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.     

Germ-Line and Somatic Recombination Induced by in Vitro Modified P Elements in Drosophila Melanogaster

The P element insertion Δ2-3(99B) has previously been shown to activate incomplete P elements elsewhere in the genome. We show that this element, in conjunction with a second incomplete P element, P[CaSpeR], also induces recombination in the male germ line. The recombination is induced preferentially in the region of the P[CaSpeR] element. Recombinant chromosomes contain the P[CaSpeR] element in more than 50% of cases, and alternative models of transposon replication and preferential chromosome breakage are put forward to explain this finding. As is the case with male recombination induced by P-M dysgenic crosses, recombination appears to be premeiotic in a high proportion of cases. The Δ2-3(99B) element is known to act in somatic cells. Correspondingly, we show that the Δ2-3(99B)-P[CaSpeR] combination elevates the incidence of somatic recombination.     

An Analysis of Male-Recombination Elements in a Natural Population of DROSOPHILA MELANOGASTER in South Texas

Genomes from a group of Drosophila melanogaster collected from a natural population at San Benito, South Texas, in March of 1975 were analyzed for the presence of male-recombination elements. All three autosomes and both sex chromosomes were examined, with emphasis placed on the two major autosomes, the second and third chromosomes. In samples of 16 second and 16 third chromosomes, at least half, but not all, of each were found to carry male-recombination elements. It is suggested, although the data are not conclusive, that some of the fourth, X, and Y chromosomes might also be associated with male-recombination elements.—When a male-recombination element, or elements, was located in the second chromosome, relatively more male recombination was induced in the second than in the third chromosome. This situation was reversed when the element(s) was located in the third chromosome.—Distortion of transmission frequency, one of the characteristics of previously studied second chromosome lines associated with male recombination, was confirmed for these second chromosomes that carried male-recombination elements. Similar, but less pronounced, distortion was observed for the third chromosome lines that carried male-recombination elements.     

Evidence for Newly Induced Genetic Activity Responsible for Male Recombination Induction in DROSOPHILA MELANOGASTER

The T-007 second chromosomal line of Drosophila melanogaster, previously shown to contain a major element, Mr, responsible for male recombination induction, also contains the genetic capability to induce male recombination activity into (nonhomologous) third chromosomes. This newly induced male recombination activity maps to the centromeric region of two third-chromosome lines that were subjected to mapping experiments. The ability of these third chromosome lines to induce male recombination accounts for previous observations concerning the ability of Mr⁺ genotypes (derived from Mr/Mr⁺ heterozygous females) to induce male recombination for only a few generations, when only second chromosomes were selected and backcrossed. The occurrence of this effect, and a similar effect induced in the homologue of T-007, suggests a possible explanation of how natural populations of D. melanogaster have come to contain such high frequencies of these "male recombination" second and third chromosomes, despite their numerous deleterious effects.     

P-Element-Induced Recombination in Drosophila Melanogaster: Hybrid Element Insertion

It has previously been shown that the combination of two deleted P elements in trans, one containing the left functional end and the second element the right functional end, can lead to high levels of male recombination. This finding strongly suggests that P-element ends from different chromosomes can become associated, followed by ``pseudo-excision.'''' We show that two different processes are involved in resolving the pseudo-excision event: (1) the excised P-element ends continue to function as a single unit (Hybrid Element) and insert at a nearby site in the chromosome or into the element itself [Hybrid Element Insertion (HEI)] and (2) free ends that do not contain P elements repair and rejoin [(Hybrid Excision and Repair (HER)]. Both types of resolution can lead to recombination, and this paper concentrates on the HEI class. One type of HEI event predicts the exact reverse complementary duplication of an 8-bp target site, and we have confirmed the existence of such a structure in six independently derived recombinant chromosomes. There is also a high tendency for insertion events to occur within a few bases of the original 8-bp target site, including six apparent cases of insertion into the exact site.     

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.     

Parameters of Male and Female Recombination Influenced by the T-007 Second Chromosome in DROSOPHILA MELANOGASTER

The T-007 second chromosome line of Drosophila melanogaster, previously shown to contain genetic elements responsible for male recombination induction, appears to affect several parameters of recombination in females. In T-007 heterozygous females, the distribution of recombination (but not the total frequency) is changed from that observed in control females; relative increases are observed in the more proximal regions of the second, third and X chromosomes, while relative decreases are observed more distally. These changes are paralleled by altered coefficient of coincidence values and in an increased nondisjunction frequency of second chromosomes. The distribution of recombination in females is strikingly similar to that observed in males as measured along the second and third chromosomes, and the frequency of nondisjunction of the X and Y chromosomes is increased in T-007 heterozygous males. Based upon these results and responses to the effect of structurally rearranged heterologues (the "interchromosomal effect"), it is suggested that T-007 affects the preconditions for meiotic exchange in females. It is not yet known if elements responsible for these effects are the same elements responsible for the numerous other traits associated with the T-007 second chromosome.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these “local” insertions was actually within ～ 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Species-specific repetitive extragenic palindromic (REP) sequences in Pseudomonas putida

Pseudomonas putida KT2440 is a soil bacterium that effectively colonises the roots of many plants and degrades a variety of toxic aromatic compounds. Its genome has recently been sequenced. We describe that a 35 bp sequence with the structure of an imperfect palindrome, originally found repeated three times downstream of the rpoH gene terminator, is detected more than 800 times in the chromosome of this strain. The structure of this DNA segment is analogous to that of the so-called enterobacteriaceae repetitive extragenic palindromic (REP) sequences, although its sequence is different. Computer-assisted analysis of the presence and distribution of this repeated sequence in the P.putida chromosome revealed that in at least 80% of the cases the sequence is extragenic, and in 82% of the cases the distance of this extragenic element to the end of one of the neighbouring genes was <100 bp. This 35 bp element can be found either as a single element, as pairs of elements, or sometimes forming clusters of up to five elements in which they alternate orientation. PCR scanning of chromosomes from different isolates of Pseudomonas sp. strains using oligonucleotides complementary to the most conserved region of this sequence shows that it is only present in isolates of the species P.putida. For this reason we suggest that the P.putida 35 bp element is a distinctive REP sequence in P.putida. This is the first time that REP sequences have been described and characterised in a group of non-enterobacteriaceae.     

