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PubMed1997

A quantum-theoretical approach to the phenomenon of directed mutations in bacteria (hypothesis).

The Darwinian paradigm of biological evolution is based on the independence of genetic variations from selection which occurs afterwards. However, according to the phenomenon of directed mutations, some genetic variations occur mostly when the conditions favorable for their growth are created. I propose that the explanation of this phenomenon should not rely on any special 'mechanism' for the appearance of directed mutations, but rather should be based on the principles of quantum theory. I consider a physical model of adaptation whereby a polarized photon, passing through a polarizer, changes its polarization according to the angle of the polarizer. This adaptation occurs by selection of the 'fitted' polarized state which exists as a component of superposition in the initial state of the photon. However, since the same state of the incoming photon should be decomposed differently depending on the angle of the polarizer, in this case the set of variations subjected to selection depends upon the selective conditions themselves. This reveals the crucial difference between this model of adaptation and canonical Darwinian selection. Based on this analogy, the capacity of a cell to grow in particular conditions is considered an observable of the cell; the plating experiments are interpreted as measurement of this observable. The only nontrivial suggestion of the paper states that the cell, analogously to the polarized photon, may be in a state of superposition of eigenfunctions of the operator which represents this observable, and with some probability can appear as a mutant upon the measurement. Alternative growth conditions correspond to the decomposition of the same state vector into a different superposition, consistent with measurement of a different observable and appearance of different mutants. Thus, consistent with the suggested analogy, directed mutations are explained as a result of random choice from the set of outcomes determined by the environment.

Adaptation, Physiological↗
PubMed1998

Hsp90 as a capacitor for morphological evolution.

The heat-shock protein Hsp90 supports diverse but specific signal transducers and lies at the interface of several developmental pathways. We report here that when Drosophila Hsp90 is mutant or pharmacologically impaired, phenotypic variation affecting nearly any adult structure is produced, with specific variants depending on the genetic background and occurring both in laboratory strains and in wild populations. Multiple, previously silent, genetic determinants produced these variants and, when enriched by selection, they rapidly became independent of the Hsp90 mutation. Therefore, widespread variation affecting morphogenic pathways exists in nature, but is usually silent; Hsp90 buffers this variation, allowing it to accumulate under neutral conditions. When Hsp90 buffering is compromised, for example by temperature, cryptic variants are expressed and selection can lead to the continued expression of these traits, even when Hsp90 function is restored. This provides a plausible mechanism for promoting evolutionary change in otherwise entrenched developmental processes.

Animals↗
PubMed1999

Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin.

RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (Ki) toward subtilisin was 2.5 microM.

Animals↗
PubMed2004

Evolution of a T7 RNA polymerase variant that transcribes 2'-O-methyl RNA.

Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior. In particular, modifications at the 2'-OH of ribose are important for enhancing the stability of RNA. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2' position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript.

DNA-Directed RNA Polymerases↗
PubMed2006

In vitro evolution of flexizymes that function under the conditions in translation system.

Flexizyme is an artificial ribozyme capable of acylating any kinds of tRNAs with virtually no limitation of acid donor substrates. Although the flexizyme system is a highly flexible tRNA acylation tool, it requires relatively high Mg(2+) concentrations (>50 mM) for the full function. For instance, lowering the Mg(2+) concentration to 10 mM, where the in vitro translation system generally functions, resulted in significant decrease in activity. To overcome this limitation, we implanted a new random domain into a part of flexizyme to aim at selecting a new functional domain that would exhibit acylation activity at lower Mg(2+) concentrations. Indeed, in vitro selection of active species from such an RNA pool afforded a new flexizyme, called mdFx, showing a 3-fold higher yield at 10 mM Mg(2+) concentration. Thus, this new flexizyme opens a new avenue to construct an mdFx-transcription-translation coupled system.

Base Sequence↗
PubMed1993

A combined molecular and cytogenetic approach to genome evolution in Drosophila using large-fragment DNA cloning.

