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PubMed2002

Genetic engineering of allergens: future therapeutic products.

Genetic engineering of allergens for specific immunotherapy should aim at the production of modified molecules with reduced IgE-binding epitopes (hypoallergens), while preserving structural motifs necessary for T cell recognition (T cell epitopes) and for induction of IgG antibodies reactive with the natural allergen (blocking antibodies). Common approaches for engineering of hypoallergens usually require knowledge of T and B cell epitopes and involve changing specific base pairs (mutated gene), introduction of a new piece of DNA into the existing DNA molecule (chimeric or hybrid gene), and deletions (truncated gene or fragments). DNA family shuffling has the advantage that it does not require a priori knowledge of structural and functional properties for efficient generation of hypoallergens. The combination of the hypoallergen concept with the Th1-inducing genetic immunization approach might be an attractive alternative for protein-based immunotherapy.

Allergens↗
PubMed2004

[Application of bioinformatics in researches of industrial biocatalysis].

Industrial biocatalysis is currently attracting much attention to rebuild or substitute traditional producing process of chemicals and drugs. One of key focuses in industrial biocatalysis is biocatalyst, which is usually one kind of microbial enzyme. In the recent, new technologies of bioinformatics have played and will continue to play more and more significant roles in researches of industrial biocatalysis in response to the waves of genomic revolution. One of the key applications of bioinformatics in biocatalysis is the discovery and identification of the new biocatalyst through advanced DNA and protein sequence search, comparison and analyses in Internet database using different algorithm and software. The unknown genes of microbial enzymes can also be simply harvested by primer design on the basis of bioinformatics analyses. The other key applications of bioinformatics in biocatalysis are the modification and improvement of existing industrial biocatalyst. In this aspect, bioinformatics is of great importance in both rational design and directed evolution of microbial enzymes. Based on the successful prediction of tertiary structures of enzymes using the tool of bioinformatics, the undermentioned experiments, i.e. site-directed mutagenesis, fusion protein construction, DNA family shuffling and saturation mutagenesis, etc, are usually of very high efficiency. On all accounts, bioinformatics will be an essential tool for either biologist or biological engineer in the future researches of industrial biocatalysis, due to its significant function in guiding and quickening the step of discovery and/or improvement of novel biocatalysts.

Biocatalysis↗
PubMed1996

Recombinant Phabs reactive with 7,8-dihydro-8-oxoguanine, a major oxidative DNA lesion.

Antibody Fabs that bind to DNA damages provide useful models for understanding DNA damage-specific protein interactions. BSA and RSA conjugates of the nucleoside and nucleotide derivatives of the oxidative DNA lesions, 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), were used to immunize mice. RNA from the responders was isolated and used to repertoire clone and phage display Fabs that bind to these haptens. Direct binding and competitive enzyme-linked immunosorbent assay (ELISA) demonstrated that phage Fabs (Phabs) specific for 8-oxopurine-BSA conjugates and 8-oxoguanine were produced although the Phabs did not react with 8-oxopurines in DNA. Amino acid sequence comparisons among clones having different binding properties suggested that a relatively small portion of the binding surfaces defined by the complementarity determining regions (CDR) accounted for hapten binding specificity, whereas other regions appeared to stabilize hapten binding by interacting with protein or DNA epitopes. Chain shuffling between 8-oxopurine-BSA binding Fabs and a DNA binding Fab showed that the heavy chain of the DNA binder conferred DNA binding capacity to the light chain of only one of the 8-oxopurine-BSA binders. Homology modeling of the 8-oxoG-specific clone g37 showed significant similarities to two previously isolated monoclonal antibodies specific for single-stranded nucleic acids. In the 8-oxoG Fab, which did not bind to DNA, the presumptive DNA binding canyon was blocked by heavy chain residues in the CDR2 region and appeared to lack part of the canyon wall due to the different placement of the light chain framework region.

Amino Acid Sequence↗
PubMed1999

Repetitive use of a phosphate-binding module in DNA polymerase beta, Oct-1 POU domain and phage repressors.

