P-element Research Articles

The 5' untranslated region of the I factor, a long interspersed nuclear element-like retrotransposon of Drosophila melanogaster, contains an internal promoter and sequences that regulate expression.

The I-R system of hybrid dysgenesis in Drosophila melanogaster is controlled by a long interspersed nuclear element-like retroposon, the I factor. Transposition of the I factor occurs at a high frequency only in the ovaries of females produced by crossing males of inducer strains that contain functional I factors with females of reactive strains that lack them. In this study, the 5' untranslated region of the I factor was joined to the chloramphenicol acetyltransferase gene, and activity was assayed in transfected D. melanogaster tissue culture cells and transformed flies. The results have identified a promoter that lies within the first 186 pb of the I factor. Deletion analysis shows that nucleotides +1 to +40 are sufficient for high promoter activity and accurate transcription initiation. This region contains sequences that are found in a class of RNA polymerase II promoters that lack both a TATA box and CpG-rich motifs. In transformed flies, high levels of expression from nucleotides +1 to +186 are confined to the ovaries of reactive females, suggesting that the promoter is involved in the tissue and cytotype specificity of transposition.

The 5' untranslated region of the I factor, a long interspersed nuclear element-like retrotransposon of Drosophila melanogaster, contains an internal promoter and sequences that regulate expression.

The I-R system of hybrid dysgenesis in Drosophila melanogaster is controlled by a long interspersed nuclear element-like retroposon, the I factor. Transposition of the I factor occurs at a high frequency only in the ovaries of females produced by crossing males of inducer strains that contain functional I factors with females of reactive strains that lack them. In this study, the 5' untranslated region of the I factor was joined to the chloramphenicol acetyltransferase gene, and activity was assayed in transfected D. melanogaster tissue culture cells and transformed flies. The results have identified a promoter that lies within the first 186 pb of the I factor. Deletion analysis shows that nucleotides +1 to +40 are sufficient for high promoter activity and accurate transcription initiation. This region contains sequences that are found in a class of RNA polymerase II promoters that lack both a TATA box and CpG-rich motifs. In transformed flies, high levels of expression from nucleotides +1 to +186 are confined to the ovaries of reactive females, suggesting that the promoter is involved in the tissue and cytotype specificity of transposition.

Characterization of the mus308 gene in Drosophila melanogaster.

Among the available mutagen-sensitive mutations in Drosophila, those at the mus308 locus are unique in conferring hypersensitivity to DNA cross-linking agents but not to monofunctional agents. Those mutations are also associated with an elevated frequency of chromosomal aberrations, altered DNA metabolism and the modification of a deoxyribonuclease. This spectrum of phenotypes is shared with selected mammalian mutations including Fanconi anemia in humans. In anticipation of the molecular characterization of the mus308 gene, it has been localized cytogenetically to 87C9-87D1,2 on the right arm of chromosome three. Nine new mutant alleles of the gene have been generated by X-ray mutagenesis and one was recovered following hybrid dysgenesis. Characterization of these new alleles has uncovered additional phenotypes of mutations at this locus. Homozygous mus308 flies that have survived moderate mutagen treatment exhibit an altered wing position that is correlated with reduced flight ability and an altered mitochondrial morphology. In addition, observations of elevated embryo mortality are potentially explained by an aberrant distribution of nuclear material in early embryos which is similar to that seen in the mutant giant nuclei.

Structure of the Drosophila melanogaster gene encoding cyclin A

A P element (PE)-induced Drosophila melanogaster mutation, hari, affects the formation of the bristle mechanosensory organ in the adult fly. In this mutation, the site of PE insertion is in the first intron of the gene ( CycA) encoding cyclin A (CycA). In order to analyze the hari mutant at the molecular level, we cloned and sequenced the cDNA and genomic DNA encoding CycA. CycA has seven exons and six introns, and its transcription unit spans 6 kb. All exon-intron junctions are compatible with the GT/AG consensus. Results of primer extension analysis and RNase protection assay indicate that CycA has major and minor transcription start points ( tsp). To our knowledge, this is the first report on the CycA genomic sequence from a multicellular organism.

