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PubMed1996

A cytoplasmic gene-shuffle system in Kluyveromyces lactis: use of epitope tagging to detect a killer plasmid-encoded gene product.

A novel gene shuffle approach has been developed for investigating the functions of genes on the cytoplasmic linear DNA killer plasmids of Kluyveromyces lactis. By transplacing k2ORF5 from larger plasmid pGKL2(k2) onto pGKL1(k1) we have shown this gene to be essential and functionally interchangeable between plasmids. Once transferred onto k1, k2ORF5 is fully able to complement a k2ORF5(0) deletion on k2 in trans, giving rise to yeast strains containing only the two recombinant plasmid forms. Additionally, the in vivo product of k2ORF5 has been identified as a 19.5 kDa protein by transplacing an epitope-tagged k2ORF5 allele from k2 to k1. The ease of detection of the tagged ORF5 product in comparison to TRF1, the gene product of k2ORF10, indicates that Orf5p is one of the most abundant k2 products, implying structural rather than regulatory function.

Amino Acid Sequence↗
PubMed2005

Application of the "codon-shuffling" method. Synthesis and selection of de novo proteins as antibacterials.

Library-based methods of non-rational and part-rational designed de novo peptides are worthy beacons in the search for bioactive peptides and proteins of medicinal importance. In this report, we have used a recently developed directed evolution method called "codon shuffling" for the synthesis and selection of bioactive proteins. The selection of such proteins was based on the creation of an inducible library of "codon-shuffled" genes that are constructed from the ligation-based assembly of judiciously designed hexamer DNA duplexes called dicodons. Upon induction with isopropyl 1-thio-beta-D-galactopyranoside, some library members were found to express dicodon-incorporated proteins. Because of this, the host cells, in our case Escherichia coli, were unable to grow any further. The bactereostatic/lytic nature of the dicodon proteins was monitored by growth curves as well as by zone clearance studies. Transmission electron microscopy of the affected cells illustrated the extent of cell damage. The proteins themselves were overexpressed as fusion partners and subsequently purified to homogeneity. One such purified protein was found to strongly bind heparin, an indication that the interaction of the de novo proteins may be with the nucleic acids of the host cell, much like many of the naturally occurring antibacterial peptides, e.g. Buforin. Therefore, our approach may help in generating a multitude of finely tuned antibacterial proteins that can potentially be regarded as lead compounds once the method is extended to pathogenic hosts, such as Mycobacteria, for example.

Amino Acid Sequence↗
PubMed1999

Killing the umpire: cooperative defects in mitotic checkpoint and BRCA2 genes on the road to transformation.

Recent findings from mouse models of BRCA2 genetic lesions have provided intriguing insights and important questions concerning modes of tumor development in familial breast and ovarian cancers. Fibroblasts from mice homozygous for the BRCA2Tr allele grow poorly and display an array of chromosomal abnormalities that are consistent with a role for BRCA2 in DNA repair. This growth defect can be overcome and cellular transformation promoted by the expression of defective, dominant negative alleles of p53 and of the mitotic checkpoint gene Bub1, both of which are known to induce chromosome instability. These findings are mirrored in the genetic lesions sustained in tumors found in the rare BRCA2Tr/Trmice that survive to adulthood, which include defects in p53 as well as the mitotic checkpoint proteins Bub1 and Mad3L. Together, these data hint that tumors in these mice evolve from an unusually intense selective pressure to remove DNA damage checkpoints, which in turn might be facilitated by chromosomal abolition of mitotic checkpoints and the consequent increase in shuffling of genetic information. How these genetic lesions co-operate to yield transformed cells and how these data relate to BRCA1 and BRCA2 defects in the human population are important questions raised by this work.

BRCA2 Protein↗
PubMed2006

Proton-shuffle mechanism of O-O activation for formation of a high-valent oxo-iron species of bleomycin.