Motifs for sequence specific-protein-DNA interactions, such as helix-turn-helix, zinc finger and leucine zipper, are now better understood as a result of extensive studies of three-dimensional (3D) structures of transcription factors. On the other hand, little attention has been paid to motifs for sequence nonspecific binding, namely DNA-phosphate binding. To address the question whether different transcription factors and DNA manipulation enzymes, that is enzymes that work on DNA, share a similar mode of phosphate binding, we surveyed interactions between DNA and protein module, a structural unit of a globular protein. We analyzed the modular organization of DNA polymerase beta and found that residues making contact with DNA phosphates were localized to five modules. Structural comparison of these phosphate-binding modules against others in transcription factors and DNA manipulation enzymes revealed that DNA polymerase beta, the Oct-1 POU domain, 434 Cro and the Arc repressor have a phosphate-binding module with 3D structures similar to one another. This newly detected module, the phosphate-binding helix-turn-helix (pbHTH) module, named for its function and 3D structure, interacts with DNA by (i) making hydrogen bonds between a DNA phosphodiester oxygen and an amino hydrogen of the main chain located at the N-terminus of a C-terminal alpha-helix, and (ii) making electrostatic interactions between DNA phosphates and side chains of lysine or arginine. Finding structurally and functionally similar phosphate-binding units in different transcription factors and DNA manipulation enzymes suggests that shuffling of modules is not limited to the DNA base-recognition motif. Phosphate-binding modules are apparently also shuffled in DNA-binding proteins.

Amino Acid Sequence↗
PubMed2003

Trans mobilization of genomic DNA as a mechanism for retrotransposon-mediated exon shuffling.

Exon shuffling, the juxtaposition and new combinations of exons from different genes, facilitates evolutionary changes by increasing protein diversity or by generating new function. Exon shuffling is generated as a consequence of segmental duplications. Long interspersed element (LINE)-1 (L1)-mediated 3' transduction is a potential pathway for exon shuffling by which L1 associates 3' flanking DNA in cis as a read-through transcript and carries the DNA to a new genomic location. In this pathway, however, the targets are limited to the regions located 3' to L1s. Here we propose that the genomic DNA distant from L1 may be mobilized by an alternative (trans) action of L1. A partial ATM sequence containing a single exon and flanking introns has been retrotransposed to a new genomic location on chromosome 7. There was no L1 around the exon of the authentic ATM locus. An unusual feature that the poly(A) tail tagged to the transposed sequence oriented oppositely to the ATM's transcriptional orientation suggests that a trans action of reverse transcriptase on antisense transcript has driven the duplication of genomic DNA without removing introns. Taking account of similar duplication events in previous studies, a certain class of segmental duplications in the human genome may be accounted for by the trans action of retrotransposon machinery.

Animals↗
PubMed1995

Evolution of the intron-exon structure of eukaryotic genes.

The origin and evolution of intron-exon structures continue to be controversial topics. Two alternative theories, the 'exon theory of genes' and the 'insertional theory of introns', debate the presence or absence of introns in primordial genes. Both sides of the argument have focused on the positions of introns with respect to protein and gene structures. A new approach has emerged in the study of the evolution of intron-exon structures: a population analysis of genes. One example is the statistical analysis of intron phases--the position of introns within or between codons. This analysis detected a significant signal of exon shuffling in the DNA sequence database containing both ancient and modern exon sequences: intron phase correlations, that is, the association together within genes of introns of the same phase. The results of this analysis suggest that exon shuffling played an important role in the origin of both ancient and modern genes.

Animals↗
PubMed1989

HLA genes associated with rheumatoid arthritis. Identification of susceptibility alleles using specific oligonucleotide probes.

We examined the association of individual HLA genes with rheumatoid arthritis (RA), using oligonucleotide probes that identified both DR4-associated and non-DR4-associated genes. Two distinct HLA-DR beta alleles (Dw4 and Dw14) were found in DR4+ RA patients compared with controls (Dw4 50% versus 17%; Dw14 35% versus 5%; total DR4 73% versus 30%), indicating that these 2 alleles are independent susceptibility genes. Remarkably, the majority of the DR4- RA patients also demonstrated a linear DNA sequence, apparently "shuffled" between different susceptibility alleles, identified with an oligonucleotide probe to a key portion of the Dw14 gene.

Adult↗
PubMed2001

Differential rates of reversibility of ecteinascidin 743-DNA covalent adducts from different sequences lead to migration to favored bonding sites.

