Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

The Relationship between P Elements and Male Recombination in Drosophila Melanogaster

P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.     

The sex determining region of Chironomus thummi is associated with highly repetitive DNA and transposable elements

The dominant male sex determiner in chromosome III of the midge Chironomus thummi thummi is closely linked to a large cluster of tandem-repetitive DNA elements, the Cla elements, which are otherwise highly repetitive and distributed over more than 200 sites on all chromosomes. Chromosome III displays a hemizygous cluster of Cla elements in males but not in females. The chromosomal location of this hemizygous Cla element cluster is in the region of the male determiner M as localized by cytogenetic analysis. With Cla elements as hybridization probe, it was possible to clone a large part of the sex determining region. Molecular analysis of the DNA of males and females in this region displayed a number of differences between the two sexes. One striking difference is an unusual transposable element associated with the male sex determining region. The sex determining region also contains several other tandem-repetitive DNA elements in addition to the Cla elements. They are interspersed with single copy DNA. The accumulation of repetitive elements in the sex determining region is interpreted as the result of a lack of recombination between the male/female heteromorphic region, although recombination in the other sections of chromosome III occurs.     

P-Element-Induced Male Recombination Can Be Produced in Drosophila Melanogaster by Combining End-Deficient Elements in Trans

Male recombination, not normally present in Drosophila melanogaster, can be produced at high rates when target P elements at homologous sites are combined in the presence of transposase protein. We have produced a set of elements by in situ deletion of a particular insertion and have found elements that have deletions stretching into either end. Elements were tested in pairs to see whether they complement each other in their ability to induce recombination. The combination of elements that are deficient for the same end produces very little recombination, but the combination of a right-end and a left-end element can generate recombination values higher than given by two complete P[CaSpeR] elements at homologous sites. This strongly suggests that ``hybrid' P elements, containing ends from two different elements, can be recognized by transposase protein. We have also examined genotypes containing a normal and an end-deficient element and found that they yield reasonably high levels of recombination. We interpret the resultant gametes from such genotypes as showing that the majority of events in this genotype derive from the association of complementary ends from the same element, whereas the complementary ends from elements in trans associate in only a minority of cases.     

Factors affecting ectopic gene conversion in mice

Duplicated genes and repetitive sequences are distributed throughout the genomes of complex organisms. The homology between related sequences can promote nonallelic (ectopic) recombination, including gene conversion and reciprocal exchange. Resolution of these events can result in translocations, deletions, or other harmful rearrangements. In yeast, ectopic recombination between sequences on nonhomologous chromosomes occurs at high frequency. Because the mammalian genome is replete with duplicated sequences and repetitive elements, high levels of ectopic exchange would cause aneuploidy and genome instability. To understand the factors regulating ectopic recombination in mice, we evaluated the effects of homology length on gene conversion between unlinked sequences in the male germline. Previously, we found high levels of gene conversion between lacZ transgenes containing 2557 bp of homology. We report here that genetic background can play a major role in ectopic recombination; frequency of gene conversion was reduced by more than an order of magnitude by transferring the transgenes from a CF1 strain background to C57BL/6J. Additionally, conversion rates decreased as the homology length decreased. Sequences sharing 1214 bp of sequence identity underwent ectopic conversion less frequently than a pair sharing 2557 bp of identity, while 624 bp was insufficient to catalyze gene conversion at significant levels. These results suggest that the germline recombination machinery in mammals has evolved in a way that prevents high levels of ectopic recombination between smaller classes of repetitive sequences, such as the Alu family. Additionally, genomic location appeared to influence the availability of sequences for ectopic recombination. Received: 12 September 1997 / Accepted: 29 December 1997     

The transformation booster sequence from Petunia hybrida is a retrotransposon derivative that binds to the nuclear scaffold

The transformation booster sequence (TBS) from Petunia hybrida enhances transformation frequencies in P. hybrida, Nicotiana tabacum and Zea mays. TBS also stimulates homologous inter- and intramolecular recombination in P. hybrida, the molecular basis for this stimulation is not known. We investigated whether TBS contains sequence elements that might contribute to the stimulation of recombination and whether its recombinogenic potential reflects a biological function of TBS. We identified a scaffold attachment region (SAR) within TBS and analysed its distribution in the genome and its homologies to other genomic sequences. A 516 by subfragment of TBS binds to the nuclear scaffold. The sequence of the TBSSAR fragment shows strong homologies to retroviral elements from plants, suggesting that TBS is an inactive derivative of a retrovirus that still promotes DNA recombination.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.     

