DNA Inversion Research Articles

Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase.

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the beta recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the beta recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the beta system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and gammadelta resolvases. The ability of the beta recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.

Inverted repeat of a heterologous 3'-untranslated region for high-efficiency, high-throughput gene silencing.

This report describes a method for the easy generation of inverted repeat constructs for the silencing of genes of unknown sequence which is applicable to high-throughput studies. This improved procedure for high-efficiency gene silencing is specific for a target gene, but does not require inverted repeat DNA of the target gene in the construct. The method employs an inverted repeat of the 3'-untranslated region (3'-UTR) of a heterologous gene, and has been demonstrated using the 3'-UTR region of the nopaline synthase (nos) gene from Agrobacterium tumefaciens, which is often used as the 3'-UTR for transgene constructs. In a population of independent tomato primary transformants harboring a stably integrated polygalacturonase (PG) transgene driven by a constitutive promoter and linked to an inverted repeat of the nos 3'-UTR, 51 of 56 primary transformants (91% of the population) showed highly effective post-transcriptional silencing of the PG gene, with PG mRNA abundance in ripe fruit reduced by 98% or more. The method was also effective in Arabidopsis, where two different, relatively uncharacterized plant transcription factors were also targeted effectively. This method has the advantage of ease and rapidity in preparation of the constructs, since a gene of interest can be inserted into a binary vector already containing the promoter and the inverted nos domain in a single-cloning step, and does not require any knowledge of the DNA sequence. The approach is suitable for high-throughput gene silencing studies, where it is necessary to investigate the function of hundreds to thousands of uncharacterized genes.

Traffic at the tmRNA gene.

A partial screen for genetic elements integrated into completely sequenced bacterial genomes shows more significant bias in specificity for the tmRNA gene (ssrA) than for any type of tRNA gene. Horizontal gene transfer, a major avenue of bacterial evolution, was assessed by focusing on elements using this single attachment locus. Diverse elements use ssrA; among enterobacteria alone, at least four different integrase subfamilies have independently evolved specificity for ssrA, and almost every strain analyzed presents a unique set of integrated elements. Even elements using essentially the same integrase can be very diverse, as is a group with an ssrA-specific integrase of the P4 subfamily. This same integrase appears to promote damage routinely at attachment sites, which may be adaptive. Elements in arrays can recombine; one such event mediated by invertible DNA segments within neighboring elements likely explains the monophasic nature of Salmonella enterica serovar Typhi. One of a limited set of conserved sequences occurs at the attachment site of each enterobacterial element, apparently serving as a transcriptional terminator for ssrA. Elements were usually found integrated into tRNA-like sequence at the 3' end of ssrA, at subsites corresponding to those used in tRNA genes; an exception was found at the non-tRNA-like 3' end produced by ssrA gene permutation in cyanobacteria, suggesting that, during the evolution of new site specificity by integrases, tropism toward a conserved 3' end of an RNA gene may be as strong as toward a tRNA-like sequence. The proximity of ssrA and smpB, which act in concert, was also surveyed.

Testing the efficiency of dsRNAi constructs in vivo: a transient expression assay based on two fluorescent proteins.

Double-stranded RNA interference technology has recently been shown to be a powerful tool to silence gene expression in various organisms, including plants. Sustained double-stranded RNA interference mediated gene silencing is normally triggered by hairpin RNAs generated by in vivo transcription of inverted repeat DNA constructs. To test the efficiency of inverted repeat constructs for their in vivo gene silencing capability, we developed an assay that is based on transient expression in single cells using two fluorescent reporter proteins with different emission spectra. We co-expressed one fluorescent protein as marker for transfected cells and the second as translational fusion with the target gene. Co-transfection of a vector mediating expression of inverted repeats of the target gene resulted in a specific decrease or undetectable fusion protein fluorescence in the majority of transfected cells.

Campylobacter surface-layers (S-layers) and immune evasion.

