Rearrangements at a hobo element inserted into the first intron of the singed gene in the unstable sn 49 system of Drosophila melanogaster

The cytological structure of the X chromosome and the DNA organisation of the singed locus were examined in five singed bristle mutants of Drosophila melanogaster. These mutants are all derived from the unstable mutant singed-49, isolated from a wild population in the Russian Far East in 1975. Rearrangements were found at a site within the first intron of the singed gene, where a hobo element is inserted in these mutants. One rearrangement, which is associated with a strong bristle phenotype, has an inversion between 2D and the location of singed at 7D, which separates the singed promoter from the singed coding region. Two phenotypically wild-type derivatives have smaller rearrangements within the first intron which do not appear to interfere with singed expression. Two derivatives with bristle phenotypes have more complex rearrangements, and one of them shows a dominant or antimorphic phenotype. DNA blotting and in situ hybridisation experiments show that, in addition to these rearrangements at a hobo element inserted at singed, other hobo elements in these strains have been mobilised. This system is therefore similar to others in which functional hobo elements continue to transpose, resulting in elevated rates of mutation and chromosome rearrangement.     

The role of the transposable element hobo in the origin of endemic inversions in wild populations of Drosophila melanogaster

Evidence from in situ hybridizations of DNA from the transposable element hobo to polytene salivary gland chromosome squashes reveals that hobo occupies both cytological breakpoints of three of four endemic inversions sampled from natural populations of Drosophila melanogaster in the Hawaiian islands. The fourth endemic inversion has a single hobo insert at one breakpoint. Cosmopolitan inversions on the same chromosomes do not show this association. Frequencies of both endemic and cosmopolitan inversions in Hawaiian populations fall in ranges typical for natural populations of D. melanogaster sampled worldwide, suggesting that these results may be typical of other regions besides Hawaii. This appears to be the first direct demonstration that transposable elements are responsible for causing specific rearrangements found in nature; consequently, it is also the first direct demonstration that chromosome rearrangements can arise in nature in a manner predicted by results of hybrid dysgenic crosses in the laboratory. Possible population genetic and evolutionary consequences are discussed.     

Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5′ splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis. Received: 20 February 1997 / Accepted: 28 August 1997     

A maize Ds transposable element containing a dihydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana

Summary A mouse dihydrofolate reductase gene (DHFR), encoding an enzyme conferring methotrexate (MTX) resistance, under the control of the cauliflower mosaic virus (CaMV) 35 S promoter, was inserted within a maize nonautonomous Ds transposable element. The presence of at least one element (Ds-DHFR) can easily be monitored using methotrexate selection in plants. This chimeric element is able to transpose at a frequency similar to its unmodified progenitor in transgenic tobacco callus containing an autonomous Ac element. The orientation of the selectable marker cassette in the Ds element does not affect relative excision frequencies. Approximately two-thirds of these elements can be detected after excision while the remaining one-third cannot. The Ds-DHFR element is useful in elucidating the mechanism by which Ac/Ds transposition occurs, and allows for a rapid identification of mutants in which methotrexate resistance cosegregates with a mutant phenotype.     

Gross chromosome rearrangements mediated by transposable elements in Drosophila melanogaster

A combination of cytogenetic and molecular analyses has shown that several different transposable elements are involved in the restructuring of Drosophila chromosomes. Two kinds of elements, P and hobo, are especially prone to induce chromosome rearrangements. The mechanistic details of this process are unclear, but, at least some of the time, it seems to involve ectopic recombination between elements inserted at different chromosomal sites; the available data suggest that these ectopic recombination events are much more likely to occure between elements in the same chromosome than between elements in different chromosomes. Other Drosophila transposons also appear to mediate chromosome restructuring by ectopic recombination; these include the retrotransposons BEL, roo, Docand I and the foldback element FB. In addition, two retrotransposons, HeT-A and TART, have been found to be associated specifically with the ends of Drosophila chromosomes. Very limited data indicate that transposon-mediated chromosome restructuring is occurring in natural populations of Drosophila. This suggests that transposable elements may help to shape the structure of the Drosophila genome and implies that they may have a similar role in other organisms.     

