The double-strand-break repair model for recombination

We have isolated and characterized several members of the P transposable element family from a Drosophila melanogaster P strain. Large 2.9 kb elements are present as multiple highly conserved copies together with smaller (0.5-1.6 kb), heterogeneous elements. The complete DNA sequences of the 2.9 kb element and four small elements (previously isolated from hybrid-dysgenesis-induced mutations of the white locus) have been determined. Each small element appears to have arisen from the 2.9 kb element by a different internal deletion. P elements have 31 bp perfect inverse terminal repeats and upon insertion duplicate an 8 bp sequence found only once at the site of insertion. Three of the insertions into the white locus occurred at the same nucleotide, indicating a high degree of local site specificity for insertion. The basis of this specificity has been investigated by DNA sequence analysis of the sites where 18 P elements are found. A revertant of one of the white locus mutants has been found to result from precise excision of the P element, restoring the wild-type DNA sequence.     

Localization of P elements, copy number regulation, and cytotype determination in Drosophila melanogaster

Seventeen highly-inbred lines of Drosophila melanogaster extracted from an M' strain (in the P/M system of hybrid dysgenesis) were studied for their cytotype and the number and chromosomal location of complete and defective P elements. While most lines were of M cytotype, three presented a P cytotype (the condition that represses P-element activity) and one was intermediate between M and P. All lines were found to possess KP elements and only eight to bear full-sized P elements. Only the lines with full-sized P elements showed detectable changes in their P-insertion pattern over generations; their rates of gain and of loss of P-element sites were equal to 0.12 and 0.09 per genome, per generation, respectively. There was no correlation between these two rates within lines, suggesting independent transpositions and excisions in the inbred genomes. The results of both Southern blot analysis and in situ hybridization of probes made from left and right sides of the P element strongly suggested the presence of a putative complete P element in region 1A of the X chromosome in the three lines with a P cytotype; the absence of P copy in this 1A region in lines with an M cytotype, favours the hypothesis that the P element inserted in 1A could play a major role in the P-cytotype determination. Insertion of a defective 2 kb P element was also observed in region 93F in 9 of the 13 M lines. The regulation of the P-element copy number in our lines appeared not to be associated with the ratio of full-length and defective P elements.     

Cytotype Control of Drosophila Melanogaster P Element Transposition: Genomic Position Determines Maternal Repression

P element transposition in Drosophila is controlled by the cytotype regulatory state: in P cytotype, transposition is repressed, whereas in M cytotype, transposition can occur. P cytotype is determined by a combination of maternally inherited factors and chromosomal P elements in the zygote. Transformant strains containing single elements that encoded the 66-kD P element protein zygotically repressed transposition, but did not display the maternal repression characteristic of P cytotype. Upon mobilization to new genomic positions, some of these repressor elements showed significant maternal repression of transposition in genetic assays, involving a true maternal effect. Thus, the genomic position of repressor elements can determine the maternal vs. zygotic inheritance of P cytotype. Immunoblotting experiments indicate that this genomic position effect does not operate solely by controlling the expression level of the 66-kD repressor protein during oogenesis. Likewise, P element derivatives containing the hsp26 maternal regulator sequence expressed high levels of the 66-kD protein during oogenesis, but showed no detectable maternal repression. These data suggest that the location of a repressor element in the genome may determine maternal inheritance of P cytotype by a mechanism involving more than the overall level of expression of the 66-kD protein in the ovary.     

Modified P Elements That Mimic the P Cytotype in Drosophila Melanogaster

Activity of the P family of transposable elements in Drosophila melanogaster is regulated primarily by a cellular condition known as P cytotype. It has been hypothesized that P cytotype depends on a P element-encoded repressor of transposition and excision. We provide evidence in support of this idea by showing that two modified P elements, each with lesions affecting the fourth transposase exon, mimic most of the P cytotype effects. These elements were identified by means of two sensitive assays capable of detecting repression by a single P element. One assay makes use of cytotype-dependent gene expression of certain P element insertion mutations at the singed bristle locus. The other measures suppression of transposase activity from the unusually stable genomic P element, delta 2-3(99B), that normally produces transposase in both germinal and somatic tissues. The P cytotype-like effects include suppression of snw germline hypermutability, snw somatic mosaicism, pupal lethality, and gonadal dysgenic sterility. Unlike P cytotype, however, there was no reciprocal cross effect in the inheritance of repression.     