Many pathogenic bacteria have evolved mechanisms for evading host immune systems. One evasion mechanism is manifest by the surface layer (S-layer), a paracrystalline protein structure composed of S-layer proteins (SLPs). The S-layer, possessed by 2 Campylobacter species (C. fetus and C. rectus), is external to the bacterial outer membrane and can have multiple functions in immune avoidance. C. fetus is a pathogen of ungulates and immunocompromised humans, in whom it causes disseminated bloodstream disease. In C. fetus, the S-layer is required for dissemination and is involved in 2 mechanisms of evasion. First, the S-layer confers resistance to complement-mediated killing in non-immune serum by preventing the binding of complement factor C3b to the C. fetus cell surface. S-layer expressing C. fetus strains remain susceptible to complement-independent killing, utilizing opsonic antibodies directed against the S-layer. However, C. fetus has also evolved a mechanism for avoiding antibody-mediated killing by high-frequency antigenic variation of SLPs. Antigenic variation is accomplished by complex DNA inversion events involving a family of multiple SLP-encoding genes and a single SLP promoter. Inversion events result in the expression of antigenically variant S-layers, which require distinct antibody responses for killing. C. rectus is implicated in the pathogenesis of periodontal disease and also possesses an S-layer that appears to be involved in evading the human system. Although studied less extensively than its C. fetus counterpart, the C. rectus S-layer appears to confer resistance to complement-mediated killing and to cause the down-regulation of proinflammatory cytokines.

A unique, bifunctional site-specific DNA recombinase from Mycoplasma pulmonis.

Site-specific DNA invertible elements often control the production of bacterial surface proteins that are subject to phase variation (ON/OFF switching). Inversion of the DNA element occurs as a result of the reciprocal exchange of DNA catalysed by a specialized enzyme (recombinase) that acts at specific sites. By continually switching the orientation of the invertible element in the chromosome, and consequently the production of the variable protein(s), the cell population remains continually responsive to environmental change such as immunological challenge. In addition to phase-variable surface proteins, Mycoplasma pulmonis has a family of phase-variable restriction-modification enzymes. We report here that a single recombinase in M. pulmonis, HvsR, catalyses independent DNA inversions at non-homologous loci, causing variations in surface lipoproteins and in the DNA recognition sequence specificity of restriction enzymes. Thus, HvsR is a site-specific DNA recombinase with dual substrate specificity.

Inversion of DNA charge by a positive polymer via fractionalization of the polymer charge

Charge inversion of a DNA double helix by an oppositely charged flexible polyelectrolyte (PE) is widely used for gene delivery. It is considered here in terms of discrete charges of DNA. We concentrate on the worst scenario case when in the neutral state of the DNA-PE complex, each of the DNA charges is locally compensated by a PE charge and show that charge inversion exists even in this case. When an additional PE molecule is adsorbed by DNA, its charge gets fractionalized into monomer charges of defects (tails and arches) on the background of the perfectly neutralized DNA. These charges spread all over the DNA eliminating the self-energy of PE. Fractionalization leads to a substantial charge inversion of DNA.

Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus.

The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5' untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5' untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the lambda integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.

The 2.1-kb inverted repeat DNA sequences flank the mat2,3 silent region in two species of Schizosaccharomyces and are involved in epigenetic silencing in Schizosaccharomyces pombe.

The mat2,3 region of the fission yeast Schizosaccharomyces pombe exhibits a phenomenon of transcriptional silencing. This region is flanked by two identical DNA sequence elements, 2.1 kb in length, present in inverted orientation: IRL on the left and IRR on the right of the silent region. The repeats do not encode any ORF. The inverted repeat DNA region is also present in a newly identified related species, which we named S. kambucha. Interestingly, the left and right repeats share perfect identity within a species, but show approximately 2% bases interspecies variation. Deletion of IRL results in variegated expression of markers inserted in the silent region, while deletion of the IRR causes their derepression. When deletions of these repeats were genetically combined with mutations in different trans-acting genes previously shown to cause a partial defect in silencing, only mutations in clr1 and clr3 showed additive defects in silencing with the deletion of IRL. The rate of mat1 switching is also affected by deletion of repeats. The IRL or IRR deletion did not cause significant derepression of the mat2 or mat3 loci. These results implicate repeats for maintaining full repression of the mat2,3 region, for efficient mat1 switching, and further support the notion that multiple pathways cooperate to silence the mat2,3 domain.

The Chapel Hill hemophilia A dog colony exhibits a factor VIII gene inversion.

