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The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. (4,19,22,23,36,43; reviewed in reference 13). These mobile elements are all retrotransposons, a group of structurally homologous elements that resemble the integrated form of mammalian retroviruses and make up the largest family of transposons in D. melanogaster (31). The major transcription products of these elements are nearly full-length, polyadenylated [poly(A)+] RNAs, and transcription of the elements is temporally regulated (24, 34). In an effort to understand the basis of suppression, genes with suppressible mutations (15,22,23,27,36,48), as well as the suppressor genes (7,25,39,46,47), are being cloned and studied at the molecular level. The transposon insertions associated with most suppressible mutations are located either in 5' untranscribed sequences or within introns. The mobile-element insertions typically interfere with synthesis of mRNA from a gene at or near the insertion site, and suppressor mutations partially or completely restore transcript accumulation. Studies of suppression by su(Hw) and su(v'") indicate that the suppressor genes encode regulators of mobile-element transcription and RNA processing, respectively (25,39,46 tagging (36), and the same sequences were cloned independently by Walker et al. (43). In using a mammalian tryptophan oxygenase cDNA probe to identify clones of the D. melanogaster v gene, Walker et al. (43) verified that v encodes tryptophan oxygenase, the enzyme that catalyzes the first step in the brown-pigment biosynthetic pathway (reviewed in reference 26). Mutations at v are clustered in a 2-kilobase (kb) region, and the v transcript is a 1.4-kb mRNA of relatively low abundance (36, 43).Three v mutations, v1, v2, and vk, can be suppressed by su(s). These alleles produce a leaky mutant phenotype and undetectable levels of tryptophan oxygenase (41). In D. melanogaster strains with a mutation at su(s) and with the 1, 2, or k mutation at v, tryptophan oxygenase activity is increased to 10 to 20% of the wild-type level (41), an amount sufficient to restore a wild-type phenotype. After the gene was cloned, the suppressible v alleles were shown to be insertions of the 7.5-kb retrotransposon 412 in the vicinity of the vermilion transcription unit (36,43). Using v as a model system, we are investigating the mechanism of suppression by slu(s). Thus, we have characterized the structure of the gene in greater detail and localized the positions of all the suppressible and several unsuppressible mutations relative to introns and exons. In addition, we have...

The nucleotide sequence and intron-exon structure of the Drosophila melanogaster vermilion (v) gene have been determined. In addition, the sites of several mutations and the effects of these mutations on transcription have been examined. The major v mRNA is generated upon splicing six exons of lengths (5' to 3') 83, 161, 134, 607, 94, and 227 nucleotides (nt). A minor species of v mRNA is initiated at an upstream site and has a 5' exon of at least 152 nt which overlaps the region included in the 83-nt exon of the major v RNA. The three v mutations, v1, v2, and vk, which can be suppressed by mutations at suppressor of sable, su(s), are insertions of transposon 412 at the same position in exon 1, 36 nt downstream of the major transcription initiation site. Despite the 7.5-kilobase insertion in these v alleles, a reduced level of wild-type-sized mRNA accumulates in suppressed mutant strains. The structure and transcription of several unsuppressible v alleles have also been examined. The v36f mutation is a B104/roo insertion in intron 4 near the splice donor site. A mutant carrying this alteration accumulates a very low level of mRNA that is apparently polyadenylated at a site within the B104/roo transposon. The v48a mutation, which deletes approximately 200 nt of DNA, fuses portions of exons 3 and 4 without disruption of the translational reading frame. A smaller transcript accumulates at a wild-type level, and thus an altered, nonfunctional polypeptide is likely to be synthesized in strains carrying this mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutation of the Drosophila melanogaster vermilion (v) gene known as v1 is caused by the insertion of a 412 retrotransposon into the 5' untranslated region of the first exon. Mutants carrying this insertion accumulate a low level of mRNA from which most of the transposon sequences have been eliminated by splicing at cryptic sites within transposon sequences. Here, we demonstrate that a revertant of the v1 allele called v+37 is caused by the insertion of a second retrotransposon, the B104/roo element, into a site near one end of the 412 element. The revertant strain accumulates a higher level of mRNA from which most of both transposons have been removed by splicing at new donor sites introduced by the B104/roo insertion and the same acceptor site within 412. Mutations at suppressor of sable [su(s)], which increase the accumulation of v1 transcripts, slightly elevate the level of v+37 RNA. In addition, we show that the first v intron downstream of the 412 insertion is not efficiently removed in the v1 mutant, and suppressor and reversion mutations increase the proportion of transcripts that are properly spliced at that downstream intron. Thus, it appears that both the suppressor and reversion mutations exert an effect at the level of pre-mRNA splicing.

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