Search

The copia insertion responsible for the wamutation is 3' to the white promotor and in the same transcriptional orientation as white. First, we have analyzed the effects of the wa copia insertion on levels of polyadenylated white transcripts and find large, developmentally programmed effects. Second, we have isolated and sequenced an LTR-excision event involving the copia insertion at wa. This represents the first documented case of an LTR-excision event in Drosophila. This single copia LTR has developmentally programmed effects on white transcript levels qualitatively similar to the intact copia element. Third, we have characterized the structures of white transcripts from wa. We find polyadenylated white transcripts apparently having 3' termini in or near the 3' LTR of the wa copia insertion, as has been reported in limited studies of wa transcription in adults by others. These earlier studies also revealed wa transcripts apparently corresponding to polyadenylated terminus formation in the 5' LTR of the copia transposon; however, our more detailed studies reveal that these transcripts probably have other origins and that little, if any, polyadenylated terminus formation for white transcripts occurs in the 5' LTR of the wa copia insertion. Moreover, we find no polyadenylated terminus formation for white transcripts occurring in the single LTR of the wa LTR-excision product. Fourth, we find that each of three mutant alleles at su(wa) produces elevated levels of several classes of RNAs apparently corresponding to transcriptional readthrough of the wa copia transposon. Elevated levels of one presumptive readthrough transcript were observed previously in one su(wa)1 mutant strain. Fifth, we have confirmed the existence of a transcript initiated in the 3' LTR of the wa copia insertion and find the levels of this transcript to be strongly influenced by developmental stage and genetic background. Lastly, we have analyzed white transcripts produced by the whd 81b11 allele, which carries an insertion of copia in the opposite transcriptional orientation and in a different position than the wa copia insertion. In contrast to the wa copia insertion allele, the whd 81b11 allele produces polyadenylated white transcript levels very similar to the w + case at the stages examined. Moreover, the whd 81b11 copia element apparently produced polyadenylated terminus formation in white transcripts and we observe no effect of the allelic state of su(wa) on apparent readthrough of this stop site. We discuss some possible implications of our results for the properties of retrotransposons as developmentally complex insertional mutagens and for the functional organization of the copia transcription unit.

The molecular nature of mutations arising in dysgenic hybrids between P and M Drosophila melanogaster strains has been investigated. Seven independent mutations at the white locus were examined, and these fell into two classes on the basis of their genetic and structural properties. The five mutations comprising the first class were caused by DNA insertions of 0.5, 0.5, 0.6, 1.2 and 1.4 kb, respectively. The DNA insertions in four of these mutations were examined in detail. Although heterogeneous in size and pattern of restriction enzyme sites, they were homologous in sequence. We refer to members of this sequence family as P elements. Mutations caused by P elements appeared to be stable in the P cytotype, but had reversion rates greater than 10 −3 in the M cytotype. Phenotypic reversion to wild-type was accompanied by excision of the P element. The two mutations comprising the second class were caused by insertion of the 5.0 kb copia element and appeared to be stable in both P and M cytotypes.

We report genetic and molecular analysis of the suppressor-of-white-apricot [su(wa)] locus, one of several retrotransposon insertion allele-specific suppressor loci in Drosophila melanogaster. First, we isolated and characterized eight new mutations allelic to the original su(wa)1 mutation. These studies demonstrated that su(wa) mutations allelic to su(wa)1 affected a conventional D. melanogaster complementation group. Second, we cloned the chromosomal region containing the su(wa) complementation group by P element transposon tagging. The ca. 14-kilobase region surrounding the su(wa) complementation group contained five distinct transcription units, each with a different developmentally programmed pattern of expression. Third, we used a modified procedure for P-mediated gene transfer to identify the transcription unit corresponding to su(wa) by gene transfer. Fourth, we found that the presumptive su(wa) transcription unit produced a family of transcripts (ranging from ca. 3.5 to ca. 5.2 kilobases) in all developmental stages, tissue fractions, and cell lines we examined, suggesting that the gene is universally expressed.

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.