Ecteinascidin 743 (Et 743), one of a series of structurally related antitumor antibiotics isolated from a marine tunicate, is currently in phase II clinical trials. Et 743 alkylates guanine N2 through the minor groove of DNA. Hydrogen-bonding networks that associate the drug with a three base pair DNA recognition site have been proposed to contribute to both the reactivity and the stability of the Et 743-DNA adduct. Here, we report that the reaction of Et 743 with DNA is reversible under nondenaturing conditions and that the rate of this reverse reaction depends critically upon the DNA-modified sequence. Quite unexpectedly, it was found that although the rates of alkylation are similar for the 5'-AGT and 5'-AGC sequences, reversal from the 5'-AGT sequence occurs faster than from the 5'-AGC sequence. Consequently, it is the differences in the rate of the reverse reaction that dictate the sequence selectivity of Et 743 toward its favored target sequence. As a direct consequence of the reversible nature of Et 743 with DNA, Et 743 can migrate from the nonfavored bonding sequence (e.g., 5'-AGT) to the favored DNA target site (e.g., 5'-AGC). The data suggest that the observed differences in the rate of reversibility arise from differences in the stability of the Et 743-DNA adduct at the 5'-AGT and 5'-AGC target sequences. On the basis of gel electrophoresis and (1)H NMR experiments, the Et 743-AGT adduct is less stable, has more dynamic motion, and produces different conformational changes in the DNA than the more stable Et 743-AGC adduct. The shuffling of Et 743-DNA adducts to the more stable alkylation sites has important implications for understanding the underlying relationship between the structural modification of DNA by Et 743 and its biological potency and efficacy in tumor cells.

Antigens, Polyomavirus Transforming↗
PubMed1999

Novel family shuffling methods for the in vitro evolution of enzymes.

It has recently been shown that shuffling of the amino acid sequences of family enzymes allows the generation of improved enzymes. Family shuffling is generally achieved by a DNase I treatment and then by PCR. Shuffling of the xylE and nahH genes, both encoding catechol 2,3-dioxygenases, was carried out by the published method. However, nahH-xylE hybrids were only formed at a very low frequency (less than 1%). Therefore, we developed improved methods for family shuffling by which DNA was cleaved by restriction enzymes instead of by DNase I. With the first improved method, five nahH fragments and five xylE fragments that had been generated by restriction enzyme digestion were subjected to the PCR reactions in two steps, the first being without a primer and the second with a set of primers. This method enabled nahH-xylE hybrid genes to be formed at a high frequency (almost 100%). With the second improved method, nahH and xylE were cleaved by several sets of restriction enzymes, and these digests were then reassembled in two steps. The nahH and xylE DNAs were each cleaved by two (or three) sets of restriction enzymes, and one type of nahH digest and one type of xylE digest were mixed, thus making four (or nine) different mixtures of the nahH and xylE digests. These mixtures were used as templates to carry out PCR without a primer. After the first PCR reaction, all the mixtures were combined, and a second PCR reaction was carried out without a primer. Following these two PCR assembly steps, a third PCR reaction was carried out with two primers to amplify the full-length nahH-xylE hybrid genes. This second method also yielded nahH-xylE hybrids at a frequency of 100%. The degree of recombination of the products with the second method was higher than that with the first method. These methods were used to isolate catechol 2,3-dioxygenases exhibiting relatively high stability at high temperature, one of them being respectively 13- and 26-fold more thermostable than XylE and NahH at 50 degrees C.

Amino Acid Sequence↗
PubMed2000

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

Base Sequence↗
PubMed2005

Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon-intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

Base Sequence↗
PubMed2006

Synthetic protein-protein interaction domains created by shuffling Cys2His2 zinc-fingers.

Cys2His2 zinc-fingers (C2H2 ZFs) mediate a wide variety of protein-DNA and protein-protein interactions. DNA-binding C2H2 ZFs can be shuffled to yield artificial proteins with different DNA binding specificities. Here we demonstrate that shuffling of C2H2 ZFs from transcription factor dimerization zinc-finger (DZF) domains can also yield two-finger DZFs with novel protein-protein interaction specificities. We show that these synthetic protein-protein interaction domains can be used to mediate activation of a single-copy reporter gene in bacterial cells and of an endogenous gene in human cells. In addition, the synthetic two-finger domains we constructed can also be linked together to create more extended, four-finger interfaces. Our results demonstrate that shuffling of C2H2 ZFs can yield artificial protein-interaction components that should be useful for applications in synthetic biology.

