Genetic Transformation of Drosophila melanogaster with an Autonomous P Element: Phenotypic and Molecular Analyses of Long-Established Transformed Lines

ABSTRACT Following transformation of a Drosophila melanogaster true M strain with an autonomous P element, six lines were established and monitored for their molecular and phenotypic properties during a 4-yr period. The number of P elements increased with time in all the lines but the rate of increase differed among lines. Furthermore, degenerate elements arose in each of the lines during propagation. By the end of the 4th yr, the total number of elements in every line was similar to that of a very strong P strain.—At the phenotypic level, all of the transformed lines evolved high P activity, but only three developed complete or nearly complete regulatory ability. The other three lines attained only intermediate levels of regulation over the 4-yr period. One of these lines was particularly noteworthy. Although it contained as many as 55 P elements per genome (20 of which were potentially complete) and had extremely high P activity potential, it continued to exhibit limited regulatory ability. In addition, when females of this line were maintained at high temperatures, the ability to suppress P activity was even further diminished. A strain with this combination of molecular and phenotypic properties, in an apparently stable configuration, has not been previously described.—The results are discussed in the context of the possible role of degenerate elements in regulating P element expression.

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The P-element-induced silencing effect of KP transposons is dose dependent in Drosophila melanogaster

Genome ◽

10.1139/g11-023 ◽

2011 ◽

Vol 54 (9) ◽

pp. 752-762 ◽

Cited By ~ 7

Author(s):

Alireza Sameny ◽

John Locke

Keyword(s):

Drosophila Melanogaster ◽

Critical Role ◽

Mutagenic Effect ◽

P Element ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

P Elements ◽

Single Well ◽

Dose Dependent

Transposable elements are found in the genomes of all eukaryotes and play a critical role in altering gene expression and genome organization. In Drosophila melanogaster, transposable P elements are responsible for the phenomenon of hybrid dysgenesis. KP elements, a deletion-derivative of the complete P element, can suppress this mutagenic effect. KP elements can also silence the expression of certain other P-element-mediated transgenes in a process called P-element-dependent silencing (PDS), which is thought to involve the recruitment of heterochromatin proteins. To explore the mechanism of this silencing, we have mobilized KP elements to create a series of strains that contain single, well-defined KP insertions that show PDS. To understand the quantitative role of KP elements in PDS, these single inserts were combined in a series of crosses to obtain genotypes with zero, one, or two KP elements, from which we could examine the effect of KP gene dose. The extent of PDS in these genotypes was shown to be dose dependent in a logarithmic rather than linear fashion. A logarithmic dose dependency is consistent with the KP products interacting with heterochromatic proteins in a concentration-dependent manner such that two molecules are needed to induce gene silencing.

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Molecular analysis of the P-M gonadal dysgenesis cline in eastern Australian Drosophila melanogaster.

Genetics ◽

10.1093/genetics/119.4.889 ◽

1988 ◽

Vol 119 (4) ◽

pp. 889-902

Author(s):

I A Boussy ◽

M J Healy ◽

J G Oakeshott ◽

M G Kidwell

Keyword(s):

Drosophila Melanogaster ◽

Transposable Element ◽

Gonadal Dysgenesis ◽

Molecular Techniques ◽

P Element ◽

Latitudinal Cline ◽

Deletion Derivative ◽

P Elements ◽

Regulatory Ability ◽

Eastern Australia

Abstract The latitudinal cline in P-M gonadal dysgenesis potential in eastern Australia has been shown to comprise three regions which are, from north to south respectively, P, Q, and M, with the P-to-Q and Q-to-M transitions occurring over relatively short distances. The P element complements of 30 lines from different regions of the cline were determined by molecular techniques. The total amount of P element-hybridizing DNA was high in all lines, and it did not correlate in any obvious way with the P-M phenotypes of individual lines. The number of potentially full-sized P elements per genome was high in lines from the P regions, but variable or low among lines from the Q and M regions, and thus declined overall from north to south. A particular P element deletion-derivative, the KP element, occurred in all the tested lines. The number of KP elements was low in lines from the P region, much higher in lines from the Q region, and highest among lines from the M region, thus forming a cline reciprocal to that of the full-sized P elements. Another transposable element, hobo, which has been described as causing dysgenic traits similar to those of P-M hybrid dysgenesis, was shown to be present in all lines and to vary among them in number, but not in any latitudinal pattern. The P-M cline in gonadal dysgenesis potential can be inferred to be based on underlying clinal patterns of genomic P element complements. P activity of a line was positively correlated with the number of full-sized P elements in the line, and negatively correlated with the number of KP elements. Among Q and M lines, regulatory ability was not correlated with numbers of KP elements.