In the Chapel Hill colony of factor VIII-deficient dogs, abnormal sequence (ch8, for canine hemophilia 8, GenBank no. ) follows exons 1-22 in the factor VIII transcript in place of exons 23-26. The canine hemophilia 8 locus (ch8) sequence was found in a 140-kb normal dog genomic DNA bacterial artificial chromosome (BAC) clone that was completely outside the factor VIII gene, but not in BAC clones containing the factor VIII gene. The BAC clone that contained ch8 also contained a homologue of F8A (factor 8 associated) sequence, which participates in a common inversion that causes severe hemophilia A in humans. Fluorescence in situ hybridization analysis indicated that exons 1-26 normally proceed sequentially from telomere to centromere at Xq28, and ch8 is telomeric to the factor VIII gene. The appearance of an "upstream" genomic sequence element (ch8) at the end of the aberrant factor VIII transcript suggested that an inversion of genomic DNA replaced factor VIII exons 22-26 with ch8. The F8A sequence appeared also in overlapping normal BAC clones containing factor VIII sequence. We hypothesized that homologous recombination between copies of canine F8A inside and outside the factor VIII gene had occurred, as in human hemophilia A. High-resolution fluorescent in situ hybridization on hemophilia A dog DNA revealed a pattern consistent with this inversion mechanism. We also identified a HindIII restriction fragment length polymorphism of F8A fragments that distinguished hemophilia A, carrier, and normal dogs' DNA. The Chapel Hill hemophilia A dog colony therefore replicates the factor VIII gene inversion commonly seen in humans with severe hemophilia A.

Evolution of nitric oxide synthase regulatory genes by DNA inversion.

DNA inversions are mutations involving major rearrangements of the genome and are often regarded as either deleterious or catastrophic to gene function and can be associated with genomic disorders, such as Hunter syndrome and some forms of hemophilia. Here, we propose that DNA inversions are also an essential and hitherto unrecognized component of gene evolution in eukaryotic cells. Specifically, we provide evidence that an ancestral neuronal nitric oxide synthase (nNOS) gene was duplicated and that one copy retained its original function, whereas an internal DNA inversion occurred in the other. Crucially, the inversion resulted in the creation of new regulatory elements required for the termination and activation of transcription. In consequence, the duplicated gene was split, and two new and independently expressed genes were created. Through its dependence on DNA inversion, this is a fundamentally new scheme for gene evolution, which we show as being of particular relevance to the generation of endogenous antisense-containing RNA molecules. Functionally, such transcripts can operate as natural negative regulators of the expression of the genes to which they are related through a common ancestor.

A Rho-dependent phase-variable transcription terminator controls expression of the FimE recombinase in Escherichia coli.

The fim switch is a 314 bp segment of invertible chromosomal DNA that is responsible for phase-variable expression of type 1 fimbriae in Escherichia coli. The switch harbours the promoter of the fimA gene. This codes for the type 1 fimbrial subunit protein and, when the promoter is directed towards fimA (phase ON), the bacteria are fimbriate and, when it is directed away, the cells are afimbriate. The switch lies immediately downstream from the fimE gene, coding for a tyrosine site-specific recombinase that catalyses inversion of the switch from the ON to the OFF phase. It has been suggested previously that, because the fim switch lies immediately downstream from the fimE gene, expression of FimE could be subject to control by antisense RNA in phase OFF bacteria in which the promoter harboured within the fim switch is oriented against the direction of transcription of the fimE gene. In this study, no effect of inducible fimE antisense RNA, expressed in cis or in trans, on FimE expression was detected. In phase ON cells, fimE mRNA extends across the switch into fimA, whereas in phase OFF cells, it terminates within the switch. This termination is Rho dependent and is abolished in a rho mutant. The extended fimE found in phase ON cells is more stable and results in an approximately fivefold increase in FimE protein compared with phase OFF bacteria. In the absence of Rho factor, fimE mRNA is equally stable in phase ON and phase OFF cells, and the levels of FimE recombinase are also equal.

