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The Drosophila suppressor-of-white-apricot [su(wa)] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(wa) expression is autoregulated at the level of pre-mRNA splicing is reported. su(wa) protein represses accumulation of the fully spliced su(wa) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(wa) pre-mRNA. Second, the fully spliced su(wa) mRNA is sufficient for all known su(wa) genetic functions indicating that it encodes the sole su(wa) protein. Third, the incompletely spliced su(wa) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(wa) expression during oogenesis allows maternal deposition exclusively of fully spliced su(wa) mRNA. Fifth, su(wa) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(wa) expression.

Regulators responsible for the pervasive, nonsex-specific alternative pre-mRNA splicing characteristic of metazoans are almost entirely unknown or uncertain. We describe here a novel family of splicing regulators present throughout metazoans. Specifically, we analyze two nematode (Caenorhabditis elegans) genes. One, CeSWAP, is a cognate of the suppressor-of-white-apricot (DmSWAP) splicing regulator from the arthropod Drosophila. Our results define the ancient, conserved SWAP protein family whose members share a colinearly arrayed series of novel sequence motifs. Further, we describe evidence that the CeSWAP protein autoregulates its levels by feedback control of splicing of its own pre-mRNA analogously to the DmSWAP protein and as expected of a splicing regulator. The second nematode gene, Ceprp21, encodes an abundant nuclear cognate of the constitutive yeast splicing protein, prp21, on the basis of several lines of evidence. Our analysis defines prp21 as a second novel, ancient protein family. One of the motifs conserved in prp21 proteins--designated surp--is shared with SWAP proteins. Several lines of evidence indicate that both new families of surp-containing proteins act at the same (or very similar) step in early prespliceosome assembly. We discuss implications of our results for regulated metazoan pre-mRNA splicing.

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.

Discovery that a process might be ancient and phylogenetically widely distributed usually leads to dramatic increases in insight as diverse, idiosyncratic sources of experimental power are simultaneously brought to bear. Addition of the Drosophila genetic system to the power of plant and fungal genetics can be expected to produce substantive new understanding of cosuppression in the next several years. Moreover, it will be of considerable interest and potential technical value to establish whether cosuppression occurs in other animal lineages, including vertebrates, as the newly expanded body of evidence clearly predicts. In this context it will be of great interest to see further analysis of the recently reported transgene silencing of a normally imprinted mammalian gene to determine if this represents cosuppression or a more specialized mechanism associated with imprinting (Hatada et al. 1997xHatada, I., Nabetani, A., Arai, Y., Ohishi, S., Suzuki, M., Miyabara, S., Nishimune, Y., and Mukai, T. J. Biol. Chem. 1997; 272: 9120–9122Crossref | PubMed | Scopus (25)See all ReferencesHatada et al. 1997).

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 white locus of Drosophila melanogaster is a genetically well-characterized locus, mutations in which alter the degree of pattern of pigmentation of the eyes. Using a previously cloned DNA segment containing a portion of the white locus of a mutant allele, we have cloned and characterized the DNA of a 48-kilobase chromosomal region of the Canton S wild-type strain. We have mapped the positions, relative to restriction endonuclease cleavage sites, of several previously characterized chromosomal rearrangement breakpoints that bracket the while locus. These results define a segment of 14 kilobase that contains all of the white locus sequences necessary for the production of a wild-type eye color phenotype. By conventional criteria, no repetitive sequences are present within this 14-kilobase segment; however, we have identified an extremely weak DNA sequence homology between a portion of this segment and a chromosomal region in the vicinity of the zeste locus.

A new mutant allele (wDZL)at the white locus of Drosophila melanogaster is dominant to the wild-type allele, but apparently only when the two alleles are synapsed. When chromosomal rearrangements prevent somatic pairing between the two white alleles, wDZL is rendered recessive to wild type. This observation suggests that the dominance of wDZL is sensitive to a synapsis (transvection) effect. On the basis of this and other properties, it is proposed that wDZL causes the repression of transcription of a synapsed w+ allele, but not of a w+ allele elsewhere in the same nucleus. One model to account for this supposes that wDzL produces a repressor of white-locus transcription. This repressor is presumed to be so unstable that other white genes, removed from wDZL but in the same nucleus, are not detectably repressed. These properties may be simply understood if it is assumed that the repressor produced by the wDZL allele is an RNA molecule.

We report sequence and transcript structures for a 6191-base chromosomal segment containing the presumptive regulatory gene from Drosophila, suppressor-of-white-apricot [su(wa)]. Our results indicate that su(wa) expression is controlled by regulating occurrence of specific splices. Seven introns are removed from the su(wa) primary transcript during precellular blastoderm development. The sequence of this mature RNA indicates that it is a conventional messenger RNA. In contrast, after cellular blastoderm the first two of these introns cease to be efficiently removed. The mature RNAs resulting from this failure to remove the first two introns have structures quite unexpected of mRNAs. We propose that postcellular blastoderm su(wa) expression is repressed by preventing splices necessary to produce a functional mRNA. Implications and mechanisms are discussed.

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.

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.

Both the white mutant allele, w DZL , and the zeste mutant allele, z 1 , reduce white transcript levels in adult head tissues, but have no effect on these levels in other tested adult tissues, w DZL results from insertion of a complex transposon approximately 5.0 kb 5′ to white . Juxtaposition of this transposon to white produces a novel transcription unit beginning in the transposon and producing a mature transcript containing both white and transposon sequences. Head specific expression of this novel transcription unit correlates with the observed repression of wild-type white transcript levels, w DZL and z 1 are known to cause synapsis-dependent repression of w 1 expression in trans as assessed by eye pigment deposition. Collectively, these results suggest an unexpected and informative correspondence between cis and synapsis-dependent trans regulatory effects. We discuss the implied mechanistic relationship between regulation of transcription and transvection effects in Drosophila.

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.

Publisher Summary This chapter provides a review of the basic elements of the origin and behavior of transposons and their relationship to their cellular hosts. It focuses on one specific aspect of the analysis of transposons in Drosophila: the molecular genetic analyses of suppressor/enhancer loci. In this case, the objective is to develop a new, detailed proposal for the role of suppressor/enhancer loci. Transposons carry on a life-style requiring very complex behaviors and decisions. In spite of this, transposons are very small (typically 3 to 10 kb in size), with limited amounts of genetic information. This paradoxical juxtaposition of genetic simplicity and a complex life-style is achieved by extensive parasitism of information produced by the cellular host. Equivalently, transposons carry only a tiny fraction of the genetic information necessary to their existence. This tiny fraction consists of a few functions idiosyncratic to transposition and whatever information is necessary to organize parasitized host functions and information. This latter “organizational” function is incompletely understood but certainly consists of cis-acting regulatory elements responsive to host regulatory information.

The past two years have seen the discovery of three independent cases in which expression of a eukaryotic protein gene product is turned on and off by controlling splicing events necessary to produce the corresponding mRNA. Various considerations suggest that such on/off regulation at the level of splicing may be unexpectedly common.

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

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.