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P-element-induced control mutations at the r gene of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2567 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2567-2574 ◽

Cited By ~ 25

Author(s):

S Tsubota ◽

M Ashburner ◽

P Schedl

Keyword(s):

Drosophila Melanogaster ◽

The Other ◽

P Element ◽

Female Sterility ◽

R Gene ◽

Temporal Expression ◽

Base Pairs ◽

P Elements ◽

Adult Females ◽

Regulatory Mutations

The P-M hybrid dysgenesis system was used to produce five putative regulatory mutations at the rudimentary locus, r. All five mutations were the result of insertions at the 5' end of the gene, upstream of the proposed start of transcription. All of the mutants displayed a leaky wing phenotype, and four of the mutants showed an uncoupling of the wing and female-sterility phenotypes, suggesting that they altered the normal spatial and temporal expression of the r gene. Four of the insertions were P elements. The fifth insertion, which was larger than an intact P element, consisted of a small P element connected to non-P-element DNA. Two of the mutants produced very little r transcript in adult females and were clustered 80 to 150 base pairs upstream of the start of transcription. The other three mutants had higher levels of r transcript in adult females and were clustered 440 to 500 base pairs upstream of the start of transcription. All of the data suggest that the insertions are in a 5' noncoding region of the r gene involved in the control of its spatial and temporal expression.

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The relationship between P elements and male recombination in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/124.2.317 ◽

1990 ◽

Vol 124 (2) ◽

pp. 317-329

Author(s):

A Duttaroy ◽

M McCarron ◽

K Sitaraman ◽

G Doughty ◽

A Chovnick

Keyword(s):

Drosophila Melanogaster ◽

Hot Spot ◽

The Other ◽

P Element ◽

Male Recombination ◽

P Elements ◽

Selective System ◽

Element Mobilization ◽

The Relationship ◽

Initial Binding

Abstract P element dysgenesis associated male recombination in Drosophila was examined with a selective system focused upon 5% of the standard female genetic map divided into eight recombination segments. We found no correspondence between P element mobilization events and recombination in males in the intervals monitored. We defined two adjacent short genetic and molecular regions, one devoid of male recombination and the other acting as a "hot spot" for exchange in the absence of supporting P element insertion and excision activity. These data suggest that, even in the presence of mobilizing P elements, transposase may be active at non-P element sites, and that the genome may harbor sequences ranging from highly responsive to completely unresponsive to transposase action. A viewpoint is presented wherein P elements, with sequences that bind transposase, serve to focus the recombination action of transposase to encompass a region of DNA radiating outward from the initial binding site. We suggest that this region is measured in terms of chromosomal segments rather than limited to P element sequences.

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P element insertions and rearrangements at the singed locus of Drosophila melanogaster.

Genetics ◽

10.1093/genetics/119.1.75 ◽

1988 ◽

Vol 119 (1) ◽

pp. 75-83

Author(s):

H Roiha ◽

G M Rubin ◽

K O'Hare

Keyword(s):

Drosophila Melanogaster ◽

High Frequency ◽

The Other ◽

Hybrid Dysgenesis ◽

P Element ◽

Wild Type ◽

Tandem Arrangement ◽

P Elements

Abstract DNA from the singed gene of Drosophila melanogaster was isolated using an inversion between a previously cloned P element at cytological location 17C and the hypermutable allele singed-weak. Five out of nine singed mutants examined have alterations in their DNA maps in this region. The singed locus is a hotspot for mutation during P-M hybrid dysgenesis, and we have analyzed 22 mutations induced by P-M hybrid dysgenesis. All 22 have a P element inserted within a 700-bp region. The precise positions of 10 P element insertions were determined and they define 4 sites within a 100-bp interval. During P-M hybrid dysgenesis, the singed-weak allele is destabilized, producing two classes of phenotypically altered derivatives at high frequency. In singed-weak, two defective P elements are present in a "head-to-head" or inverse tandem arrangement. Excision of one element results in a more extreme singed bristle phenotype while excision of the other leads to a wild-type bristle phenotype.

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P-element-induced control mutations at the r gene of Drosophila melanogaster

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2567-2574.1985 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2567-2574

Author(s):

S Tsubota ◽

M Ashburner ◽

P Schedl

Keyword(s):

Drosophila Melanogaster ◽

The Other ◽

P Element ◽

Female Sterility ◽

R Gene ◽

Temporal Expression ◽

Base Pairs ◽

P Elements ◽

Adult Females ◽

Regulatory Mutations

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Modified P elements that mimic the P cytotype in Drosophila melanogaster.

Genetics ◽

10.1093/genetics/123.4.815 ◽

1989 ◽

Vol 123 (4) ◽

pp. 815-824 ◽

Cited By ~ 2

Author(s):

H M Robertson ◽

W R Engels

Keyword(s):

Drosophila Melanogaster ◽

Reciprocal Cross ◽

Somatic Mosaicism ◽

The Other ◽

P Element ◽

Cross Effect ◽

P Elements ◽

Somatic Tissues ◽

Insertion Mutations ◽

Cellular Condition

Abstract 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.

