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Results for “DNA Shuffling”

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PubMed2003

Diversification of exogenous genes in vivo in Neurospora.

We have adapted the meiotic recombination hotspot cog of Neurospora crassa for shuffling exogenous DNA, providing a means of generating novel genes in situ from sequences introduced into chromosomes. Genes to be diversified are inserted between the his-3 locus and cog. Diversification crosses are heterozygous both for alleles of the exogenous DNA and for auxotrophic alleles of his-3. Progeny selected for ability to grow without histidine supplementation are enriched for exchange events within the exogenous DNA. Exchange events initiated by cog can propagate past DNA sequences mismatched for more than 370 bp and complete exchanges in patches of matched sequence as short as 24 bp, parameters that make the system suited for use in the directed evolution of genes for protein engineering. Here we demonstrate the system by shuffling human immunoglobulin kappa chain genes and also endoglucanase genes derived from different species of fungi.

Cellulase↗
PubMed1996

Most frequent scenario for recurrent Candida vaginitis is strain maintenance with "substrain shuffling": demonstration by sequential DNA fingerprinting with probes Ca3, C1, and CARE2.

The following three basic scenarios have emerged for the genetic relatedness of strains in recurrent vaginal candidiasis: strain maintenance without genetic variation, strain maintenance with minor genetic variation, and strain replacement. To test the frequency of each of the three scenarios, the genetic relatedness of Candida albicans isolates from each of 18 patients with recurrent infections was assessed by sequential DNA fingerprinting with the following three probes: the Ca3 probe; the C1 probe, a subfragment of the Ca3 probe which hybridizes to hypervariable genomic fragments; and the unrelated CARE2 probe. In each of the 18 patients with recurrent infections, the same strain was responsible for sequential infections, suggesting that the predominant scenario is strain maintenance. However, in 56% of these patients, the strain exhibited minor genetic variations in sequential infections. These changes were not found to be progressive. Rather, the changes suggest that substrains of an established infecting strain are shuffled in sequential infections. Results are also presented that in 45% of patients with recurrent infections, oral and vulvovaginal isolates were identical, in 35% they were highly related but not identical, and in 20% they were unrelated. These results differ markedly from those for commensal isolates simultaneously cultured from the oral cavity and vulvovaginal region of healthy individuals. Finally, it is demonstrated that in all eight cases in which C. albicans was isolated from both the male sexual partner of the patient with a recurrent infection and the patient, an isolate from the male partner was identical or highly related to the vulvovaginal strain. These results demonstrate that in patients with recurrent vulvovaginitis, a single strain usually dominates both in the different body locations of the patient and in the male partner and that it is maintained through sequential infections. However, in patients with recurrent infections, different substrains of the established clone dominate in an apparently random fashion, a process that we refer to as "substrain shuffling".

Candida albicans↗
PubMed2005

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system.

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors.

Cloning, Molecular↗
PubMed2004

Genome shuffling improves degradation of the anthropogenic pesticide pentachlorophenol by Sphingobium chlorophenolicum ATCC 39723.

Pentachlorophenol (PCP), a highly toxic anthropogenic pesticide, can be mineralized by Sphingobium chlorophenolicum, a gram-negative bacterium isolated from PCP-contaminated soil. However, degradation of PCP is slow and S. chlorophenolicum cannot tolerate high levels of PCP. We have used genome shuffling to improve the degradation of PCP by S. chlorophenolicum. We have obtained several strains that degrade PCP faster and tolerate higher levels of PCP than the wild-type strain. Several strains obtained after the third round of shuffling can grow on one-quarter-strength tryptic soy broth plates containing 6 to 8 mM PCP, while the original strain cannot grow in the presence of PCP at concentrations higher than 0.6 mM. Some of the mutants are able to completely degrade 3 mM PCP in one-quarter-strength tryptic soy broth, whereas no degradation can be achieved by the wild-type strain. Analysis of several improved strains suggests that the improved phenotypes are due to various combinations of mutations leading to an enhanced growth rate, constitutive expression of the PCP degradation genes, and enhanced resistance to the toxicity of PCP and its metabolites.

Base Sequence↗
PubMed2001

Comparative analysis for expressed genes by polymerase chain reaction using module-shuffling primers.