Bleomycins (BLMs) can utilize H2O2 to cleave DNA in the presence of ferric ions. DFT calculations were used to study the mechanism of O-O bond cleavage in the low-spin FeIII-hydroperoxo complex of BLM. The following alternative hypotheses were investigated using realistic structural models: (a) heterolytic cleavage of the O-O bond, generating a Compound I (Cpd I) like intermediate, formally BLM-FeV=O; (b) homolytic O-O cleavage, leading to a BLM-FeIV=O species and an OH* radical; and (c) a direct O-O cleavage/H-abstraction mechanism by ABLM. The calculations showed that (a) is a facile and viable mechanism; it involves acid-base proton reshuffle mediated by the side-chain linkers of BLM, causing thereby heterolytic cleavage of the O-O bond and generation of Cpd I. Formation of Cpd I is found to involve a barrier of 13.3 kcal/mol, which is lower than the barriers in the alternative mechanisms (b and c) that possess respective barriers of 31 and 17 kcal/mol. The so-formed Cpd I species with a radical on the side-chain linker, methylvalerate (V), adjacent to the BLM-FeIV=O complex, resembles the formation of the active species of cytochrome c peroxidase in the Poulos-Kraut proton-shuffle mechanism in heme peroxidases (Poulos, T. L.; Kraut, J. J. Biol. Chem. 1980, 255, 8199-8205). Experimental data are discussed and shown to be in accord with this proposal. It suggests that the high-valence Cpd I species of BLM participates in the DNA cleavage. This is an alternative mechanistic hypothesis to the exclusive reactivity scenario based on ABLM (FeIII-OOH).

Bleomycin↗
PubMed2003

Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.

Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >10(24) possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.

Amino Acid Sequence↗
PubMed2002

Proteinase detection, DNA typing and antimycotic susceptibility of Candida isolates from Colombian women with vulvovaginal candidiasis.

Forty non-pregnant Colombian women (ages 18-45) with vulvovaginal candidiasis diagnosis (VVC) were enrolled in a blinded study to compare the efficacy of Itraconazole (ITRA) 400 mg vs. Fluconazole (FLU) 150 mg. Sexual partners received similar therapy. Proteinase detection by the Staib method and minimal inhibitory concentration (MIC) for FLU and ITRA by Etest method were performed in all Candida isolates. Patients were followed one year to determine clinical evolution and recurrence of VVC (RVVC). The strain identity of the RVVC isolates was determined by contour-clamped homogeneous electric field (CHEF) gel electrophoresis karyotyping and restriction fragment length polymorphism (RFLP). Thirty patients (75%) had one or two episodes of VVC/year, 83% of these were due to Candida albicans, while ten patients (25%) developed RVVC (three or more episodes/year); seven of them were treated with FLU. Non-C. albicans Candida species were detected in five of 30 (17%) of the patients with VVC and in seven of ten (70%) patients with RVVC (p=0.003). Isolates from nineteen patients were proteinase positive. Proteinase production and type of treatment were not related to recurrence of VVC (p>0.05). DNA typing revealed that in this population RVVC might be due to the same strain, substrain shuffling or different strains and species.

Journal Article↗
PubMed2006

Aptamer selection based on inhibitory activity using an evolution-mimicking algorithm.

In order to efficiently select aptamers that bind to and inhibit proteins, we developed a method that involves screening DNA aptamers based on their inhibitory activities using an evolution-mimicking algorithm after the pre-selection by SELEX. The value of this method was demonstrated by the identification of an inhibitor of Taq DNA polymerase in a unique single-stranded DNA library, which was expected to form a G-quartet structure. This method consists of selection via an inhibition assay, sequence shuffling, and mutation in silico. After six rounds of selection, the inhibitory activities of the aptamers had evolved significantly. This demonstrates the utility of this strategy for screening aptamers based on their inhibitory actions.

Algorithms↗
PubMed1995

Epstein-Barr virus latent messages with shuffled leader exons: remnants of circumgenomic transcription?

The EBNA transcription unit which is active in Epstein-Barr virus-immortalized latently infected B lymphocytes covers approximately 60% of the 172-kb genome. Since the genome exists as a circular double-stranded DNA molecule in latently infected cells, it is conceivable that complete copies are made during transcription. Rather than attempt to detect gigantic RNA molecules directly, we used RNA-PCR to detect incorporation of leader exons into mRNA in a shuffled order. The downstream U leader exon was detected spliced upstream of the internal repeat leader exons W1 and W2 in the polyadenylated RNA fraction of spontaneous lymphoblastoid cell lines, restricted phenotype BL cell lines Wanyanyi and Wewak2, and in B95-8, Raji, and Akata cells. Quantitative competitive RNA-PCR showed that the ratio of U exon-containing EBNA1 messages to U exon-shuffled leader messages was approximately 10:1, with large variation from cell line to cell line, and was not affected by induction of the lytic cycle in B95-8, Raji, or Akata cells. Messages with shuffled exons contained only the C2W1 alternative splice, which does not produce an initiator AUG methionine codon for EBNA4 gene expression. The results provide evidence for long-range exon skipping and imply that genome-length transcripts may occur and participate in viral gene expression in latency.

