(H)) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three-dimensional structures to evaluate changes in compactness of the V(L)-V(H) interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium-sized nonprotein antigens cause an opening of the V(L)-V(H) interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V(L)-V(H) interface. The largest changes in the atomic buried contact surface area at the V(L)-V(H) interface occur in residue pairs providing two 'shock absorbers' between the edge beta-strands of the V(L) and V(H) beta-sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V(L)-V(H) interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V(L)-V(H) interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V(L)-V(H) interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity.

Mobility of the primary electron-accepting plastoquinone QA of photosystem II in a Synechocystis sp. PCC 6803 strain carrying mutations in the D2 protein.

Upon introduction of random mutations in a region of the psbDI gene that encodes the D2 protein in the cyanobacterium Synechocystis sp. PCC 6803, an obligate photoheterotrophic mutant was isolated that contained three mutations: V247M, A249T, and M329I. This mutant evolved oxygen in the absence of added electron acceptors, but oxygen evolution was inhibited by micromolar concentrations of several artificial quinones. Complementation analysis showed that the V247M and/or A249T mutations were responsible for this phenotype. Using fluorescence induction and decay measurements, the site of inhibition by the quinones was found to be at the level of the primary electron-accepting quinone in photosystem II, QA. Duroquinone inhibited by blocking reduction of QA, and in the presence of other quinones such as 2,5-dichloro-p-benzoquinone, 2, 5-dimethyl-p-benzoquinone, and p-benzoquinone, QA could be reduced but could not efficiently transfer an electron to QB. To distinguish the effects of the V247M and A249T mutations, single mutants were created. V247M was photoautotrophic and had an essentially normal phenotype. The A249T mutant, although photoautotrophic, was affected by artificial quinones, but less than the mutant carrying both the V247M and A249T changes. The results indicate a decreased plastoquinone affinity at the QA site in the strains carrying a A249T mutation, such that after dark-adaptation a significant percentage of the QA sites is empty or is occupied by an artificial quinone. In light, the percentage of photosystem II centers with plastoquinone bound at the QA site appears to increase, which may be due in part to an increased affinity of the semiquinone versus that of the quinone at the QA site.

Optimal sequence selection in proteins of known structure by simulated evolution.

Rational design of protein structure requires the identification of optimal sequences to carry out a particular function within a given backbone structure. A general solution to this problem requires that a potential function describing the energy of the system as a function of its atomic coordinates be minimized simultaneously over all available sequences and their three-dimensional atomic configurations. Here we present a method that explicitly minimizes a semiempirical potential function simultaneously in these two spaces, using a simulated annealing approach. The method takes the fixed three-dimensional coordinates of a protein backbone and stochastically generates possible sequences through the introduction of random mutations. The corresponding three-dimensional coordinates are constructed for each sequence by "redecorating" the backbone coordinates of the original structure with the corresponding side chains. These are then allowed to vary in their structure by random rotations around free torsional angles to generate a stochastic walk in configurational space. We have named this method protein simulated evolution, because, in loose analogy with natural selection, it randomly selects for allowed solutions in the sequence of a protein subject to the "selective pressure" of a potential function. Energies predicted by this method for sequences of a small group of residues in the hydrophobic core of the phage lambda cI repressor correlate well with experimentally determined biological activities. This "genetic selection by computer" approach has potential applications in protein engineering, rational protein design, and structure-based drug discovery.