Recombination Signals In The rpoC1 Gene Indicate Gene‐Flow Between Planktothrix (Cyanoprokaryota) Species

The delineation of species boundaries in the potentially harmful cyanobacterium Planktothrix Anagnostidis et Komárek 1988 is particularly tangled. Genetic recombination has been invoked to explain the occurrence of overlapping biological traits among recognized species. Although horizontal gene transfer is shown as a driver of diversification in this genus, clear evidence for homologous recombination at the single gene level is still lacking. Several Planktothrix strains (n = 244) sampled in eight fresh water lakes in north Italy were characterized by sequencing the rpoC1 gene, a molecular marker previously proposed to discriminate between species. Six haplotypes were detected, four of which are newly described. A relevant number of rpoC1 sequences (n = 54) showed evidence of homologous recombination. By comparing the sequences produced in the work presented here to those available on GenBank for the genus, multiple recombination events were tracked between haplotypes associated to P. rubescens, P. suspensa and P. agardhii, the latter being a species not found in our survey. Recombination signals were in form of (i) a vast mosaic structure present in the alignment of rpoC1 haplotypes, (ii) multiple and statistically significant paths in the split decomposition network connecting these haplotypes and (iii) many individual crossing‐over events detected by means of recombination detection tests. Data suggest that the molecular evolution of the rpoC1 gene in the genus Planktothrix appears as strongly influenced by homologous recombination. In addition, rpoC1 diversity effectively tracks recombinational processes among species in the complex made up by P. rubescens, P. agardhii and P. suspensa, which are not isolated in terms of gene‐flow.     

Presynaptic filament dynamics in homologous recombination and DNA repair

Homologous recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA (ssDNA). Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we reviewed the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments; some intrinsic such as recombinase ATP-binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examined dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examined the biochemical properties of recombination proteins from four model systems (T4 phage, Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We proposed that the presynaptic filament has evolved to rely on multiple external factors for increased multilevel regulation of HR processes in genomes with greater structural and sequence complexity.     

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.     

Whole‐genome patterns of linkage disequilibrium across flycatcher populations clarify the causes and consequences of fine‐scale recombination rate variation in birds

Recombination rate is heterogeneous across the genome of various species and so are genetic diversity and differentiation as a consequence of linked selection. However, we still lack a clear picture of the underlying mechanisms for regulating recombination. Here we estimated fine‐scale population recombination rate based on the patterns of linkage disequilibrium across the genomes of multiple populations of two closely related flycatcher species (Ficedula albicollis and F. hypoleuca). This revealed an overall conservation of the recombination landscape between these species at the scale of 200 kb, but we also identified differences in the local rate of recombination despite their recent divergence (<1 million years). Genetic diversity and differentiation were associated with recombination rate in a lineage‐specific manner, indicating differences in the extent of linked selection between species. We detected 400–3,085 recombination hotspots per population. Location of hotspots was conserved between species, but the intensity of hotspot activity varied between species. Recombination hotspots were primarily associated with CpG islands (CGIs), regardless of whether CGIs were at promoter regions or away from genes. Recombination hotspots were also associated with specific transposable elements (TEs), but this association appears indirect due to shared preferences of the transposition machinery and the recombination machinery for accessible open chromatin regions. Our results suggest that CGIs are a major determinant of the localization of recombination hotspots, and we propose that both the distribution of TEs and fine‐scale variation in recombination rate may be associated with the evolution of the epigenetic landscape.     

A cointegrate of the bacteriophage P1 genome and the conjugative R plasmid R100

Restriction cleavage analysis identified a P1CmSmSuTc plasmid isolated by Mise and Arber (1976) (Virology 69, 191–205) as a cointegrate between bacteriophage P1 and the R plasmid R100. Cointegration occurred by reciprocal recombination between the IS1 element of P1 and IS1b of R100. It involved neither gain nor loss of genetic material, so that the cointegrate carries three IS1 elements in the same orientation. The cointegrate propagates with relatively high stability as plasmid in Escherichia coli host bacteria. It displays the Tra⁺ functions of R100, incompatibility FII of R100, and incompatibility Y of P1, Res⁺ (P1), Mod⁺ (P1) functions of P1 and P1 immunity. Production of P1 phage particles is inducible as for wild type P1. However, because of the large genome size of 180 kb, progeny phage particles contain only a fraction (about 100 kb) of the cointegrate genome. Because of cyclic permutation all genome regions are equally represented in a population of the phage particles of an induced lysate. Occasionally, reciprocal recombination between IS1 elements allows the restoration of the P1 genome. These segregants are found as plaque formers at a rate of about 1 per 300 phage particles in induced lysates.     