Adult↗
PubMed2000

Scapharca inaequivalvis A and B miniglobin genes: promoter activity of the 5' flanking regions and in vivo transcription.

Globin genes of the bivalve mollusk Scapharca inaequivalvis have the two intron/three exon organization typical of vertebrate and many invertebrate globins, with introns in highly conserved positions. Sequence studies on the A and B globin genes of the mollusk tetrameric hemoglobin gave evidence for the existence of 'minigenes' spanning part of the first and second intron, in-frame with the heme binding domain encoded by the central exon. Putative promoter and regulatory sequences flanking these minigenes were identified in the 3' regions of intron I. Here we report cloning and functional analysis of these regions ( approximately 400bp) and their promoter activity, which was assessed in K562 cells by transient transfection, was established. Moreover, in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) on total cytoplasmatic RNA demonstrated that the A and B minigenes are transcriptionally active in the erythrocytes of S. inaequivalvis. Thus, the present results lead to the conclusion that the present-day organization of the globin genes of S. inaequivalvis tetrameric hemoglobin is still reminiscent of an ancestral globin gene before exon shuffling.

Animals↗
PubMed1997

Parallel overlap assembly for the construction of computational DNA libraries.

Algorithms for computing with DNA currently require the construction of pools of molecules in which each distinct molecule represents a different starting point for the calculation. We have begun building such pools using the technique of parallel overlap assembly that is already used for the generation of diversity in biologically useful combinatorial search techniques such as gene shuffling. Unlike these applications, a pool in a molecular computer must be complete, containing all possible strands, and ordered, having minimal contamination from incorrectly assembled DNA. We present an experiment in which parallel overlap assembly is used to construct a computational pool and an experiment in which this pool is used to solve the NP-complete maximal-clique problem.

Algorithms↗
PubMed1998

Correlation of chi orientation with transcription indicates a fundamental relationship between recombination and transcription.

Cross-over hot-spot instigator (Chi) sequences (5'-GCTGGTGG-3') are abundant, strand-specific, sequences, which locally increase recombination in Escherichia coli. Located within G-rich 'recombination islands', Chi orientations correlate with the orientations both of DNA replication and of transcription. Consistent with evidence from eukaryotic systems for a fundamental relationship between recombination and transcription, we find for E. coli Chi sequences, and for Haemophilus influenzae Chi-like sequences, that orientations correlate better with transcription than with replication. Complying with Szybalski's transcription direction rule, open reading frames in these prokaryotes have purine-rich mRNA-synonymous DNA strands. Hence, the G-richness of 'recombination islands' may reflect their correspondence with 'transcriptional islands' (genes). Comparison of a natural with the corresponding shuffled sequence, indicates a base order-dependent island unit of approx. 1kb. 1998 Elsevier Science B.V.

Base Composition↗
PubMed1999

Evolution of DNA organization in hypotrichous ciliates.

Profound changes have been introduced into the germline (micronuclear) and somatic (macronuclear) genomes of hypotrichous ciliates during evolution. First, multiple, short, unique, noncoding sequences, called IESs, have been inserted into micronuclear genes. IESs are spliced out of each gene, and the gene segments, called MDSs, are ligated during conversion of the micronuclear genome to a macronuclear genome after cell mating. The IESs in a particular gene can change dramatically in number, position, length, and sequence during speciation. Once inserted, IESs can shift along the DNA of a gene 1 or 2 bp at a time by a mutational mechanism that does not alter the coding sequence of the gene. Second, the MDSs in the same genes have been rearranged into random or nonrandom, scrambled disorder. The origin of nonrandom scrambling patterns can be explained by a model of simultaneous insertion of multiple IESs into a germline gene during evolution. Subsequent recombinations among the IESs may change the MDS arrangement from a nonrandomly scrambled to a randomly scrambled pattern, including inversions of MDSs. Third, during the conversion of a micronucleus to a macronucleus after cell mating, MDSs are ligated in the unscrambled, orthodox order in association with IES excision, and the genes are then removed from the chromosomes as individual, short DNA molecules. These gene-size molecules are amplified many-fold to produce a mature macronucleus. All of these phenomena attest to a remarkable fluidity of the hypotrich genome both over evolutionary time and in the conversion of a germline genome to a somatic genome. The significance of this fluidity for the life and evolution of these organisms is still obscure. Recombination among IESs could shuffle MDSs and facilitate faster evolution of new genes.