High levels of recombination induced by homologous P elements in Drosophila melanogaster

Summary P element transposons in Drosophila melanogaster are capable of mobilizing incomplete P elements elsewhere in the genome, and of inducing recombination. This recombination is usually only of the order of 1% or less. We show that two P elements, located at exactly homologous sites, induce levels of recombination of 20% or higher. The recombination appears to be exact, as determined by the lack of phenotypic effects in recombinant products and the lack of size changes detectable by Southern hybridization. Female recombination is increased, but to a lesser extent than male recombination. Somatic recombination levels are also elevated. Alternative explanations for the high recombination levels are given in terms of the consequences of repair of an excision site and in terms of recombination as part of the replicative transposition process.     

Elements Causing Male Crossing over in DROSOPHILA MELANOGASTER

A second chromosome line of Drosophila melanogaster (Symbol: T-007) has previously been shown to be responsible for the induction of male recombination. In the present investigation, the genetic elements responsible for this phenomenon have been partially identified and mapped. A major element (Symbol: Mr, for Male recombination) locates on the second chromosome between the pr (2L-54.4) and c (2R-75.5) loci and is responsible for the large majority of male recombination. In addition, there appear to be "secondary elements" present which have the ability to induce male recombination in much reduced frequencies and which are diluted out through successive backcross generations when Mr is removed by recombination. The possible nature of these "secondary elements" is discussed.     

Flanking Duplications and Deletions Associated with P-Induced Male Recombination in Drosophila

We studied P element-induced recombination in germline mitotic cells by examining the structure of the recombinant chromosomes. We found that most recombinants retain a mobile P element at the site of the recombination, usually with either a deletion or a duplication immediately adjacent to the P end at which the crossover occurred. The sizes of these deletions and duplications ranged from a few base pairs to well over 100 kb. These structures fit the ``hybrid element insertion' (HEI) model of male recombination in which the two P-element copies on sister chromatids combine to form a ``hybrid element' whose termini insert into a nearby position on the homologue. The data suggest that P-induced recombination can be used as an efficient means of generating flanking deletions in the vicinity of existing P elements. These deletions are easily screened using distant flanking markers, and they can be chosen to extend in a given direction depending on which reciprocal recombinant type is selected. Furthermore, the retention of a mobile P element allows one to extend the deletion or generate additional variability at the site by subsequent rounds of recombination.     

Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot

In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases) called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.     

Chaos of Rearrangements in the Mating-Type Chromosomes of the Anther-Smut Fungus Microbotryum lychnidis-dioicae

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures.     

A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase.     

Recombination between chloroplast genomes of Trachystoma ballii and Brassica juncea following protoplast fusion

We document here the presence of a recombinant plastome in a cytoplasmic male sterile (CMS) line of Brassica juncea developed from the somatic hybrid Trachystoma ballii?+?B. juncea. Restriction endonuclease digestion of the chloroplast (cp) DNA has revealed that the recombinant plastome gives rise to novel fragments in addition to the parent-specific fragments. Analysis of the 16S rRNA region by Southern hybridization shows no variation between B. juncea, T. ballii and the CMS line. The rbcL gene region of the recombinant plastome is identical to that in T. ballii. Analysis with probes for psbA and psbD using single and double DNA digests indicates that the hybridization patterns of the recombinant plastome are identical to those of the parents in digests obtained with some restriction enzymes, while novel bands hybridize to probes in other digests. In the psbA region, a B. juncea-specific PstI site and a T. ballii-specific EcoRI site are found in the recombinant plastome. The psbD region of the recombinant plastome contains a B. juncea-specific HindIII site and T. ballii-specific BamHI and HpaII sites. These results indicate the occurrence of intergenomic recombination between the chloroplasts of T. ballii and B. juncea in the somatic hybrid from which the CMS line was developed. The recombined plastome appears to be a mosaic of fragments specific to both parents and the recombination event has occurred in the single-copy regions. These recombinational events have not caused any imbalance in the recombinant plastome in terms of chloroplast-related functions, which have remained stable over generations.     

Studies on the Rate and Site-Specificity of P Element Transposition

A single genetically marked P element can be efficiently mobilized to insertionally mutagenize the Drosophila genome. We have investigated how the structure of the starting element and its location along the X chromosome influenced the rate and location of mutations recovered. The structure of two P[rosy+] elements strongly affected mobilization by the autonomous "Jumpstarter-1" element. Their average transposition rates differed more than 12-fold, while their initial chromosomal location had a smaller effect. The lethal and sterile mutations induced by mobilizing a P[rosy+] element from position 1F were compared with those identified previously using a P[neoR] element at position 9C. With one possible exception, insertion hotspots for one element were frequently also targets of the other transposon. These experiments suggested that the genomic location of a P element does not usually influence its target sites on nonhomologous chromosomes. During the course of these experiments, Y-linked insertions expressing rosy+ were recovered, suggesting that marked P elements can sometimes insert and function at heterochromatic sites.     

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.     

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.