Chromosome rearrangement patterns of an SD chromosome (SDKona-2) in Drosophila melanogaster caused by hybrid dysgenesis.

The types and frequencies of spontaneous chromosome rearrangements caused by hybrid dysgenesis were studied in a second chromosome autosome of Drosophila melanogaster. This second chromosome, being an SD chromosome, had two important advantages over other autosomes for this study: (i) it had the two inversions characteristic of a standard SD-72 chromosome type, which distinguished it from its homolog in polytene chromosome spreads, and (ii) because of the meiotic drive associated with the segregation distorter system, it was preferentially transmitted to the next generation. The chromosome mutation frequency of this chromosome (given the name SDKona-2) was 8.3 and 11.7% in the F2 and F3 generations, respectively. The types of new chromosome rearrangements observed in the first four generations included paracentric inversions, pericentric inversions, duplications, deletions, reciprocal translocations (involving the third chromosome), and transpositions. Small paracentric inversions were the most common type of new rearrangement. Later, over 35 generations, some of these new rearrangements changed, either by becoming more complex or by being replaced with yet another new chromosome rearrangement. Duplications were unstable and were replaced by paracentric inversions whose breakpoints were on either side of the duplication. Transpositions arose both from a single multibreak event and from a series of two-break events.

Visible mutations induced by P-M hybrid dysgenesis in Drosophila melanogaster result predominantly from P element insertions

This study supplied no evidence that P-M hybrid dysgenesis is a general release mechanisms for transposon movement. Newly induced mutations (23 singed, three yellow, and one white) were generated by hybrid dysgenesis and were molecularly analyzed for the presence or absence of P element insertions. Only one dysgenically-induced insertion mutation out of 27 analyzed was the result of a non- P insert; this frequency is not statistically above the non-dysgenic control level. Thus, it appears that individual transposable elements families are independently regulated.

I-R hybrid dysgenesis in Drosophila melanogaster. use of in situ hybridization to show the association of I factor DNA with induced sex-linked recessive lethals

The purpose of this paper is the genetic visualization by in situ hybridization of 130 sex-linked recessive lethals plus a non-lethal induced by I-R dysgenesis. This collection of lethals involves inducer strains which differ in the position of the I elements on the X chromosomes. The I-R interaction was strong. Our previous results have shown that about 30% of the induced recessive lethals are associated with cytologically visible chromosomal rearrangements. 1. (1) The rearrangements induced by I-R-type hybrid dysgenesis often exhibit homology with the I factor at the level of one or both junction points, depending on the types of chromosome rearrangements. These results suggest that the chromosome rearrangements arise directly from the transposition of I elements. However, the breakpoints of some types of cytologically non-visible deficiencies and of 2 small cytologically visible deficiencies do not present detectable homology with the I factor. 2. (2) The majority of rearrangements do not involve the I elements already present on the paternal X chromosome. 3. (3) The hybridization signal distributions on the X chromosome are not uniform. They present peaks of various heights which may correspond to specific anchoring areas of copies of I in the course of integration. 4. (4) The data presented here agree with the literature with respect to the mean number of copies of I per X chromosome and to the excess of copies of I at locus 1A. Two rearrangement formation mechanisms are envisaged: crossing-over and ‘target’ exchanges.

A particular P-element insertion is correlated to the P-induced hybrid dysgenesis repression in Drosophila melanogaster.

Transposable P elements in Drosophila melanogaster cause hybrid dysgenesis if their mobility is not repressed. The ability to regulate the dysgenic activity of the P elements depends on several mechanisms, one of which hypothesized that a particular deleted P element (the KP element) results in a non-susceptibility which is biparentally transmitted. In this study totally non-susceptible lines, and susceptible lines containing exclusively KP elements (IINS2 line and IIS2 line) were isolated from a M' strain. We show that non-susceptibility is correlated with a particular insertion of one KP element located at the cytological site 47D1. The repression ability of the GD sterility is determined by a recessive chromosomal factor, and cannot be due to the KP-element number. Here the repression of the P mobility is associated with reduction of the P transcripts and the inhibition of P promoter activity.

The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding.

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.

