Spread of the autonomous transposable element hobo in the genome of Drosophila melanogaster

The transposable element hobo has been introduced into the previously empty Drosophila melanogaster strain Hikone so that its dynamics can be followed and it can be compared with the P element. Five transformed lines were followed over 58 generations. The results were highly dependent on the culture temperature, the spread of hobo element being more efficient at 25 degrees C. The multiplication of hobo sequences resulted in a change in the features of these lines in the hobo system of hybrid dysgenesis. The number of hobo elements remained low (two to seven copies) and the insertions always corresponded to complete sequences. Our findings suggest that, despite their genetic similarities, P and hobo elements differ in many aspects, such as mobility and regulation mechanisms.      

Hobo Enhancer Trapping Mutagenesis in Drosophila Reveals an Insertion Specificity Different from P Elements

P element enhancer trapping has become an indispensable tool in the analysis of the Drosophila melanogaster genome. However, there is great variation in the mutability of loci by these elements such that some loci are relatively refractory to insertion. We have developed the hobo transposable element for use in enhancer trapping and we describe the results of a hobo enhancer trap screen. In addition, we present evidence that a hobo enhancer trap element has a pattern of insertion into the genome that is different from the distribution of P elements in the available database. Hence, hobo insertion may facilitate access to genes resistant to P element insertion.     

Wanderings of Hobo: A Transposon in Drosophila Melanogaster and its Close Relatives

The transposon hobo is present in the genomes of Drosophila melanogaster and Drosophila simulans (and D. mauritiana and probably D. sechellia, based on Southern blots) as full-size elements and internally deleted copies. The full-size melanogaster, simulans and mauritiana hobo elements are 99.9% identical at the DNA sequence level, and internally deleted copies in these species essentially differ only in having deletions. In addition to these, hobo-related sequences are present and detectable with a hobo probe in all these species. Those in D. melanogaster are 86-94% identical to the canonical hobo, but with many indels. We have sequenced one that appears to be inserted in heterochromatin (GenBank Acc. No. AF520587). It is 87.6% identical to the canonical hobo, but quite fragmented by indels, with remnants of other transposons inserted in and near it, and clearly is defunct. Numerous similar elements are found in the sequenced D. melanogaster genome. It has recently been shown that some are fixed in the euchromatic genome, but it is probable that still more reside in heterochromatic regions not included in the D. melanogaster genome database. They are probably all relics of an earlier introduction of hobo into the ancestral species. There appear to have been a minimum of two introductions of hobo into the melanogaster subgroup, and more likely three, two ancient and one quite recent. The recent introduction of hobo was probably followed by transfers between the extant species (whether 'horizontally' or by infrequent interspecific hybridization).     

DNA sequence requirements for hobo transposable element transposition in Drosophila melanogaster

We have conducted a structure and functional analysis of the hobo transposable element of Drosophila melanogaster. A minimum of 141 bp of the left (L) end and 65 bp of the right (R) end of the hobo were shown to contain sequences sufficient for transposition. Both ends of hobo contain multiple copies of the motifs GGGTG and GTGGC and we show that the frequency of hobo transposition increases as a function of the copy number of these motifs. The R end of hobo contains a unique 12 bp internal inverted repeat that is identical to the hobo terminal inverted repeats. We show that this internal inverted repeat suppresses transposition activity in a hobo element containing an intact L end and only 475 bp of the R end. In addition to establishing cis-sequences requirements for transposition, we analyzed trans-sequence effects of the hobo transposase. We show a hobo transposase lacking the first 49 amino acids catalyzed hobo transposition at a higher frequency than the full-length transposase suggesting that, similar to the related Ac transposase, residues at the amino end of the transposase reduce transposition. Finally, we compared target site sequences of hobo with those of the related Hermes element and found both transposons have strong preferences for the same insertion sites.     