We have developed a method for comparative analysis of gene expression. It is based on competitive PCR amplification with module-shuffling primers, followed by gel electrophoresis in a fluorescent DNA sequencer. In this method, tagged-cDNA restriction fragments derived from different sources were amplified in one tube at the same amplification efficiency. The method can detect different amounts of each expressed gene, up to difference in amounts of 30%. The method was successfully used for comparative analysis of expressed genes in yeast.

DNA Primers↗
PubMed2004

Module shuffling.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1999

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.

Antibodies↗
PubMed2001

Random multi-recombinant PCR for the construction of combinatorial protein libraries.

Development of a new methodology to create protein libraries, which enable the exploration of global protein space, is an exciting challenge. In this study we have developed random multi-recombinant PCR (RM-PCR), which permits the shuffling of several DNA fragments without homologous sequences. In order to evaluate this methodology, we applied it to create two different combinatorial DNA libraries. For the construction of a 'random shuffling library', RM-PCR was used to shuffle six DNA fragments each encoding 25 amino acids; this affords many different fragment sequences whose every position has an equal probability to encode any of the six blocks. For the construction of the 'alternative splicing library', RM-PCR was used to perform different alternative splicings at the DNA level, which also yields different block sequences. DNA sequencing of the RM-PCR products in both libraries revealed that most of the sequences were quite different, and had a long open reading frame without a frame shift or stop codon. Furthermore, no distinct bias among blocks was observed. Here we describe how to use RM-PCR for the construction of combinatorial DNA libraries, which encode protein libraries that would be suitable for selection experiments in the global protein space.

Alternative Splicing↗
PubMed2002

Extensive domain shuffling in transcription regulators of DNA viruses and implications for the origin of fungal APSES transcription factors.

BACKGROUND: Viral DNA-binding proteins have served as good models to study the biochemistry of transcription regulation and chromatin dynamics. Computational analysis of viral DNA-binding regulatory proteins and identification of their previously undetected homologs encoded by cellular genomes might lead to a better understanding of their function and evolution in both viral and cellular systems. RESULTS: The phyletic range and the conserved DNA-binding domains of the viral regulatory proteins of the poxvirus D6R/N1R and baculoviral Bro protein families have not been previously defined. Using computational analysis, we show that the amino-terminal module of the D6R/N1R proteins defines a novel, conserved DNA-binding domain (the KilA-N domain) that is found in a wide range of proteins of large bacterial and eukaryotic DNA viruses. The KilA-N domain is suggested to be homologous to the fungal DNA-binding APSES domain. We provide evidence for the KilA-N and APSES domains sharing a common fold with the nucleic acid-binding modules of the LAGLIDADG nucleases and the amino-terminal domains of the tRNA endonuclease. The amino-terminal module of the Bro proteins is another, distinct DNA-binding domain (the Bro-N domain) that is present in proteins whose domain architectures parallel those of the KilA-N domain-containing proteins. A detailed analysis of the KilA-N and Bro-N domains and the associated domains points to extensive domain shuffling and lineage-specific gene family expansion within DNA virus genomes. CONCLUSIONS: We define a large class of novel viral DNA-binding proteins and their cellular homologs and identify their domain architectures. On the basis of phyletic pattern analysis we present evidence for a probable viral origin of the fungus-specific cell-cycle regulatory transcription factors containing the APSES DNA-binding domain. We also demonstrate the extensive role of lineage-specific gene expansion and domain shuffling, within a limited set of approximately 24 domains, in the generation of the diversity of virus-specific regulatory proteins.

Amino Acid Sequence↗
PubMed1992

DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae.

Two temperature-sensitive DNA polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.

Amino Acid Sequence↗
PubMed2005

Prevalence of quadruplexes in the human genome.

Guanine-rich DNA sequences of a particular form have the ability to fold into four-stranded structures called G-quadruplexes. In this paper, we present a working rule to predict which primary sequences can form this structure, and describe a search algorithm to identify such sequences in genomic DNA. We count the number of quadruplexes found in the human genome and compare that with the figure predicted by modelling DNA as a Bernoulli stream or as a Markov chain, using windows of various sizes. We demonstrate that the distribution of loop lengths is significantly different from what would be expected in a random case, providing an indication of the number of potentially relevant quadruplex-forming sequences. In particular, we show that there is a significant repression of quadruplexes in the coding strand of exonic regions, which suggests that quadruplex-forming patterns are disfavoured in sequences that will form RNA.

Algorithms↗
PubMed2005

Identification of peptide ligands to the chemokine receptor CCR5 and their maturation by gene shuffling.