Distribution and structure of cloned P elements from the Drosophila melanogaster P strain pi 2.

P transposable elements of Drosophila melanogaster cloned from the strong P strain pi 2 have been analysed. The structures and chromosomal locations of 26 of the 30-50 elements estimated to be present in pi 2 have been determined. At one location two elements are inserted 100 base pairs (bp) apart, and in a second location two elements are only separated by the 8 bp duplicated upon P-element insertion. In addition to 2.9 kilobase-pair (kbp) elements, elements with 14 different internal deletions from 1.3 to 2.3 kbp in size have been isolated. There are 7 copies of the 2.9 kbp element, 2 copies each of 5 internally deleted elements and a single copy of 9 internally deleted elements. One of the elements found twice is the KP element, which may play a role in the regulation of hybrid dysgenesis in strains which contain many copies of this element. Apart from internal deletions the elements are extremely homogeneous in DNA sequence, with only 2 single base polymorphisms detected twice each in over 16 kbp of P-element sequence. Although transpositions are infrequent in an inbred P cytotype strain such as pi 2, the distribution of these cloned elements indicates that when the genomic library was made, the strain was polymorphic with respect to element location. The distribution and structures of the element are discussed with respect to models for regulation of P-element transposition.

Spatial and temporal expression of the I factor during oogenesis in Drosophila melanogaster.

The I factor is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. Its mobility is associated with the I-R hybrid dysgenesis. In order to study the expression pattern of this LINE in vivo, a translational fusion between the first ORF of the I factor and the lacZ gene of Escherichia coli has been carried out and introduced in the genome of reactive (R) flies. Homozygous transgenic Drosophila lines have been established and analysed. ORF1 expression is limited to germ-line cells (nurse cells and oocyte) between stage 2 and 10 of oogenesis. No somatic expression is found. Position effects may limit the level of expression of a given transgene but do not modify its basic pattern of expression during the development of the fly. This reproducible control demonstrates both that I factor is driven by its own promoter, probably the internal one suggested by Mizrokhi et al. (Mizrokhi, L.J., Georgevia, S.G. and Ilying, Y.V. (1988). Cell 54, 685-691), and that tissue-specific regulatory sequences are present in the 5' untranslated part of the I factor. The nuclear localization of the fusion protein reveals the presence of nuclear localization signals (NLS) in the ORF1-encoded protein correlating with the possible structural and/or regulatory role of this protein. This expression is restricted to dysgenic and reactive females, and is similar in the two conditions. All the results obtained in this work suggest that I factor transposition occurs as a meiotic event, between stage 2 and 10 of the oogenesis and is regulated at the transcriptional level. It also appears that our transgene is an efficient marker to follow I factor expression.

Characterization of the progeny of X-ray irradiated males from two Drosophila virilis strains differing in genetic instability

Significant effects of X-ray treatment on the increase in the number of phenotypic variations, two visible mutations, and chromosome aberrations were found in the progeny of irradiated males from the D. virilis laboratory stock that is capable of hybrid dysgenesis syndrome induction. This effect is much more pronounced than in the progeny of irradiated males from strong wild-type strains studied. A correlation between genetic instability and chromosome radiosensitivity was outlined. The mechanism of this phenomenon and the possibilities of using the property of genome instability for the productive induction of gene and chromosome damage in radiation mutagenesis experiments are discussed.

Radiation and transposon-induced genetic damage in Drosophila melanogaster: X-ray dose-response and synergism with DNA-repair deficiency