Transpositions of mobile elements mdg4 (gypsy) and hobo in somatic and germ cells of a genetically unstable mutator strain of Drosophila melanogaster]

Analysis of distribution of the several families of mobile genetic elements has been performed. The analysis dealt with the X chromosomes of male progeny from the crosses of individual males of Mutator strain (MS) with attached-X females. The experimental results demonstrated different localization of the elements gypsy and hobo in the salivary gland squashes of different males-brothers. Location of other elements under study--mdg1, 412, mdg3, copia, 297, 17.6, Beagle, BS, Doc, FB, Springer--was invariant in all larvae. The analysis is equal to the study of transposition events at the level of gametes. Thus, doubtless, the capability of gypsy and hobo to transpose in germ cells of the MS individuals has been detected. Mobilization of the elements occurs at premiotic stages of gametes' development, as indicated by appearance of the clusters of transpositions. In the process of studies on coincidence of gypsy and hobo transposition acts, independent character of the elements' movement has been revealed. It has been detected in the same experiment that the distribution of the gypsy copies in different cells of the same salivary gland varies strongly. All hybridization sites were divided into two groups: "constant" sites common for all cells and "additional" ones, whose locations did not coincide in neighbouring cells of salivary gland. The existence of additional sites is major evidence of gypsy transpositions in somatic cells of MS. Transposition events have been as well discovered for hobo in somatic cells.     

Participation of mobile element hobo in transposition events in the long-term instability system of Drosophila melanogaster]

The lines with an active hobo elements as well as those without any hobo fragments were hybridized with the y2sc1waG line. This resulted in the appearance of a number of mutations at the white, miniature, and some other loci. The authors analysed, in which way the hobo transposable elements take part in mutagenesis in these crosses. Most of the white mutants obtained were analysed and transpositions of hobo and Stalker elements were demonstrated. Both independent and simultaneous transpositions were found. It was shown by means of the Southern blot analysis that additional hobo or Stalker insertion into or close to the parental unknown waG insertion resulted in mutant white phenotype's shift toward both extreme and partial reversion. Possible participation in mutagenesis of other mobile elements is also under debate.     

Behavior of hobo and P transposons in yellow2-717 unstable line of Drosophila melanogaster and its derivatives after crossing with a laboratory strain

Using fluorescent in situ hybridization technique (FISH), the frequency of hobo and P mobile elements transpositions on X chromosomes from the y2-717, isolated from the Uman' population of Drosophila melanogaster, as well as from its phenotypically normal and mutant derivatives, obtained as a result of crosses the males examined with the C(I)DX, ywf/Y females, was evaluated. It was demonstrated that the maximum frequency of hobo transpositions on X chromosomes of the males from derivative strains, subjected to repeated hobo-dysgenic crosses reached a value of 1.2 x 10(-2) per site per X chromosome per generation. The number of hobo copies in male X chromosomes from derivative strains was 3 times higher than in the original initial strain. Furthermore, the "old" hobo sites remained unchanged. In derivative strains, the frequency of hobo insertions was higher than that of excisions. One of the derivative strains, y1t-717alk3-2, was characterized by high intra-strain instability of hobo element localization. In the y2-717a1k3 and y1t-717alk3-2 strains a large inversion, In(1)1B; 13CD, was described. At the absence of the full-sized P element in the strains involved in crosses, maximum frequency of P element transpositions in the derivative strains reached a value of 1.2 x 10(-2) per site per X chromosome per generation.     

Identification of a full-size hobo element and deletion-derivatives in Korean populations of Drosophila melanogaster.

We have isolated and characterized several members of the hobo transposable element family from Korean populations of Drosophila melanogaster. All of the Korean lines tested appeared to have 3.0 kb hobo elements and a high copy number of smaller derivatives of the element. To determine whether a 3.0 kb hobo element of these populations is consistent with the role of an autonomous hobo element, we cloned and sequenced this hobo element. Based on the result of the entire DNA sequence, a cloned 3.0 kb element called HKN96, it was found to be the same as a fully-functional 2959 bp HFL1-type sequence. Each small element appeared to have arisen from the HFL1 element by a different internal deletion. A specific 1.7 kb Kh hobo element, which is the most abundant in the Korean lines tested, seems to have originated from the HFL1 hobo element by an internal deletion of 1253 bp by the removal of nucleotides between positions 939 and 2191. The sequences of the Th1 and Th2 elements appeared to be identical to that of the HFL1 with the exception of internal deletions of 1442 bp and 1455 bp removing nucleotides 940-2381 and 923-2377, respectively. Based on the number of TPE repeats, all of the members of the hobo element family in Korean lines tested have three perfect S repeats. The widespread presence of identical copies of the Kh deletion derivative suggests that it might have a role in the regulation of hobo-induced hybrid dysgenesis.     