The determination of protein-protein interactions and their role in diverse pathophysiological processes is a promising approach to the identification of molecules of therapeutic potential. This paper describes the identification of peptidic CCR5 receptor ligands as potential drug leads against HIV-1 infection using in vitro evolution based on phage display. A phage-displayed peptide library was used to select for anti-CCR5 peptide. Further in vitro evolution of the peptide by exon shuffling was performed to identify peptides with optimized characteristics for CCR5 receptor. This peptide inhibited HIV coreceptor activity in a cell fusion assay with an IC50 of 5 microM. It did not exhibit either agonistic or antagonistic activity on CCR5 in the concentration range used. To our knowledge, this is a first report that describes the identification of peptide ligands specific to the CCR5 receptor from a phage-displayed library and the maturation of the selected peptide sequence by gene shuffling.

Animals↗
PubMed1990

The nucleotide sequence of a nodule-specific gene, Nms-25 of Medicago sativa: its primary evolution via exon-shuffling and retrotransposon-mediated DNA rearrangements.

We present the primary structure of a nodule-specific gene, Nms-25 from Medicago sativa L. cultivar Nagyszénási. Analysis of the nucleotide sequence of Nms-25 revealed that this gene shows all the characteristics of an interrupted plant gene consisting of 13 exons and 12 introns. The promoter region of Nms-25 contains the common promoter elements of plant genes as well as motifs which are supposed to be involved in nodule-specific expression. There are two exon-like sequences in the gene named PE1 and PE2 which are not present in the cDNA clones of Medicago sativa cultivar Cardinal. Intron 9 carries a retrotransposon-like element, Tms1, which might be responsible for downstream deletion events in which a heptanucleotide, ATTAGCT, might have been involved. Most of the exons, except 1, 12 and 13, are similar to each other both in length (54 bp) and sequence (up to 94% sequence similarity). All exons are interrupted by introns in the same phase (type I). It is suggested that exon-shuffling based on illegitimate recombination in which the ATTAGCT motif might have played an active role, and retrotransposon-mediated DNA rearrangements were the primary events in the molecular evolution of the Nms-25 gene.

Amino Acid Sequence↗
PubMed2026

Whole-Genome Analysis of Bacillus Licheniformis Ali5 and Synthesis of Lichenysin via Genome Shuffling.

Whole-genome sequencing of Bacillus licheniformis Ali5 was performed via MGI-seq PE150 and Nanopore single-molecule real-time sequencing. The strain has a 4,114,664 bp circular genome encoding 4030 protein-coding genes. Functional annotation across NR, COG, GO, KEGG, CARD, BacMet, and CAZy databases identified 4025, 2812, 988, 1242, 72, 69, and 94 corresponding genes, respectively, and antiSMASH 6.0 revealed multiple antimicrobial biosynthetic gene clusters, including intact lichenysin and lichenicidin VK21 A1/A2 gene clusters. Three rounds of recursive protoplast fusion-based genome shuffling, paired with a dual-index screening system, significantly improved strain growth and lichenysin biosynthesis. Recombinants exhibited shortened lag phase, enhanced proliferation, improved stationary-phase stability, and higher diauxic peak biomass. PP3-176 and PP3-186 showed 4.6%-8.1% higher 12-h shake-flask titer and 3.1%-4.0% higher maximum titer than the parental average, with excellent fermentation stability. 1-L bioreactor validation confirmed strong scale-up potential. PP3-186 achieved 27.2% and 31.6% titer increases at 12 h and 20 h, while PP3-176 yielded 20.4% and 14.6% improvements with robust metabolic performance. This study validates genome shuffling as an effective strategy for enhancing lichenysin production, providing candidate strains and technical support for industrial application.

Bacillus licheniformis↗
PubMed2004

Dealing with complexity: evolutionary engineering and genome shuffling.

Comparative analysis of the growing number of microbial genome sequences has shown a high plasticity of genomes and several mechanisms for the adaptation of microbial cells to changing environmental conditions have been discovered. By contrast, the underlying metabolic networks of microorganisms are under strict control and relatively rigid, which poses a significant challenge for rational metabolic engineering approaches. Recursive shuffling of whole genomes has recently been demonstrated as an effective new evolutionary whole-cell engineering approach for the rapid improvement of industrially important microbial phenotypes.

Bacterial Proteins↗
PubMed2004

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol.

3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.

Catechols↗
PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