The interaction of X-ray-induced and transposon-induced damage was investigated in P-M hybrid dysgenesis in Drosophila melanogaster. The X-ray dose-response of 330–1320 rad was monitored for sterility, fecundity and partial X/Y chromosome loss among F 2 progeny derived from the dysgenic cross of M strain females ×P strain males (cross A) and its reciprocal (cross B), using a weaker and the standard Harwich P strain subline. The synergistic effect of P element activity and X-rays on sterility was observed only in cross A hybrids and the dose-response was nonlinear in hybrids derived from the strong standard reference Harwich subline, H w. This finding suggests that the lesions induced by both mutator systems which produce the synergistic effect are two-break events. The effect of increasing dose on the decline of fecundity was synergistic, but linear, in hybrids of either subline. There was no interaction evident and thus no synergism in X/Y nondisjunction and in partial Y chromosome loss measured by the loss of the B s marker alone or together with the y + marker. Interaction was detected in the loss of the y + marker alone from the X and Y chromosomes. The possible three-way interaction of X-rays (660 rad), post-replication repair deficiency and P element mobility was assessed by measuring transmission distortion in dysgenic males derived from the Π 2 P strain. X-Irradiation of spermatids significantly increased the preferential elimination of the P-element-bearing second chromosome in mei-41, DNA-repair-deficient dysgenic males, but had no effect in their DNA-repair-proficient brothers. These findings indicate that the post-replication repair pathway is required for processing lesions induced by the combined effect of P element mobility and X-rays, and that the unrepaired lesions ultimately lead to chromosome loss.

Sequence instability and functional inactivation of murine Y chromosomes can occur on a specific genetic background.

When the Y chromosome from Mus. poschiavinus (YPos) is backcrossed onto the C57BL/6J laboratory strain, testicular dysfunction occurs at high frequencies. When five different multicopy probes from the recombinationally suppressed region of the Y chromosome were used, genomic DNAs from sibling female progeny of C57BL/6J YPos males were found to contain YPos-specific sequences ranging from trace levels to levels consistent with an intact Y chromosome. Females with a high copy number of YPos-specific sequences had a karyotype of XYPos and were sterile. Females with trace levels of these sequences were XX and fertile. Repeated sequences in the testis-determining-region (Sxr) of inactive YPos chromosomes were unstable relative to sequences in non-Sxr regions. In contrast, the YPos chromosome was stable and functioned normally in other inbred laboratory strains such as 129/Sv. The frequency and extent of YPos chromosome instability increased with successive backcrosses from stable (129/Sv) to unstable (C57BL/6J) genetic backgrounds. Traces of YPos-specific sequences were first detected in N2 female offspring of F1 males. Therefore, sequences were deleted from YPos chromosomes in the F1 male germ line and were transmitted to N2 females; inactive YPos chromosomes (XYPos females) were first detected in the N3 generation. The mouse line being derived by backcrossing the YPos chromosome onto C57BL/6J inbred strains ended in the N7 generation, since all XYPos offspring were sterile. Even stable repeated sequences from the non-Sxr regions of their inactive YPos chromosomes were precisely rearranged in these N7 offspring at high frequencies. These data are consistent with hybrid dysgenesis in mammals.

Super-unstable mutations associated with P-M hybrid dysgenesis in Drosophila melanogaster.

Super-unstable mutations occasionally appear either in natural populations of Drosophila melanogaster or in P-M hybrid dysgenesis. We found that they may be reproducibly obtained with a high frequency from crosses between males from the pi 2 strain and females from the waG* strain or its derivatives. Super-unstable mutations in the ocelliless, singed, white, yellow and other loci have been obtained. Each super-unstable mutation gives rise to a large family of new super-unstable mutations with a wide range of phenotypic expression. Mutations with the same phenotype often differ in the specificity of their potential for further mutation. As a rule, a super-unstable mutation is associated with a specific reversible mutation and paired alleles are formed in this way. Other mutations are usually irreversible, but new mutations of these may also form paired alleles. Active transposase encoded by transposable P elements is necessary to maintain super-instability. Finally, some preliminary molecular data are discussed which suggest that this type of super-instability is a result of interaction between P elements and a novel mobile element, designated as X.

Mutations in the glutamine synthetase I (gsI) gene produce embryo-lethal female sterility in Drosophila melanogaster.

A female-sterile mutation (fs(2) PM11-19) was recovered in a screen for P-M hybrid dysgenesis induced mutations uncovered by a deletion of region 21B and was identified as an allele of the gene encoding the Drosophila glutamine synthetase I (GSI) mitochondrial isozyme. Molecular analysis has shown that fs(2)PM11-19 contains a 5 kb insert within 500 bp upstream of the transcriptional start site of the gsI gene. Mutant flies have extremely low levels of gsI transcription and GSI activity. A pre-existing deficiency (Df(2L) netPM1) with a breakpoint near the transcription start site was also found to be a female-sterile allele of gsI. All eggs laid by PM11-19 homozygous females, as well as by females heterozygous for this mutation and a deletion or any of several recessive lethal alleles of the gsI gene, fail to hatch. We conclude that an adequate level of maternally supplied GSI activity is necessary in the early stages of Drosophila embryonic development.