Patterns of transposable elements distribution on the Drosophila melanogaster polytene chromosomes before and after selection for a quantitative trait

The effect of selection for radius vein length on the distribution of hybridization sites of the P and hobo transposons and the mdgl and mdg2 retrotransposons on polytene chromosomes of Drosophila melanogaster salivary glands was studied. The patterns of these transposable elements (TEs) distribution were polymorphic in both the parental strain and selected strains. The similarity in mdg1 and mdg2 patterns between strains selected in one direction was closer than between strains selected in opposite directions, but the selected strains were closer to each other than to the parental strain regardless of selection direction. No mdg2 hybridization sites that would be absent in the control were found in the selected strains. There were more mdg2 and hobo hybridization sites in the strains selected in the (+) direction than in the (-) direction. The mobility of hobo copies in the strains studied correlated with the presence of its full-sized copy in the genome. The polymorphism of all TEs studied except for mdgl was greater for strains selected in the (+) direction that in the (-) direction. These facts suggest that some TEs migrate over the genome independently of selection, and others are markers of evolutionary events rather than their causes.     

Identification of a fully-functional hobo transposable element and its use for germ-line transformation of Drosophila.

The transposable element hobo can be mobilized to induce a variety of genetic abnormalities within the germ-line of Drosophila melanogaster. Strains containing hobos have 3.0 kb elements and numerous smaller derivatives of the element. By analogy with other transposable element systems, it is likely that only the 3.0 kb elements are capable of inducing hobo mobilization. Here, we report that a cloned 3.0 kb hobo, called HFL1, is able to mediate germ-line transformation and therefore is an autonomous (fully-functional) transposable element. Germ-line transformation was observed when HFL1 and a marked hobo element were co-injected into recipient embryos devoid of endogenous hobos. Integration did not occur in the absence of the 3.0 kb element. A single copy of the marked hobo transposon inserted at each site, and the target sites were widely distributed throughout the genome. Integration occurred at (or very near) the termini of hobo, without internal rearrangement of the hobo or marker gene sequences. The hobo transformation system will allow us to determine the structural and regulatory features of hobo responsible for its mobilization and will provide novel approaches for the molecular and genetic manipulation of the Drosophila genome.     

Ancient allopolyploid speciation in Geinae (Rosaceae): evidence from nuclear granule-bound starch synthase (GBSSI) gene sequences

A nuclear low-copy gene phylogeny provides strong evidence for the hybrid origin of seven polyploid species in Geinae (Rosaceae). In a gene tree, alleles at homologous loci in an allopolyploid species are expected to be sisters to orthologues in the ancestral taxa rather than to each other. Alleles at a duplicated locus in an autopolyploid, however, are expected to be more closely related to each other than they are to any orthologous copies in closely related species. We cloned and sequenced about 1.9 kilobases from the 5' end of the GBSSI-1 gene from two diploid, one tetraploid, and six hexaploid species. Each of the three loci in the hexaploid species forms a separate group, two of which are more closely related to copies in other species than they are to each other. This finding indicates that the hexaploid lineage evolved through two consecutive allopolyploidization events. Based on the GBSSI-1 gene tree, we hypothesized that there was an initial hybridization between a diploid species from the ancestral lineage of Coluria and Waldsteinia and an unknown diploid species to form the tetraploid Geum heterocarpum lineage. Backcrossing of G. heterocarpum with a representative of the unknown diploid lineage then resulted in a hexaploid lineage that has radiated considerably since its origin, comprising at least 40 extant species with various morphologies. A penalized likelihood analysis indicated that Geinae may be about 17 million years old, implying that the hypothesized allopolyploid speciation events are relatively ancient. Six of the 22 cloned Geinae GBSSI-1 copies in this study, which all are duplicate copies in polyploid taxa, may have become pseudogenes. We compared the GBSSI-1 phylogeny with one from chloroplast data and explored implications for the evolution of some fruit characters.     