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A COMPARISON OF MUTATION RATES FOR SPECIFIC LOCI AND CHROMOSOME REGIONS IN DYSGENIC HYBRID MALES OF DROSOPHILA MELANOGASTER

Genetics ◽

10.1093/genetics/106.1.85 ◽

1984 ◽

Vol 106 (1) ◽

pp. 85-94

Author(s):

Michael J Simmons ◽

John D Raymond ◽

Nancy A Johnson ◽

Thomas M Fahey

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

The Other ◽

Hybrid Dysgenesis ◽

P Element ◽

Mutation Rates ◽

P Elements ◽

White Locus ◽

Insertion Mutations ◽

Chromosome Regions

ABSTRACT The mutation rates of specific loci and chromosome regions were estimated for two types of dysgenic hybrid males. These came from crosses between P or Q males and M females in the P-M system of hybrid dysgenesis. The M × P hybrids were the more mutable for each of the loci and chromosome regions tested. The Beadex locus was highly mutable in these hybrids but did not mutate at all in the sample of gametes from the M × Q hybrids. The singed locus had 75% of the mutability of Beadex in the M × P hybrids; it was also mutable in the M × Q hybrids. The white locus was only slightly mutable in the M × P hybrids and not at all mutable in the M × Q hybrids. The mutations in singed and white probably arose from the insertion of P elements into these loci; the mutations at Beadex probably involved the action of a P element located near this locus on the X chromosome of the P strain that was used in the experiments. Mutations in two chromosome regions, one including the zeste-white loci and the other near the miniature locus, were much more frequent in the M × P hybrids than in the M × Q hybrids. These mutations also probably arose from P element insertions. The implication is that insertion mutations occur infrequently in the M × Q hybrids, possibly because most of the P elements they carry are defective. In M × P hybrids, there is variation among loci with respect to P elements mutagenesis, indicating that P elements possess a degree of insertional specificity.

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A hobo Transgene That Encodes the P-Element Transposase in Drosophila melanogaster: Autoregulation and Cytotype Control of Transposase Activity

Genetics ◽

10.1093/genetics/161.1.195 ◽

2002 ◽

Vol 161 (1) ◽

pp. 195-204 ◽

Cited By ~ 1

Author(s):

Michael J Simmons ◽

Kevin J Haley ◽

Craig D Grimes ◽

John D Raymond ◽

Jarad B Niemi

Keyword(s):

Drosophila Melanogaster ◽

Gonadal Dysgenesis ◽

P Element ◽

Hsp70 Gene ◽

Alternate Splicing ◽

Male And Female ◽

P Elements ◽

Male Germline ◽

Female Germline ◽

Transposase Expression

Abstract Drosophila were genetically transformed with a hobo transgene that contains a terminally truncated but otherwise complete P element fused to the promoter from the Drosophila hsp70 gene. Insertions of this H(hsp/CP) transgene on either of the major autosomes produced the P transposase in both the male and female germlines, but not in the soma. Heat-shock treatments significantly increased transposase activity in the female germline; in the male germline, these treatments had little effect. The transposase activity of two insertions of the H(hsp/CP) transgene was not significantly greater than their separate activities, and one insertion of this transgene reduced the transposase activity of P(ry+, Δ2-3)99B, a stable P transgene, in the germline as well as in the soma. These observations suggest that, through alternate splicing, the H(hsp/CP) transgene produces a repressor that feeds back negatively to regulate transposase expression or function in both the somatic and germline tissues. The H(hsp/CP) transgenes are able to induce gonadal dysgenesis when the transposase they encode has P-element targets to attack. However, this ability and the ability to induce P-element excisions are repressed by the P cytotype, a chromosomal/cytoplasmic state that regulates P elements in the germline.

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The Regulatory Properties of Autonomous Subtelomeric P Elements Are Sensitive to a Suppressor of variegation in Drosophila melanogaster

Genetics ◽

10.1093/genetics/143.4.1663 ◽

1996 ◽

Vol 143 (4) ◽

pp. 1663-1674 ◽

Cited By ~ 3

Author(s):

Stéphane Ronsseray ◽

Monique Lehmann ◽

Danielle Nouaud ◽

Dominique Anxolabéhère

Keyword(s):

Drosophila Melanogaster ◽

Genetic Recombination ◽

Natural Populations ◽

P Element ◽

Single Element ◽

Heterochromatin Protein 1 ◽

X Chromosomes ◽

P Elements ◽

Associated Sequences ◽

Regulatory Properties

Abstract Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site 1A) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is sitedependent and could involve the structure of the chromatin.

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