Molecular analysis of radiation-induced thyroid carcinomas in humans

Correlations have been found between radiation exposure and thyroid carcinoma development, particularly in children. Recent studies on a large cohort of radiation-induced papillary thyroid carcinomas (PTC) after the Chernobyl reactor accident disclosed a common type of underlying genetic alteration. A high prevalence of rearrangements of the receptor tyrosine kinase (TK) c-RET was observed, besides some rearrangements involving NTRK1. Radiation-induced RET rearrangements in PTC consist most frequently of fusions to the H4 gene (RET/PTC1) or to the ELE1 (ARA70) gene (RET/PTC3). Both fusions are formed by balanced paracentric inversions on chromosome 10. An analysis of the fused genes in ELE1/RET rearrangements revealed DNA double-strand breaks spread over a distance of about 2.3 kb in two introns and the interposed exon of ELE1, exon 11 and intron 11 of RET, without significant clustering in these parts of the genes. Topoisomerase I sites were found exactly at or in close vicinity to all breakpoints, suggesting a role for this enzyme in formation of DNA strand breaks or inversions. The genes fuse at short regions of sequence homology and short direct or inverted repeats (microhomology-mediated DNA end joining). A minority of PTC cases contain novel types of RET rearrangement, with RIα, GOLGA2, HTIF, HTIF homolog, RFG8, ELKS, KTN1 and PCM-1 as the 5′-fused genes. These novel types of gene fusions are formed by interchromosomal translocation. The formation of these rare types of rearrangement seems to be highly related to radiation as they have rarely been found in sporadic PTC. All RET gene fusions seem to act similarly on RET function: The strict physiological control of RET TK activity is suspended through constitutive activation by 5′-fused parts of genes containing coiled-coil domains with dimerization potential. RET expression in thyrocytes, which under normal conditions, lack RET TK activity apparently triggers clonal expansion and early invasion of the affected cells. RET-fused genes, some of which are transcriptional coactivators, are important determinators of the peculiar phenotype of the tumour and for its clinical course. This is most significant in RET/PTC3 rearrangements with ELE1 as the RET-fused gene: this type of rearrangement leads more often to the phenotype of a solid variant of PTC, and to rapid tumour development and early lymph node metastasis. Up to now, no other genetic aberration has more frequently been observed in PTC than RET rearrangement, thus suggesting that RET rearrangement represents a genetic marker lesion of radiation history in the development of a PTC.

Symmetric DNA sites are functionally asymmetric within Flp and Cre site-specific DNA recombination synapses.

Flp and Cre-mediated recombination on symmetrized FRT and loxP sites, respectively, in circular plasmid substrates yield both DNA inversion and deletion. However, upon sequestering three negative supercoils outside the recombination complex using the resII-resIII synapse formed by Tn3 resolvase and the LER synapse formed by phage Mu transposase in the case of Flp and Cre, respectively, the reactions are channeled towards inversion at the expense of deletion. The inversion product is a trefoil, its unique topology being conferred by the external resolvase or LER synapse. Thus, Flp and Cre assign their symmetrized substrates a strictly antiparallel orientation with respect to strand cleavage and exchange. These conclusions are supported by the product profiles from tethered parallel and antiparallel native FRT sites in dilution and competition assays. Furthermore, the observed recombination bias favoring deletion over inversion in a nicked circular substrate containing two symmetrized FRT sites is consistent with the predictions from Monte Carlo simulations based on antiparallel synapsis of the DNA partners.

Identification and characterization of a site-specific tyrosine recombinase within the variable loci of Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae

Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis. Each of the mycoplasmal proteins are members of the λ integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci.

Plant chromosomal HMGB proteins efficiently promote the bacterial site-specific beta-mediated recombination in vitro and in vivo.

In the presence of an accessory DNA bending protein, the bacterial site-specific beta recombinase catalyzes resolution and DNA inversion. Five different maize high mobility group B (HMGB) proteins were examined for their potential to facilitate beta recombination in vitro using DNA substrates with different intervening distances (73-913 bp) between two directly oriented recombination (six) sites. All analyzed HMGB proteins (HMGB1 to HMGB5) could promote beta recombination, but depending on the DNA substrate with different efficiencies. The HMGB1 protein displayed an activity comparable to that of the natural promoting protein Hbsu, whereas the other HMGB proteins were less effective. Phosphorylation of the HMGB1 protein resulted in an increased efficiency of HMGB1 to promote beta recombination. Analyses of DNA substrates with closely spaced six sites demonstrated that in the presence of HMGB1 the recombination rate was correlated to the distance between the six sites, but independent of the helical orientation of the six sites. Using a Bacillus subtilis strain defective in Hbsu, the coexpression of beta recombinase and HMGB1 (or a truncated HMGB1 derivative) revealed that a plant HMG-box domain protein is sufficient for assisting beta to catalyze recombination in vivo. Our results using beta recombination as a model system suggest that the various plant HMGB proteins (and their posttranslationally modified versions) have the potential of forming a repertoire of different DNA structures, which is compatible with the idea that the HMGB proteins can act as architectural factors in a variety of nucleoprotein structures.