Homogeneity in the structure of the medaka fish transposable element Tol2

The hAT family is a group of transposable elements of the terminal inverted repeat class, which includes Ac of maize, hobo of Drosophila and Tam3 of Antirrhinum (snapdragon). All the members of this family so far examined are known to comprise complete and defective copies, with a good correspondence to autonomous and non-autonomous elements, respectively. Internal deletion is the most common cause of defective copies. Tol2, a transposable element of the medaka fish Oryzias latipes, is a member of the hAT family. We examined, mainly by the genomic Southern blot analysis, variation in the structure of copies of this element, and revealed that there are few or no internally deleted copies. This situation is unusual in a member of the hAT family. Possible causes of this anomaly are discussed.     

Distribution of hobo transposable elements in the genus Drosophila

This study describes the distribution of hobo-hybridizing sequences in the genus Drosophila. Southern blot analysis of 134 species revealed that hobo sequences are limited to the melanogaster and montium subgroups of the melanogaster-species group. Of the hobo-bearing species, only D. melanogaster and two of its sibling species, D. simulans and D. mauritiana, were found to contain potentially complete hobo elements. The distribution of hobo sequences is one of the narrowest distributions thus far described for any Drosophila transposable element.     

Identification of a new hobo element in the cabbage moth, Mamestra brassicae (Lepidoptera)

A complete hobo-like element, called Mbhobo, was identified in the cabbage moth, Mamestra brassicae. This element has a high sequence similarity to the HFL1 hobo element of Drosophila melanogaster. Amplification of Mbhobo termini indicated that transposition occurred into a 5'-GTGGGTAC-3' target sequence that was duplicated upon insertion. This target site conforms to the consensus sequence established for the insertion sites of insect hAT elements. Mbhobo has a single 1935 bp long ORF with significant homology to the D. melanogaster HFL1 hobo transposase. FISH experiments evidenced Mbhobo clusters located in heterochromatic regions of Z and W sex chromosomes and in heterochromatic areas of chromosome pair 10.     

The instability of the TE-like mutation Dp(2:2)GYL of Drosophila melanogaster is intimately associated with the hobo element

We have characterized molecularly several derivatives of the TE-like element Dp(2:2)GYL of Drosophila melanogaster. This highly unstable mutation occurred in a dysgenic cross involving the 23.5 MRF chromosome, and represents an inverted insertional duplication of approximately 130 polytene bands of the paternal 2L, at 50AB of the right arm of the maternal 2R. The instability of this mutation is characterized by deletion of some of duplicated material, by the induction of rearrangements in its vicinity and by the transposition of parts of the original element. We have found that the mobile element hobo is present at , or very near, the breakpoints of all GYL derivatives analysed, demonstrating that hobo is not only active in dysgenic crosses, but also that it can promote genetic instability reminiscent of transposable elements (TE).     

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.     

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.     

Germline Transformation of Drosophila Virilis Mediated by the Transposable Element Hobo

A laboratory strain of Drosophila virilis was genetically transformed with a hobo vector carrying the miniwhite cassette using a helper plasmid with an hsp70-driven hobo transposase-coding sequence. The rate of transformation was 0.5% per fertile G0 animal. Three transgenic insertions were cloned and characterized and found to be authentic hobo insertions. These results, together with the known wide-spread distribution of hobo in diverse insect species, suggest that hobo and related transposable elements may be of considerable utility in the germline transformation of insects other than D. melanogaster.     

The Hobo Transposable Element Excises and Has Related Elements in Tephritid Species

Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobo-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.