Methods of genome analysis, including the cloning and manipulation of large fragments of DNA, have opened new strategies for uniting molecular evolutionary genetics with chromosome evolution. We have begun the development of a physical map of the genome of Drosophila virilis based on large DNA fragments cloned in bacteriophage P1. A library of 10,080 P1 clones with average insert sizes of 65.8 kb, containing approximately 3.7 copies of the haploid genome of D. virilis, has been constructed and characterized. Approximately 75% of the clones have inserts exceeding 50 kb, and approximately 25% have inserts exceeding 80 kb. A sample of 186 randomly selected clones was mapped by in situ hybridization with the salivary gland chromosomes. A method for identifying D. virilis clones containing homologs of D. melanogaster genes has also been developed using hybridization with specific probes obtained from D. melanogaster by means of the polymerase chain reaction. This method proved successful for nine of ten genes and resulted in the recovery of 14 clones. The hybridization patterns of a sample of P1 clones containing repetitive DNA were also determined. A significant fraction of these clones hybridizes to multiple euchromatic sites but not to the chromocenter, which is a pattern of hybridization that is very rare among clones derived from D. melanogaster. The materials and methods described will make it possible to carry out a direct study of molecular evolution at the level of chromosome structure and organization as well as at the level of individual genes.

Animals↗
PubMed2006

mAMSA resistant human topoisomerase IIbeta mutation G465D has reduced ATP hydrolysis activity.

Type II Human DNA Topoisomerases (topos II) play an essential role in DNA replication and transcription and are important targets for cancer chemotherapeutic drugs. Topoisomerase II causes transient double-strand breaks in DNA, forming a gate through which another double helix is passed, and acts as a DNA dependent ATPase. Mutations in topoII have been linked to atypical multi-drug resistance. Both human Topoisomerase II isoforms, alpha and beta, are targeted by amsacrine. We have used a forced molecular evolution approach to identify mutations conferring resistance to acridines. Here we report mutation betaG465D, which was selected with mAMSA and DACA and is cross-resistant to etoposide, ellipticine and doxorubicin. Resistance to mAMSA appears to decrease over time indicating a previously unreported resistance mechanism. G465D lies within the B' domain in the region that contacts the cleaved gate helix. There is a 3-fold decrease in ATP affinity and ATP hydrolysis and an altered requirement for magnesium in decatenation assays. The decatenation rate is decreased for the mutated G465D protein. And we report for the first time the use of fluorescence anisotropy with intact human topoisomerase II.

Acridines↗
PubMed2006

Culture of Spirulina platensis in human urine for biomass production and O(2) evolution.

Attempts were made to culture Spirulina platensis in human urine directly to achieve biomass production and O(2) evolution, for potential application to nutrient regeneration and air revitalization in life support system. The culture results showed that Spirulina platensis grows successfully in diluted human urine, and yields maximal biomass at urine dilution ratios of 140 approximately 240. Accumulation of lipid and decreasing of protein occurred due to N deficiency. O(2) release rate of Spirulina platensis in diluted human urine was higher than that in Zarrouk medium.

Biomass↗
PubMed2006

Design and evolution of new catalytic activity with an existing protein scaffold.

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.

Amino Acid Sequence↗
PubMed2006

Metabolic analysis of adaptive evolution for in silico-designed lactate-producing strains.

Experimental evolution is now frequently applied to many biological systems to achieve desired objectives. To obtain optimized performance for metabolite production, a successful strategy has been recently developed that couples metabolic engineering techniques with laboratory evolution of microorganisms. Previously, we reported the growth characteristics of three lactate-producing, adaptively evolved Escherichia coli mutant strains designed by the OptKnock computational algorithm. Here, we describe the use of (13)C-labeled experiments and mass distribution measurements to study the evolutionary effects on the fluxome of these differently designed strains. Metabolic flux ratios and intracellular flux distributions as well as physiological data were used to elucidate metabolic responses over the course of adaptive evolution and metabolic differences among strains. The study of 3 unevolved and 12 evolved engineered strains as well as a wild-type strain suggests that evolution resulted in remarkable improvements in both substrate utilization rate and the proportion of glycolytic flux to total glucose utilization flux. Among three strain designs, the most significant increases in the fraction of glucose catabolized through glycolysis (>50%) and the glycolytic fluxes (>twofold) were observed in phosphotransacetylase and phosphofructokinase 1 (PFK1) double deletion (pta- pfkA) strains, which were likely attributed to the dramatic evolutionary increase in gene expression and catalytic activity of the minor PFK encoded by pfkB. These fluxomic studies also revealed the important role of acetate synthetic pathway in anaerobic lactate production. Moreover, flux analysis suggested that independent of genetic background, optimal relative flux distributions in cells could be achieved faster than physiological parameters such as nutrient utilization rate.