An Acetobacter xylinum insertion sequence element associated with inactivation of cellulose production.

An insertion sequence (IS) element, IS1031, caused insertions associated with spontaneous cellulose deficient (Cel-) mutants of Acetobacter xylinum ATCC 23769. The element was discovered during hybridization analysis of DNAs from Cel- mutants of A. xylinum ATCC 23769 with pAXC145, an indigenous plasmid from a Cel- mutant of A. xylinum NRCC 17005. An IS element, IS1031B, apparently identical to IS1031, was identified on pAXC145. IS1031 is about 950 bp. DNA sequencing showed that the two elements had identical termini with inverted repeats of 24 bp containing two mismatches and that they generated 3-bp target sequence duplications. The A. xylinum ATCC 23769 wild type carries seven copies of IS1031. Southern hybridization showed that 8 of 17 independently isolated spontaneous Cel- mutants of ATCC 23769 contained insertions of an element homologous to IS1031. Most insertions were in unique sites, indicating low insertion specificity. Significantly, two insertions were 0.5 kb upstream of a recently identified cellulose synthase gene. Attempts to isolate spontaneous cellulose-producing revertants of these two Cel- insertion mutants by selection in static cultures were unsuccessful. Instead, pseudorevertants that made waxlike films in the liquid-air interface were obtained. The two pseudorevertants carried new insertions of an IS1031-like element in nonidentical sites of the genome without excision of the previous insertions. Taken together, these results suggest that indigenous IS elements contribute to genetic instability in A. xylinum. The elements might also be useful as genetic tools in this organism and related species.     

Recovery and characterization of hybrid dysgenesis-induced mei-9 and mei-41 alleles of Drosophila melanogaster

X-Linked methyl methanesulfonate (MMS)-sensitive mutations were induced with hybrid dysgenesis using four P strains: pi 2, Harwich, T-007 and OK-1. Mutations were identified after two generations of backcrosses to M strain females to replace the autosomes. Among 51,471 X-chromosomes examined 10 carried stable MMS-sensitive mutations representing 8 independent events. Males of the mutant strains failed to induce gonadal dysgenesis in crosses to Oregon-R females at 28.5 degrees C. Complementation tests showed that 3 of the induced mutations were mei-9 alleles, 2 were mei-41 alleles, 1 was a mus102 allele, and 2 were alleles at a newly identified MMS-sensitive locus, mus112 (map position: 1-32.8). As assayed by in situ hybridization on polytene chromosomes, each X-chromosome had no more than four P element insertions. 4 of the 8 mutations recovered in this study proved to have P element insertions at or very close to sites to which MMS sensitivity has been mapped. Hybrid dysgenesis-induced reversion of 2 mutants, mei-9RT1 and mei-41RT2, is associated with the loss of the P element from regions 4B and 14C respectively.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region,the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Plasmid pHH1 of Halobacterium salinarium: characterization of the replicon region, the gas vesicle gene cluster and insertion elements

The DNA sequence of the 5.7 kb plasmid pHH9 containing the replicon region of the 150 kb plasmid pHH1 from Halobacterium salinarium was determined. The minimal region necessary for stable plasmid maintenance lies within a 2.9 kb fragment, as defined by transformation experiments. The DNA sequence contained two open reading frames arranged in opposite orientations, separated by an unusually high AT-rich (60–70% A + T) sequence of 350 bp. All H. salinarium strains (H. halobium, H. cutirubrum) investigated harbour endogenous plasmids containing the pHH1 replicon; however, these pHH1-type plasmids differ by insertions and deletions. Adjacent to the replicon, and separated by a copy of each of the insertion elements ISH27 and ISH26, is the 9 kb p-vac region required for gas vesicle synthesis. Analysis of these and other ISH element copies in pHH1 revealed that most of them lack the target DNA duplication usually found with recently transposed ISH elements. These results underline the plasticity of plasmid pHH1.     