Binding Sites↗
PubMed1984

Kinetic evidence for redistribution of actinomycin molecules between potential DNA-binding sites.

The kinetics of interaction between actinomycin D and DNA have been measured by stopped-flow and detergent-dissociation methods. The results are consistent with a model in which the antibiotic initially binds to many sequences on the heterogeneous DNA lattice and subsequently 'shuffles' between the available sites until a thermodynamically determined optimal state of binding is attained. The amplitudes of the two slowest components in the reaction with calf thymus DNA do not vary in parallel as the total level of antibiotic binding is increased; they appear to reflect directly the redistribution of antibiotic molecules along the DNA lattice. The dissociation profile is shown to depend upon the time for which the antibiotic and DNA are premixed, so that for short mixing times a higher proportion of the decay is represented by faster-dissociating species. The rate of appearance of the slowest-dissociating species correlates well with the slowest optical change in the association reaction. Stopped-flow experiments indicate that the antibiotic first binds to sites on natural DNA with an average association constant of 4 X 10(3) M-1 and that it subsequently migrates to sites with higher affinity. Similar experiments performed with poly(dG-dC) are less easily interpreted and seem to indicate that conformational changes or cooperative effects can also occur.

Binding Sites↗
PubMed1985

Kinetic evidence that echinomycin migrates between potential DNA binding sites.

The hypothesis that echinomycin locates its preferred nucleotide sequences in DNA by a process of "shuffling" between potential binding sites has been tested. Immediately after reacting with calf thymus DNA the antibiotic is relatively weakly bound inasmuch as the complex dissociates quite rapidly when detergent is added. If the complex is allowed to equilibrate for various periods of time after mixing, an increasing proportion of the bound antibiotic dissociates slowly on addition of detergent. The kinetics of appearance of the slowly-dissociating form, and its dependence upon ionic strength, are fully consistent with the shuffling model. In contrast the dissociation profiles from poly(dG-dC) and poly(dA-dT) are independent of mixing time.

Base Sequence↗
PubMed2003

Fluorescent microplate-based analysis of protein-DNA interactions. II: Immobilized DNA.

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Adsorption↗
PubMed2005

Monitoring the DNA binding kinetics of a binuclear ruthenium complex by energy transfer: evidence for slow shuffling.

The semirigid binuclear ruthenium complex Delta,Delta-[mu-(11,11'-bidppz)(phen)(4)Ru(2)](4+) has been shown to rearrange slowly from an initial groove-bound nonluminescent state to a final intercalated emissive state by threading one of its bulky Ru(phen)(2) moieties through the DNA base stack. When this complex binds to poly[d(A-T)(2)], a further increase in emission from the complex is observed after completion of the intercalation, assigned to reorganization of the intercalated complex. We here report a study of the threading process in poly[d(A-T)(2)], in which the minor groove binding dye DAPI is used as an energy transfer probe molecule to assess the distribution of ruthenium complex during and also after the actual threading phase. The emission from DAPI is found to change with the same rate as the emission from the ruthenium complex, and furthermore, DAPI does not disturb the binding kinetics of the latter, justifying it as a good probe of both the threading and the reorganization processes. We conclude from the change in the emission from both DAPI and the ruthenium complex with time that DAPI-ruthenium interactions are most pronounced during the process of threading of the complex, suggesting that the complexes are initially threaded slightly anticooperatively and thereafter redistribute along the DNA to reach their thermodynamically most favorable distribution. The final distribution is characterized by a small but significant binding cooperativity, probably as a result of hydrophobic interactions between the complex ions despite their tetravalent positive charges. The mechanism of "shuffling" the complex along the DNA chain is discussed, i.e., whether the ruthenium complex remains threaded (requiring sequential base-pair openings) or if unthreading followed by lateral diffusion within the ionic atmosphere of the DNA and rethreading occurs.

DNA↗