We report results indicating that the temperature-sensitive white-blood (wbl) mutation has a novel molecular basis, involving the formation of RNA transcripts of the affected gene rather than the behavior of the polypeptide product. First, we show that the temperature-sensitive mutant phenotype of wbl correlates with (and presumably results from) a temperature-dependent effect on levels of the 2.6-kb polyadenylated white transcript. Second, DNA sequence analysis and other studies show that wbl is associated with the insertion into the second white intron of a previously uncharacterized retrotransposon (designated the blood transposon). We discuss the potential origins of the novel temperature-sensitive molecular phenotype of wbl and the prospects for exploiting wbl to engineer temperature-sensitive mutations in other genes.

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, Rpll C4 , caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the Rpll locus. The non-P sequences in this clone (λDmRpll-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of Rpll C4 , and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila.

We have shown previously that four of five white mutant alleles arising in P-M dysgenic hybrids result from the insertion of strongly homologous DNA sequence elements. We have named these P elements. We report that P elements are present in 30–50 copies per haploid genome in all P strains examined and apparently are missing entirely from all M strains examined, with one exception. Furthermore, members of the P family apparently transpose frequently in P-M dysgenic hybrids; chromosomes descendant from P-M dysgenic hybrids frequently show newly acquired P elements. Finally, the strain-specific breakpoint hotspots for the rearrangement of the π 2 P X chromsome occurring in P-M dysgenic hybrids are apparently sites of residence of P elements. These observations strongly support the P factor hypothesis for the mechanistic basis of P-M hybrid dysgenesis.

We have identified and sequenced the portion of the white locus affected by an idiosyncratic set of white mutant alleles (the wsp alleles). The affected white locus portion (wsp region) extends from ca. 590 base pairs (bp) to ca. 1270 bp 5' to the apparent start site for the major white transcription unit. Based on the properties of these mutant alleles, we infer the existence of two distinct cis-acting regulatory elements in the wsp region and a third element mapping 3' to this region (3' to position ca. -670). Our analysis allows us to define the apparent position of one of the two wsp region elements with substantial precision. Examination of the DNA sequences in this region suggests that it is functionally similar to the enhancers identified in vertebrates. This same element participates in synapsis-dependent genetic interactions, suggesting a largely unexpected relationship between enhancer-like, cis-acting genetic elements and the genetic elements responsible for the synapsis-dependent genetic interactions in trans revealed by the existence of transvection effects. Our results further suggest that a presumptive regulatory locus (suppressor-of-white-spotted) regulates white transcription in adult tissues and is not involved in regulating white expression in larvae. We discuss the regulation of white expression in light of our studies. We also demonstrate unusual structures for an X-ray-induced deletion and a spontaneous deletion.

G-C leads to A-T transitions are induced by heat, and arise from the deamination of cytosine (5-hydroxymethylcytosine in the case of bacteriophage T4) generating uracil. The reaction is proton-catalyzed, and is also characteristic of acid mutagenesis. Mutation rates and activation energies of mutation are site-specific, and are presumably influenced by neighboring bases. Rates of heat-induced mutation in bacteriophage T4 under conditions of temperature, pH, and ionic strength similar to those prevailing in higher eukaryotic cells suggest that heat mutagenesis may present a serious challenge to organisms with large genomes, and may comprise an important determinant of the rates of spontaneous mutation.

Expression of the presumptive regulatory gene, suppressor-of-white-apricot [su(wa)], is controlled at the level of splicing. Results reported here indicate that this control represents autorepression of su(wa) expression. Specifically, reverse genetic studies demonstrate that the 3.5 kb mature su(wa) RNA (produced by removal of seven introns) is a message essential for su(wa)+ function and indicate that the abundant 4.4 kb and 5.2 kb mature su(wa) RNAs (resulting when the first or first and second of the seven introns are not removed) are, unexpectedly, byproducts of repression of production of the functional 3.5 kb RNA. Moreover, several experiments indicate that this repression of splices necessary to produce the 3.5 kb RNA is dependent on the translation product of the 3.5 kb RNA itself. We propose that this regulatory gene autoregulates its expression by controlling splicing of its primary transcript.