FOR, a Novel Orphan Nuclear Receptor Related to Farnesoid X Receptor

We have identified and characterized a new amphibian orphan member of the nuclear receptor superfamily and termed it FOR1 (farnesoid X receptor (FXR)-like Orphan Receptor) because it shares the highest amino acid identity with the mammalian FXR. We also identified a variant of FOR1, called FOR2, which has 15 additional C-terminal amino acids. Both variants include an unusual insertion of 33 amino acids in the helix 7 region of the canonical ligand binding domain sequence, suggesting a unique structure for FOR. Northern blot analysis demonstrates that the FOR gene is highly expressed in adult and tadpole liver, kidney, and tail bud stage of the embryo. Detailed expression analysis using in situ hybridization indicates that FOR expression is first detectable at stage 30/31 in the presumptive liver region lasting until stage 41 with a peak level evident at stage 35/36. FOR forms heterodimeric complexes with retinoid X receptor (RXR) as demonstrated by biochemical and mammalian two-hybrid approaches. Gel mobility shift assays demonstrate that FORs form specific DNA-protein complexes on an FXR binding element consisting of an inverted repeat DNA element with 1 nucleotide spacing (IR1) from the phospholipid transfer protein gene promoter. Finally, although FORs do not exhibit constitutive transcriptional activity, frog gallbladder extract significantly augments the transcriptional activities of FORs.

Inversion of DNA charge by a positive polymer via fractionalization of the polymer charge

Charge inversion of a DNA double helix by an oppositely charged flexible polyelectrolyte (PE) is widely used for gene delivery. It is considered here taking into account the discreteness of the charges of DNA. We concentrate on the worst scenario case when in the neutral state of the DNA-PE complex, each of the DNA charges is locally compensated by a PE charge and show that charge inversion exists even in this case. When an additional PE molecule is adsorbed by DNA, its charge gets fractionalized into monomer charges of defects (tails and arches) on the background of the perfectly neutralized DNA. These charges spread all over the DNA eliminating the self-energy of PE. Fractionalization leads to a substantial charge inversion of DNA. We show that fractionalization mechanism of charge inversion works also for non-linear polymers such as dendrimers. Remarkably, such fractionalization happens for adsorption of both PE or dendrimers on a two-dimensional charged lattice as well. Relation of fractionalization to other mechanisms of charge inversion is discussed.

Fifteen minutes of fim: control of type 1 pili expression in E. coli.

Pili are used by Escherichia coli to attach to and invade mammalian tissues during host infection and colonization. Expression of type 1 pili, believed to act as virulence factors in urinary tract infections, is under control of the 'firm' genetic network. This network is able to sense the environment and actuate phase variation control. It is a prime exemplar of an integrative regulatory system because of its role in mediating a complex infection process, and because it instantiates a number of regulatory motifs, including DNA inversion and stochastic variation. With the help of a mathematical model, we explore the mechanisms and architecture of the fim network. We explain (1) basic network operation, including the roles of the recombinase and global regulatory protein concentrations, their DNA binding affinities, and their switching rates in observed phase variation behavior; (2) why there are two recombinases when one would seem to suffice; (3) the source of on-to-off switching specificity of FimE; (4) the role of fimE orientational control in switch dynamics; and (5) how temperature tuning of piliation is achieved. In the process, we identify a general regulatory motif that tunes phenotype to an environmental variable, and explain a number of apparent experimental inconsistencies.

8 Homology-dependent gene silencing and host defense in plants

Analyses of transgene silencing phenomena in plants and other organisms have revealed the existence of epigenetic silencing mechanisms that are based on recognition of nucleic acid sequence homology at either the DNA or RNA level. Common triggers of homology-dependent gene silencing include inverted DNA repeats and double-stranded RNA, a versatile silencing molecule that can induce both degradation of homologous RNA in the cytoplasm and methylation of homologous DNA sequences in the nucleus. Inverted repeats might be frequently associated with silencing because they can potentially interact in cis and in trans to trigger DNA methylation via homologous DNA pairing, or they can be transcribed to produce double-stranded RNA. Homology-dependent gene silencing mechanisms are ideally suited for countering natural parasitic sequences such as transposable elements and viruses, which are usually present in multiple copies and/or produce double-stranded RNA during replication. These silencing mechanisms can thus be regarded as host defense strategies to foreign or invasive nucleic acids. The high content of transposable elements and, in some cases, endogenous viruses in many plant genomes suggests that host defenses do not always prevail over invasive sequences. During evolution, slightly faulty genome defense responses probably allowed transposable elements and viral sequences to accumulate gradually in host chromosomes and to invade host genes. Possible beneficial consequences of this "foreign" DNA buildup include the establishment of genome defense-derived epigenetic control mechanisms for regulating host gene expression and acquired hereditary immunity to some viruses.