Animals↗
PubMed2000

Bacterial mode of replication with eukaryotic-like machinery in a hyperthermophilic archaeon.

Despite a rapid increase in the amount of available archaeal sequence information, little is known about the duplication of genetic material in the third domain of life. We identified a single origin of bidirectional replication in Pyrococcus abyssi by means of in silico analyses of cumulative oligomer skew and the identification of an early replicating chromosomal segment. The replication origin in three Pyrococcus species was found to be highly conserved, and several eukaryotic-like DNA replication genes were clustered around it. As in Bacteria, the chromosomal region containing the replication terminus was a hot spot of genome shuffling. Thus, although bacterial and archaeal replication proteins differ profoundly, they are used to replicate chromosomes in a similar manner in both prokaryotic domains.

Archaeal Proteins↗
PubMed2004

The structure and evolution of centromeric transition regions within the human genome.

An understanding of how centromeric transition regions are organized is a critical aspect of chromosome structure and function; however, the sequence context of these regions has been difficult to resolve on the basis of the draft genome sequence. We present a detailed analysis of the structure and assembly of all human pericentromeric regions (5 megabases). Most chromosome arms (35 out of 43) show a gradient of dwindling transcriptional diversity accompanied by an increasing number of interchromosomal duplications in proximity to the centromere. At least 30% of the centromeric transition region structure originates from euchromatic gene-containing segments of DNA that were duplicatively transposed towards pericentromeric regions at a rate of six-seven events per million years during primate evolution. This process has led to the formation of a minimum of 28 new transcripts by exon exaptation and exon shuffling, many of which are primarily expressed in the testis. The distribution of these duplicated segments is nonrandom among pericentromeric regions, suggesting that some regions have served as preferential acceptors of euchromatic DNA.

Animals↗
PubMed1987

A sensitive procedure to compare amino acid sequences.

Methods are discussed that provide sensitive criteria for detection of weak sequence homologies. They are based on the Dayhoff relatedness odds amino acid exchange matrix and certain residue physical characteristics. The search procedure uses several residue probe lengths in comparing all possible segments of two protein sequences, and search plots are shown with peak values displayed over the entire search length. Alignments are automatically effected using the highest search matrix values and without the necessity of gap penalties. Tests for significance are derived from actual protein sequences rather than a random shuffling procedure.

Alcohol Dehydrogenase↗
PubMed1993

Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production.

A cloning and expression system allowing display of functional cDNAs or other gene products on the surface of filamentous phage has been developed, exploiting the high-affinity interaction of the Jun and Fos leucine zippers. Gene jun was expressed from a lacZ promoter as a fusion protein with the viral coat protein, pIII, thereby being structurally incorporated into phage particles during infection with a helper phage. Using a second lacZ promoter of the phagemid, gene fos was co-expressed as an N-terminal fusion peptide to cDNA library gene products, so that the resulting Fos-fusion proteins could become associated with the Jun-decorated phage particles. To avoid interphage exchange of fos-cDNA fusion products, cysteines were engineered at the N- and C-termini of each of the leucine zippers, providing a covalent link of the cDNA gene product to the genetic instructions required for its production. Dissociation between phage and cDNA gene products was readily achieved using reducing agents. Phages displaying gene products covalently anchored on their surface via the modified leucine zippers can be selectively enriched 10(4)-10(6)-fold over nonspecific phages using antibodies. Thus, this cloning system allows rapid isolation of rare mRNA products from complex cDNA libraries by enrichment with appropriate ligands. This approach should allow the expression and cloning of dimeric proteins by cDNA shuffling.

Amino Acid Sequence↗