Evidence of P-element-induced sister-chromatid exchange in a ring-X chromosome in Drosophila, with implication for a high rate of formation of hybrid elements

Activation of a single incomplete P element induces recombination at a rate of approximately 0.5-1% in the male germline of Drosophila. Male recombination rises by an order of magnitude to approximately 20% if homologous P elements are involved. The high rate of recombination suggests the possibility that sister-chromatid exchange (SCE) might be elevated to a similar extent, since homologous P elements must always be present in sister chromatids. This possibility was tested by recombining a single P element onto a ring-X chromosome and using sex-ratio distortion to measure the loss of the ring-X due to SCE in the male germline. The results confirmed a rate of loss comparable to that expected with homologous elements, although the rate of loss was variable. Both SCE and recombination results are consistent with the "hybrid element insertion" model, in which the left and right ends from different elements associate, providing that insertion occurs preferentially in the vicinity of a P-element end. For autosomes, hybrid element formation may thus occur at a much higher rate than the 0.5-1% implied by single element recombination, with only a small minority of hybrid element excision events being resolved by recombination.     

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.     

Local Transposition of P Elements in Drosophila Melanogaster and Recombination between Duplicated Elements Using a Site-Specific Recombinase

The transposase source Δ2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic while gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.     

The influence of heterochromatin, inversion-heterozygosity and somatic pairing on x-ray induced mitotic recombination in Drosophila melanogaster

Summary Mitotic recombination has been induced with X-rays in Drosophila melanogaster larvae and assayed later as twin mosaic spots in the adult eyes. When the X-chromosomes are marked with zeste and white and the third chromosomes with roughoid and sepia, the frequency of twin spots was about 20 times higher for the X-chromosome than for the third chromosome. The greater amount of heterochromatin in the X-chromosome was considered responsible for the difference.Experiments with different inversion heterozygotes support this interpretation. Euchromatic inversions of different lengths have, when heterozygous, little or no influence on the twin spot frequency. The shorter the heterochromatic segment between the kinetochore and the proxomal break point of the inversion the stronger is the reduction of the twin spot frequency.The heterozygotes for the long sc ⁸ and sc ^S1 inversions gave exceptionally low twin spot frequencies. It seems possible that potential twin spot daughter cells die after recombination because of genetic imbalance and/or lack of proper cell separation resulting from the persistence of the dikinetic chromosome elements.To test whether inaccurate somatic pairing in inversion heterozygotes could help explain the low twin spot frequencies in those of sc ⁸ and sc ^S1, neuroblast chromosomes were investigated. They show that chromosomal arrangement during metaphase is determined exclusively by the location of the kinetochore, which always points, irrespective of earlier somatic pairing, toward the center of the metaphase plate. It is possible that there is a lack of proper chromosome alignment at the X-ray sensitive stage for mitotic recombination.     

A molecular map of the apomixis-control locus in <Emphasis Type="Italic">Paspalum procurrens</Emphasis> and its comparative analysis with other species of <Emphasis Type="Italic">Paspalum</Emphasis>

Since apomixis was first mapped in Paspalum, the absence of recombination that characterizes the related locus appeared to be the most difficult bottleneck to overcome for the dissection of the genetic determinants that control this trait. An approach to break the block of recombination was developed in this genus through an among-species comparative mapping strategy. A new apomictic species, P. procurrens (Q4094) was crossed with a sexual plant of P. simplex and their progeny was classified for reproductive mode with the aid of morphological, embryological and genetic analyses. On this progeny, a set of heterologous rice RFLP markers strictly co-segregating in coupling phase with apomixis was identified. These markers were all located on the telomeric region of the long arm of the chromosome 12 of rice. In spite of the lack of recombination exhibited by the apomixis-linked markers in P. procurrens, a comparative mapping analysis among P. simplex, P. malacophyllum, P. notatum and P. procurrens, allowed us to identify a small group of markers co-segregating with apomixis in all these species. These markers bracketed a chromosome region that likely contains all the genetic determinants of apomictic reproduction in Paspalum. The implications of this new inter-specific approach for overcoming the block of recombination to isolate the genetic determinants of apomixis and gain a better comprehension of genome structure of apomictic chromosome region are discussed.     

Characterization of the 16S–23S internal transcribed spacer among 34 higher plants: suitability for interspecific plastid transformation

Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S–23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S–23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified ∼2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3′ terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S–23S region as a target for interspecific transformation, and describe a ‘primer-to-plastid’ system to rapidly generate species-specific flanking regions for transformation vectors.