Hybrid Dysgenesis in Drosophila melanogaster: Evidence from Sterility and Southern Hybridization Tests That P Cytotype Is Not Maintained in the Absence of Chromosomal P Factors

A two-generation crossing program was used to replace the entire chromosome complement of P strains by M strain chromosomes, the maternal contribution being from the P strain. The cytotype of chromosomally substituted females was indistinguishable from M strain cytotype, judged by the sterility of offspring from the cross of such females to P strain males. In addition, following replacement of the chromosomes, the level of DNA homologous to the P factor was sufficiently low to be explicable by low levels of P factor transposition. These results are consistent with immediate chromosomal control for the switching from P to M cytotype. However, the reverse chromosome substitution, replacing all chromosomes of an M strain with P chromosomes, did not usually lead to immediate change of cytotype properties, showing that there is a true maternal effect in the M to P direction. The absence of true maternal inheritance for P cytotype argues against models of P factor repression which depend on autonomous replication of a nonchromosomal element. The repression could still be explained by nonchromosomal copies of the P factor, provided that these are replenished from chromosomal P factors. A model is put forward in which P cytotype is due to the presence of circular P factors carrying a P factor target sequence, leading to preferential transposition of chromosomal P factors to nonchromosomal target sites.     

Absence of insertions among spontaneous mutants of Salmonella typhimurium

Summary While insertion sequences (IS) in Escherichia coli transpose frequently to generate spontaneous insertion mutants, such mutations are rare in Salmonella typhimurium: the only documented insertion mutation is a hisD mutation caused by the Salmonella-specific IS element IS200. To obtain more examples of IS200 insertion mutations and to seek additional types of IS elements in Salmonella, we selected and characterized 422 independent, spontaneous His^– mutants and some 2100 additional mutants that are not necessarily independent. None of the mutants showed the absolute polar effect characteristic of insertion mutations or the reversion properties characteristic of insertions (low spontaneous reversion frequency and no reversion induction by chemical mutagens). A few mutants, showing a high spontaneous reversion frequency, were screened physically. No insertion mutations were found. Thus insertion mutations appear to be rare in S. typhimurium, in strong contrast to E. coli and despite the possession in Salmonella of at least one type of insertion element (IS200). These results suggest that in Salmonella transposition of the endogenous elements has been controlled. The transposition ability of the elements may have been reduced or favored target sites removed from the host genome.     

Hybrid Dysgenesis in DROSOPHILA MELANOGASTER: Nature and Inheritance of P Element Regulation

The genetic determination of the control of resistance or susceptibility to germ line changes mediated by P elements was studied in two strains and in derivatives of crosses between them. One strain, characterized as true M, completely lacked P elements. The second strain, pseudo-M (M'), carried a number of P elements, but these did not have the potential to induce the gonadal sterility that is associated with P-M hybrid dysgenesis. Individuals from the true M strain were invariably unable to suppress P factor activity (i.e., all daughters of outcrosses of M females and P males were sterile). In contrast, individuals from the M' strain showed variable degrees of suppression that were manifested in a wide range of gonadal sterility frequencies in standard tests. This continuous distribution pattern was reproducible for more than 25 generations.--The results of the genetic analysis indicate that a strain with a variable degree of suppression of gonadal dysgenesis is not necessarily in a transient state between the extreme conditions of P and M cytotype. A large variance in the ability to suppress gonadal dysgenesis with a mean value intermediate between the extremes of P and M cytotype may be a relatively stable strain characteristic. No reciprocal cross effect was observed in the suppression of sterility of F1 females from M X M' matings. Thus, the existence of M' strains indicates a Mendelian component in P element regulation and suggests that cytotype, which has an extrachromosomal aspect, may be only one of perhaps several mechanisms involved in regulation. Analysis of the effects of individual chromosomes from the M' strain showed that each chromosome contributed to the reduction of gonadal dysgenesis in the progeny of test matings. The results are consistent with a one-component titration model for P element regulation.     

Maternal inheritance of P cytotype in Drosophila melanogaster: a “pre-P cytotype” is strictly extra-chromosomally transmitted

In Drosophila melanogaster, transposition of the P element is under the control of a cellular state known as cytotype. The P cytotype represses P transposition whereas the M cytotype is permissive for transposition. In the long-term, the P cytotype is determined by chromosomal P elements but over a small number of generations it is maternally inherited. In order to analyse the nature of this maternal inheritance, we tested whether a maternal component can be transmitted without chromosomal P elements. We used a stable determinant of P cytotype, linked to the presence of two P elements at the tip of the X chromosome (IA site) in a genome devoid of other P elements. We measured P repression capacity using two different assays: gonadal dysgenic sterility (GD) and P-lacZ transgene repression. We show that zygotes derived from a P cytotype female (heterozygous for P (1A)/balancer devoid of P copies) and which inherit no chromosomal P elements from the mother, have, however, maternally received a P-type extra-chromosomal component: this component is insufficient to specify the P cytotype if the zygote formed does not carry chromosomal P elements but can promote P cytotype determination if regulatory P elements have been introduced paternally. We refer to this strictly extra-chromosomally inherited state as the “pre-P cytotype”. In addition, we show that a zygote that has the pre-P cytotype but which has not inherited any chromosomal P elements, does not transmit the pre-P cytotype to the following generation. The nature of the molecular determinants of the pre-P cytotype is discussed.     

