Interactions among modifiers of retrotransposon-induced alleles of the white locus of Drosophila melanogaster

Mutations in five loci that modify the phenotype of whiteapricot (wa), caused by the retrotransposon, copia, were examined in two-way combinations to determine whether their effects were additive or epistatic. All two-way combinations of mutations in these five loci, mottler of white (mw), suppressor of forked (su(f], suppressor of white apricot (su(wa], Enhancer of whiteapricot, (E(wa] and Darkener of apricot (Doa), are additive in their effects on wa, implying that each second-site modifier locus affects a different process. Three other copia-induced mutations, HwUa, whd81b25 and ctns were also examined for responsiveness to mutations in these modifier loci. None clearly responded. Mutations associated with B104 insertions, including Gl, vgni, ctn and wric were also examined for responsiveness to mw mutations, which have specificity for this element as well. Both vgni and wric respond to mutations in mw. The former interaction demonstrates that mw is capable of interacting with B104 elements in loci other than white. The significance of the results with respect to the nature of second-site modifier loci is discussed.     

A dosage-sensitive modifier of retrotransposon-induced alleles of the Drosophila white locus.

The apricot allele of the white locus results from the insertion of the retrotransposon copia. Mutations in a newly discovered locus, the Darkener-of-apricot (Doa), suppress wa and some of its revertants. Of 44 other white alleles tested, only wsp55 is affected by Doa, although, in contrast, it is enhanced by Doa mutations. The Doa locus modulates wa and wsp55 expression as a function of its own dosage. Mutations in Doa are dominant suppressors or enhancers and are recessive lethals. Rare Doa mutant homozygotes escaping lethality demonstrate extreme phenotypic suppression of wa and enhancement of wsp55. RNA from wa is substantially wild-type in structure in escapers, although reduced in quantity.     

A copy of the copia transposable element is very tightly linked to the Wa allele at the white locus of D. melanogaster

Results are described demonstrating that several X chromosomes of Drosophila melanogaster carrying the Wa (white-apricot) mutant allele also carry homology to the copia transposable element in distal 3C of the polytene chromosome map as assessed by situ hybridization. The locus of the Wa mutation, white, resides in distal 3C. We further show, using fine scale genetic mapping techniques, that the copia homology in distal 3C in Wa-bearing chromosomes is very tightly linked to the Wa mutation. Both the Wa mutation and the copia homology associated with it map to the central portion of the white locus.     

On the Identification of the ROSY Locus DNA in DROSOPHILA MELANOGASTER: Intragenic Recombination Mapping of Mutations Associated with Insertions and Deletions

DNA extracts of several rosy-mutation-bearing strains were associated with large insertions and deletions in a defined region of the molecular map believed to include the rosy locus DNA. Large-scale, intragenic mapping experiments were carried out that localized these mutations within the boundaries of the previously defined rosy locus structural element. Molecular characterization of the wild-type recombinants provides conclusive evidence that the rosy locus DNA is localized to the DNA segment marked by these lesions. One of the mutations, ry2101, arose from a P-M hybrid dysgenesis experiment and is associated with a copia insertion. Experiments are described which suggest that copia mobilizes in response to P-M hybrid dysgenesis. Relevance of the data to recombination in higher organisms is considered.     

Partial Revertants of the Transposable Element-Associated Suppressible Allele White-Apricot in Drosophila Melanogaster: Structures and Responsiveness to Genetic Modifiers

The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers.     

P Element-Mediated in Vivo Deletion Analysis of White-Apricot: Deletions between Direct Repeats Are Strongly Favored

We have isolated and characterized deletions arising within a P transposon, P[hsw(a)], in the presence of P transposase. P[hsw(a)] carries white-apricot (w(a)) sequences, including a complete copia element, under the control of an hsp70 promoter, and resembles the original w(a) allele in eye color phenotype. In the presence of P transposase, P[hsw(a)] shows a high overall rate (approximately 3%) of germline mutations that result in increased eye pigmentation. Of 234 derivatives of P[hsw(a)] with greatly increased eye pigmentation, at least 205 carried deletions within copia. Of these, 201 were precise deletions between the directly repeated 276-nucleotide copia long terminal repeats (LTRs), and four were unique deletions. High rates of transposase-induced precise deletion were observed within another P transposon carrying unrelated 599 nucleotide repeats (yeast 2μ FLP; recombinase target sites) separated by 5.7 kb. Our observation that P element-mediated deletion formation occurs preferentially between direct repeats suggests general methods for controlling deletion formation.     

Genotype-environment interactions and the maintenance of polygenic variation

The Enhancer of wa [E(wa)] mutation was shown to interact strongly with 4 of 41 tested alleles of the white (w) eye color locus. All four of the affected w alleles result from the insertion of a transposable element. E(wa) was further localized cytogenetically. The locus lies between the breakpoints of T(Y;2)L11 and T(Y;2)H137 (section 60) in 2R. The original mutation was shown to be antimorphic on the basis of its action in the presence of additional normal copies and the ability to revert the original allele to one that mimics the effect of a deficiency for the locus. The RNA transcribed from wa was analyzed from flies segregating for E(wa) and normal. The low level of normal functional messenger RNA present in white-apricot is reduced further in Enhancer homozygotes. Total copia RNA was also examined on Northern analyses from the segregating population but no quantitative change in the major copia RNA was produced by E(wa) homozygotes compared to normal.     

Terminal repeats of the Drosophila transposable element copia: nucleotide sequence and genomic organization

We have determined the nucleotide sequence of the terminal regions of two members of the copia sequence family of D. melanogaster. The first 276 bp at one end of a copia element are repeated in direct orientation at its other end. The direct repeats on a single copia element are identical to each other, but they differ by two nucleotide substitutions between the two elements which were examined; this suggests that during transposition only one direct repeat of the parent element is used as a template for both direct repeats of the transposed element. Each direct repeat itself contain a 17 bp imperfectly matched inveted terminal repetition. The ends of copia show significant sequence homology both to the yeast Ty1 element and to the integrated provirus of avian spleen necrosis virus, two other eucaryotic elements known to insert at many different chromosomal locations. Analysis of the genomic organization of the direct repeat sequence demonstrates that it seldom, if ever, occurs unlinked to an entire copia element.     

Accumulation of transposable elements in laboratory lines of Drosophila melanogaster

It is recognized that a stable number of transposable element (TE) copies per genome is maintained in natural populations of D. melanogaster as a result of the dynamic equilibrium between transposition to new sites and natural selection eliminating copies. The force of natural selection opposing TE multiplication is partly relaxed in inbred laboratory lines of flies. The average rate of TE transposition is from 2.6 × 10 ^-4 to 5.0 ×10 ^-4 per copy per generation, and the average rate of excision is at least two orders of magnitude lower; therefore inbred lines accumulate increasing numbers of copies with time. Correlations between the rate of transposition and TE copy number have been determined for copia, Doc, roo, and 412 and found to be either zero or positive. Because the rate of transposition is not a decreasing function of TE copy number, TE accumulation in inbred lines is self-accelerating. Transpositions cause a substantial fraction of mutations in D. melanogaster, therefore the mutation rate should increase with time in laboratory lines of this species. Inferences about the properties of spontaneous mutations from studies of mutation accumulation in laboratory lines should be reevaluated, because they are based on the assumption of a constant mutation rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Two Classes of Tn10 Transposase Mutants That Suppress Mutations in the Tn10 Terminal Inverted Repeat

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named ``SEM'''' for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.