Isolation and analysis of functional homologues of the secretion-related SAR1 gene of Saccharomyces cerevisiae from Aspergillus niger and Trichoderma reesei

The Aspergillusniger and Trichodermareesei genes encoding the functional homologues of the small GTP-binding protein SAR1p, which is involved in the secretion pathway in Saccharomyces cerevisiae, have been cloned and characterised. The A. niger gene (sarA) contains five introns, whereas the T. reesei gene (sar1) has only four. In both cases the first intron is at the same position as the single S. cerevisiae SAR1 intron. The encoded proteins show 70–80% identity to the SAR1 protein. Complementation of S. cerevisiaesar1 and sec12 mutants by expression vectors carrying the A. nigersarA and T. reesei sar1 cDNA clones confirmed that the cloned genes are functional homologues of the S. cerevisiae SAR1 gene. Three mutant alleles of the A. nigersarA gene (D29G, E109K, D29G/E109K), generated by site-directed mutagenesis, revealed a thermosensitive dominant-negative phenotype in the presence of the wild-type sarA allele. This result contrasts with the situation in S. cerevisiae, where similar mutations have a thermosensitive phenotype. Taken together, our results indicate that the sarA gene is involved in an essential function in A. niger. Received: 21 January 1997 / Accepted: 21 June 1997     

Sesqui-Ds , the chromosome-breaking insertion at bz-m1 , links double Ds to the original Ds element

The bz-m1 mutation in maize was one of the first to arise by direct transposition of the chromosome-breaking Ds element from its original or `standard' location in chromosome 9S to a known locus in the same chromosome arm. Thus, elucidation of its structure should shed light on the nature of the original Ds element described by McClintock in 1948. The Ds insertion in bz-m1 has been reported to be only 1.2 kb long – much shorter than other chromosome-breaking Ds elements that have been described. We have characterized here the Ds element in our bz-m1 stocks and have confirmed by genetic and molecular tests that, in the presence of Ac, it acts as a chromosome breaker. The Ds insertion at bz-m1 is 1260 bp long. Besides its normal 5′ and 3′ ends, it contains an internal 3′ end at the same junction as the chromosome-breaking double Ds element that has been found in several sh mutations. Thus, it appears to have arisen from the 4.1-kb double Ds by internal deletion of 2.9 kb. Because the element has lost one internal 5′ end, but retains the chromosome-breaking properties of double Ds, we have named it sesqui-Ds (sDs). The origin, structure and properties of sDs vis-à-vis double Ds support the hypothesis that double Ds corresponds to the chromosome-breaking Ds element at the `standard' location in 9S. Received: 10 March 1997 / Accepted: 2 May 1997     

Analysis of secondary, integration sites for IS117 in Streptomyces lividans and their role in the generation of chromosomal deletions

IS117, the 2.6 kb mini-circle of Streptomyces coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome. When introduced into S. lividans, IS117 integrates into one preferred chromosomal site, but when this site was artificially deleted, IS117 integrated into many secondary sites. Nucleotide sequence analysis of several secondary integration sites revealed varying degrees of similarity with the preferred site, but no consensus sequence. Nevertheless, sites more similar to the preferred site tended to be occupied more often than those that are less similar. Insertion of IS117 into secondary sites in the chromosome of S. lividans sometimes mediated chromosomal rearrangements. It was shown that some strains containing IS117 integrated into secondary sites had suffered deletions of chromosomal DNA. Deletions were adjacent to the inserted element and were at least several kilobases long. The proposed model implicates homologous recombination between IS117 copies integrated into two different secondary sites in the same chromosome as a cause of the deletions.     