Analysis of a contiguous 211 kb sequence in diploid wheat (Triticum monococcum L.) reveals multiple mechanisms of genome evolution

In plant species with large genomes such as wheat or barley, genome organization at the level of DNA sequence is largely unknown. The largest sequences that are publicly accessible so far from Triticeae genomes are two 60 kb and 66 kb intervals from barley. Here, we report on the analysis of a 211 kb contiguous DNA sequence from diploid wheat (Triticum monococcum L.). Five putative genes were identified, two of which show similarity to disease resistance genes. Three of the five genes are clustered in a 31 kb gene-enriched island while the two others are separated from the cluster and from each other by large stretches of repetitive DNA. About 70% of the contig is comprised of several classes of transposable elements. Ten different types of retrotransposons were identified, most of them forming a pattern of nested insertions similar to those found in maize and barley. Evidence was found for major deletion, insertion and duplication events within the analysed region, suggesting multiple mechanisms of genome evolution in addition to retrotransposon amplification. Seven types of foldback transposons, an element class previously not described for wheat genomes, were characterized. One such element was found to be closely associated with genes in several Triticeae species and may therefore be of use for the identification of gene-rich regions in these species.     

Hot spot for Tn1000 insertions in cloned repressor gene of the L phage

Insertions of Tn1000 into a cloned fragment of the L-phage genome comprising the repressor gene were prepared. Repressor activities produced by plasmids with insertions were assayed in vivo. As a result, the repressor gene was localized within 0.5 kb near one end of the cloned fragment. Transposon insertions were nonrandomly clustered within the repressor gene and in its close vicinity. An analysis of supercoiled plasmid DNA with the nuclease S1 revealed no distortion of the secondary structure of DNA in this region.     

Dynamic equilibrium between insertion and excision of P elements in highly inbred lines from an M′ strain of Drosophila melanogaster

Six highly inbred lines of Drosophila melanogaster extracted from an M strain (in the P/M system of hybrid dysgenesis) were studied for the evolution of the number and chromosomal location of complete and defective P elements through generations 52–200. These lines possessed full-sized P elements but differed in their cytotype (M or P). Three lines with P cytotype and full-sized P elements at site 1A had a constant P copy number over generations with low rates of insertion and excision. Three lines with M cytotype and at least one full-sized P element accumulated P copies over the generations and reached a plateau near generation 196, at which rates of transposition and excision were equal to 1.2 × 10^–3 to 3 × 10^–3 events per element per generation. At that time these three lines still presented an M cytotype, produced transposase, and were able to regulate P copy number. The similarity at equilibrium between insertion and excision rates was exactly what was expected from theoretical models for a self-regulated element. The large number of generations necessary to attain the equilibrium in copy number indicates, however, that caution may be de rigueur when testing theoretical models of copy-number containment based on transposition and excision-rate comparison.     

Mutations in the CCGTTCACA DnaA box of Mycobacterium tuberculosis oriC that abolish replication of oriC plasmids are tolerated on the chromosome

The origin of replication (oriC) region in some clinical strains of Mycobacterium tuberculosis is a hot spot for IS6110 elements. To understand how clinical strains with insertions in oriC can replicate their DNA, we characterized the oriC regions of some clinical strains. Using a plasmid-based oriC-dependent replication assay, we showed that IS6110 insertions that disrupted the DnaA box sequence CCGTTCACA abolished oriC activity in M. tuberculosis. Furthermore, by using a surface plasmon resonance technique we showed that purified M. tuberculosis DnaA protein binds native but not mutant DnaA box sequence, suggesting that stable interactions of the DnaA protein with the CCGTTCACA DnaA box are crucial for replication of oriC plasmids in vivo. Replacement by homologous recombination of the CCGTTCACA DnaA box sequence of the laboratory strain M. tuberculosis H37Ra with a mutant sequence did not result in nonviability. Together, these results suggest that M. tuberculosis strains have evolved mechanisms to tolerate mutations in the oriC region and that functional requirements for M. tuberculosis oriC replication are different for chromosomes and plasmids.