Acetate-CoA Ligase↗
PubMed2003

Preferred RNA binding sites for a threading intercalator revealed by in vitro evolution.

In pursuit of small molecules capable of controlling the function of RNA targets, we have explored the RNA binding properties of peptide-acridine conjugates (PACs). In vitro evolution (SELEX) was used to isolate RNAs capable of binding the PAC Ser-Val-Acr-Arg, where Acr is an acridine amino acid. The PAC binds RNA aptamers selectively and with a high degree of discrimination over DNA. PAC binding sites contain the base-paired 5'-CpG-3' sequence, a known acridine intercalation site. However, RNA structure flanking this sequence causes binding affinities to vary over 30-fold. The preferred site (K(D) = 20 nM) contains a base-paired 5'-CpG-3' step flanked on the 5' side by a 4 nt internal loop and the 3' side by a bulged U. Several viral 5'- and 3'-UTR RNA sequences that likely form binding sites for this PAC are identified.

Acridines↗
PubMed2000

Design and evolution of artificial M13 coat proteins.

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.

Amino Acid Sequence↗
PubMed2000

Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif.

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 microM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.

Base Sequence↗
PubMed2001

In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates.

The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity.

Directed Molecular Evolution↗
PubMed2001

RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension.

The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.

Base Sequence↗
PubMed2005

Degenerate oligonucleotide gene shuffling (DOGS) and random drift mutagenesis (RNDM): two complementary techniques for enzyme evolution.

Improvement of the biochemical characteristics of enzymes has been aided by misincorporation mutagenesis and DNA shuffling. Many gene shuffling techniques result predominantly in the regeneration of unshuffled (parental) molecules. We describe a procedure for gene shuffling using degenerate primers that allows control of the relative levels of recombination between the genes that are shuffled, and reduces the regeneration of unshuffled parental genes. This shuffling procedure avoids the use of endonucleases for gene fragmentation prior to shuffling and allows the inclusion of random mutagenesis of selected portions of the chimeric genes as part of the procedure. We illustrate the use of the shuffling technique with a family of beta-xylanase genes that possess widely different G+C contents. In addition, we introduce a new method (RNDM) for rapid screening of mutants from libraries where no adaptive selection has been imposed on the cells. They are identified only by their retention of enzymatic activity. The combination of RNDM followed by DOGS allows a comprehensive exploration of a protein's functional sequence space.

Bacterial Proteins↗
PubMed1999

In vitro evolution of thermostable p53 variants.

The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53delta30 variant, which lacks the self-inhibiting carboxy terminal domain, has a half-life of only 8 min at 37 degrees C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53delta30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53delta30 variant has a half-life of 100 min at 37 degrees C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild-type protein.

DNA Mutational Analysis↗
PubMed1998

An evolutionary approach to the design of glutathione-linked enzymes.

Studies of protein structure provide information about principles of protein design that have come into play in natural evolution. This information can be exploited in the redesign of enzymes for novel functions. The glutathione-binding domain of glutathione transferases has similarities with structures in other glutathione-linked proteins, such as glutathione peroxidases and thioredoxin (glutaredoxin), suggesting divergent evolution from a common ancestral protein fold. In contrast, the binding site for glutathione in human glyoxalase I is located at the interface between the two identical subunits of the protein. Comparison with the homologous, but monomeric, yeast glyoxalase I suggests that new domains have originated through gene duplications, and that the oligomeric structure of the mammalian glyoxalase I has arisen by 'domain swapping'. Recombinant DNA techniques are being used for the redesign of glutathione-linked proteins in attempts to create binding proteins with novel functions and catalysts with tailored specificities. Enzymes with desired properties are selected from libraries of variant structures by use of phage display and functional assays.

Binding Sites↗