The hobo transposable element has transposase-dependent and-independent excision activity in drosophilid species

Mobility of the hobo transposable element was determined for several strains of Drosophila melanogaster and several Drosophila species. Mobility was assessed by use of an in vivo transient assay in the soma of developing embryos, which monitored hobo excision from injected indicator plasmids. Excision was detected in a D. melanogaster strain (cn; ry ⁴²) devoid of endogenous hobo elements only after co-injection of a helper plasmid containing functional hobo transposase under either heat shock or normal promoter regulation. Excision was also detected in D. melanogaster without helper in strains known to contain genomic copies of hobo. In Drosophila species confirmed not to contain hobo, hobo excision occurred at significant rates both in the presence and absence of co-injected helper plasmid. In four of the seven species tested, excision frequencies were two- to fivefold lower in the presence of plasmid-borne hobo. hobo excision donor sites were sequenced in indicator plasmids extracted from D. melanogaster cn; ry ⁴² and D. virilis embryos. In the presence of hobo transposase, the predominant excision sites were identical in both species, having breakpoints at the hobo termini with an inverted duplication of proximal insertion site DNA. However, in the absence of hobo transposase in D. virilis, excision breakpoints were apparently random and occurred distal to the hobo termini. The data indicate that hobo is capable of functioning in the soma during embryogenesis, and that its mobility is unrestricted in drosophilids. Furthermore, drosophilids not containing hobo are able to mobilize hobo, presumably by a hobo-related cross-mobilizing system. The cross-mobilizing system in D. virilis is not functionally identical to hobo with respect to excision sequence specificity.     

Dap ( D efective a leurone p igmentation) mutations affect maize aleurone development

Dap (Defective aleurone pigmentation) is the designation for mutations in maize that give rise to a characteristic dappled endosperm phenotype, consisting of patches of purple tissue, of variable size and shape, on a yellow background. Features shared by all Dap mutants are: dominant expression when they are maternally derived, lack of expression or transmission when they originate from pollen, failure to recover homozygous Dap genotypes, reduced frequency of Dap seeds in the progeny of outcrosses of Dap/+ females, association of the dappled phenotype with reduction in seed size. The mutants so far tested, six in all, can be grouped into two classes, one including male-transmissible (MT) isolates not expressed in the endosperm if their contribution is paternal, and a second class of isolates (NMT) that are permanently lost following paternal transmission. We suggest that the NMT mutations are on a chromosome that carries an intercalary deletion. Assuming linkage between the mutant and the deletion, selection against the deficient chromosome during male gametogenesis would account for the failure to recover Dap seeds in the progeny of Dap/+ male parents. We have obtained genetic evidence supporting this hypothesis. This interpretation, however, does not apply to MT alleles. For these, other mechanisms, such as imprinting and/or dosage effects may be proposed. The mutable pattern in the endosperm to which all Dap mutants give rise is an intriguing phenotype which remains to be clarified. An unexpected finding is that aleuronic and subaleuronic cells corresponding to the colourless areas are abnormal in shape and anthocyanin biosynthesis is blocked in these cells. This finding calls for further investigation in light of a possible connection between flavonoid precursors and cell shape. Received: 2 January 1997 / Accepted: 26 May 1997     

Mobilization of a Hobo-related Sequence in the Genome of Drosophila simulans

The hobo transposable element can occur under three forms in the Drosophila genome: as a complete element (also called canonical), as internally deleted copies, or as hobo-related sequences (relics). Some evidence indicated that canonical elements and internally deleted copies are recent acquisitions of Drosophila genomes, while the “relics” are old components, normally degenerated and immobile. Here we present the characterization of a hobo-related sequence, found in the genome of a hypermutable strain of D. simulans, which insertion into the white locus raised a de novo white mutation. It is a shorter hobo related element presenting, overall, roughly 18% of divergence at the DNA level from the canonical hobo, with many indels that make clear this element is defective. However, its ITRs and flanking regions are extremely conserved. This is the first hobo “relic” showed to be mobilizable. We suggest, and point up some evidences, toward the idea that this sequence could have been mobilized by the canonical element. The presence of a similar “relic” element in D. sechellia allows us to suggest that these elements have been maintained mobilizable since the time of divergence between these species.     

Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene

A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336 bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tc1-like transposases, and a residual sequence of about 2 kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus. Received: 12 February 1997 / Accepted: 2 May 1997     

Characterization of permissivity for hobo -mediated gonadal dysgenesis in Drosophila melanogaster

The hobo transposon is responsible for one of the three hybrid dysgenic systems that have been described in Drosophila melanogaster. Most studies on the hobo dysgenic system have been carried out using the PM system as a reference. However, these two systems differ significantly. In particular, several studies have failed to find any correlation between the molecular structures of hobo elements, the instability of the transposon and the incidence of gonadal dysgenic (GD) sterility. On the other hand, no study of the ability of females to permit hobo activity in their progeny when they are crossed with males harboring active hobo elements (permissivity) has yet been reported. In order to investigate the parameters involved in hobo permissivity, four E strains were studied with regard to the molecular nature of their hobo sequences and the GD sterility induced by a controlled source of hobo transposase. We show that hobo permissivity varies both within and between E strains. Moreover, permissivity decreases with age in E females. Our results are discussed with respect to similar phenomena that have been described in relation to the reactivity of the IR dysgenic system. Received: 17 August 1998 / Accepted: 17 December 1998     

Interplasmid transposition of Drasopbila hobo elements in non-drosophilid insects

A modified hobo element from Drosophila melanogaster was introduced into embryos of the housefly, Musca domestica (family Muscidae) and the Queensland fruitfly, Bactrocera tryoni (family Tephritidae) to assess its ability to transpose. Hobo was capable of transposition in these species and transposition products had all of the hallmarks of hobo transposition products recovered from D. melanogaster, including the movement only of sequences precisely delimited by the inverted terminal repeats of hobo, the creation of an 8 by duplication of the insertion site and an absolute requirement for hobo-encoded transposase. Transposition of hobo into the target gene resulted in a non-random distribution of insertion sites, with 10 of 38 independent insertions into the same nucleotide position. The results indicate that hobo can transpose in heterologous species, further demonstrating the similarty of hobo to Ac (Activator) of Zea mays and Tam3 of Antirrhinum majus. Hobo has excellent potential to act as a gene vector or gene tagging agent in nondrosophilid insects.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Involvement of the rolled/MAP kinase gene in Drosophila mitosis: interaction between genes for the MAP kinase cascade and abnormal spindle

We have found that mutations that lead to loss of rolled/MAP kinase function result in a reduced mitotic index in the larval central nervous system, consistent with an interphase block to cell cycle progression, associated with a low frequency of cells showing chromosome over-condensation in mitosis and abnormal anaphase figures. In contrast to wild-type tissue, such rolled mutants do not show a significant increase in accumulation of mitotic cells when treated with colchicine. We have studied double mutant combinations between mutations affecting the activity of rolled/MAP kinase and several genes that are essential to the establishment of a bipolar spindle during progression through mitosis, and find no interactions with mutations in polo, mgr,or aurora. However, partial loss-of-function mutations in rolled enhance the abnormal spindle (asp) phenotype, whereas gain-of function mutations in rolled or in the gene encoding its activating kinase Dsor1, act as suppressors. We discuss these findings in relation to the proposed role of MAP kinase in mediating the spindle integrity checkpoint. Received: 27 October 1997 / Accepted: 18 December 1997     

Comparison of genetic behaviour of the transposable element Tam3 at two unlinked pigment loci in Antirrhinum majus

Summary In Antirrhinum majus the transposable element Tam3 has been described at two unlinked loci pallida and nivea, both of which are required for the production of anthocyanin pigment in flowers. In each case the element is inserted in the promoter region and gives a variegated phenotype. We show that the rate of Tam3 excision at both loci is greatly affected by temperature, being approximately 1000-fold higher at 15°C compared with 25°C. Tam3 is also controlled by an unlinked gene Stabiliser, which considerably reduces excision rate. We show that the high degree of sensitivity to temperature and Stabiliser is an intrinsic property of Tam3 which is not shared by an unrelated element, Tam1. The Tam3 insertion at nivea gives rise to a series of alleles which confer reduced pigmentation, novel spatial patterns and changed instability. These are probably a result of imprecise excision and rearrangements of the Tam3 element.     

prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns

Summary We have generated a bank of temperature-sensitive (ts) Schizosaccharomyces pombe mutant strains. About 150 of these mutants were transformed with a ura4 gene containing an artificial intron. We screened these is mutants for mutants deficient in splicing of the ura4 intron. With this approach three mutants were isolated which have a general defect in the splicing process. Two of these mutants fall into the prp1 complementation group and one defines a new complementation group, prp4.