I elements and the Drosophila genome.

LINEs are a large class of transposable elements in eukaryotes. They transpose by reverse transcription of an RNA intermediate. I elements of Drosophila melanogaster belong to this class and are responsible for the I-R system of hybrid dysgenesis. Many results indicate that at the beginning of the century natural populations of this species were devoid of active I elements and that they were invaded by functional I elements in the last decades. Many Drosophila species contain both defective and active I elements. It seems that the latter were lost in Drosophila melanogaster before its spread throughout the world, and that the recent invasion results from the spread of functional elements originating either from another species by horizontal transfer or from an isolated population of the same species. These data are discussed, as well as their significance in evolutionary processes.

The threat of extinction to native populations experiencing spawning intrusions by cultured Atlantic salmon

I examined the influence of spawning intrusions by cultured Atlantic salmon, Salmo salar, on the persistence of native populations. The probability of interbreeding between cultured and native salmon depends on the existence of behavioural, developmental, and morphological reproductive isolating mechanisms and on the relative competitive abilities of cultured and native salmon. I modelled the influence of spawning intrusions on population size by varying the proportional representation of cultured salmon in the spawning population, the frequency of spawning intrusions, and the difference in fitness between the two forms for three mating situations (no interbreeding and interbreeding with and without hybrid dysgenesis). The extinction probability of native genomes is greatest when interbreeding occurs between native salmon and relatively large numbers of frequently intruding cultured salmon. The threat of extinction is greatest for small populations, is influenced by genetic (interbreeding between forms) and non-genetic (increased density) factors, and is inextricably linked with the probability of co-occurrence of the two forms.

Gonadal dysgenesis reveals sexual dimorphism in the embryonic germline of Drosophila.

In hybrid dysgenesis, sterility can occur in both males and females. At 27.5 degrees, however, we found that P element-induced germline death was restricted to females. This sex-specific gonadal dysgenesis (GD) is complete by the first larval instar stage. As such, GD at 27.5 degrees reveals the sexually dimorphic character of the embryonic germline. The only other known dimorphic trait of the embryonic germline is the requirement for ovo. ovo is required for germline development in females only and has been implicated in germline sex determination. Dominant mutations of ovo partially suppressed female GD. Although embryonic germ cells are undifferentiated and morphologically indistinguishable between males and females, the functional dimorphism seen in ovo requirement and GD at 27.5 degrees indicates that sexual identity in Drosophila germ cells is established in embryogenesis.

The synergistic effect of X-rays and deficiencies in DNA repair in P-M hybrid dysgenesis in Drosophila melanogaster.

X-rays and deficiencies in DNA repair had a synergistic effect on genetic damage associated with P-element mobility in Drosophila melanogaster. These interactions, using sterility and fecundity as endpoints, were tested in dysgenic males deficient in either excision or post-replication DNA repair. Three sublines of the Harwich P strain were used for the construction of hybrid males. These sublines differ in P-induction ability based on gonadal dysgenesis sterility (GD) and snw mutability tests, in P-element insertion site pattern, and in the types of defective P-elements, such as KP elements, they possess. A lower degree of gonadal dysgenesis was correlated with the presence of KP elements. GD sterility and snw mutability were not always correlated. Dysgenic hybrids originating from the standard reference subline, Harwich(white), were much more sensitive to the post-replication repair than the excision repair defect. In contrast, sterility of hybrids derived from the weak subline was least affected by, and that of hybrids of the strongest subline was most affected by either DNA repair deficiency. The exacerbation by X-rays of the effects of DNA repair deficiencies on genetic damage indicates that both repair mechanisms are required for processing DNA lesions induced by the combined effect of P activity and ionizing radiation.