Dumpy Gene Research Articles

A Molecular Analysis of Mutations at the Complex dumpy Locus in Drosophila melanogaster

The Drosophila dumpy gene consists of seventy eight coding exons and encodes a huge extracellular matrix protein containing large numbers of epidermal growth factor-like (EGF) modules and a novel module called dumpy (DPY). A molecular analysis of forty five mutations in the dumpy gene of Drosophila melanogaster was carried out. Mutations in this gene affect three phenotypes: wing shape, thoracic cuticular defects, and lethality. Most of the mutations were chemically induced in a single dumpy allele and were analyzed using a nuclease that cleaves single base pair mismatches in reannealed duplexes followed by dHPLC. Additionally, several spontaneous mutations were analyzed. Virtually all of the chemically induced mutations, except for several in a single exon, either generate nonsense codons or lesions that result in downstream stop codons in the reading frame. The remaining chemically induced mutations remove splice sites in the nascent dumpy message. We propose that the vast majority of nonsense mutations that affect all three basic dumpy phenotypes are in constitutive exons, whereas nonsense mutants that remove only one or two of the basic functions are in alternatively spliced exons. Evolutionary comparisons of the dumpy gene from seven Drosophila species show strong conservation of the 5′ ends of exons where mutants with partial dumpy function are found. In addition, reverse transcription PCR analyses reveal transcripts in which exons marked by nonsense mutations with partial dumpy function are absent.

Dumpy interacts with a large number of genes in the developing wing of Drosophila melanogaster

The complex Drosophila dumpy gene encodes a gigantic protein located in the apical extracellular matrix of epithelial cells. It has been shown to interact with several proteins notably during embryonic tracheal development. Here we examine Dumpy's interactions in vivo with mutations in 20 genes previously recovered in a screen for recessive lethals that generate blisters when somatic clones are produced by mitotic crossing over during wing development. Primarily using double mutants, we looked for both dominant effects of the wing blister mutants and the effects of blister mutant clones on dumpy expression. Sixteen of the mutants either suppressed or enhanced dumpy mutant phenotypes indicating the large Dumpy protein is a very important component of the epithelial extracellular matrix in the wing. Dumpy also interacts strongly with held out wings, which is involved in RNA localization and possibly alternative splicing.

The Rate of Unequal Crossing Over in the dumpy Gene from Drosophila melanogaster

The PIGSFEAST (PF) exon of the Drosophila dumpy gene is undergoing concerted evolution by the process of unequal crossing over. We have developed a long-range PCR-based assay to amplify the approximately 12 kb long exon which contains variable numbers of 303 or 306 nt long repeats in a tandem array. We applied this procedure to mutation accumulation lines of Drosophila melanogaster established by M. Wayne and L. Higgins. Nine new repeat length variants were found in these lines allowing us to measure the rate of unequal crossing over in the PF exon. The rate, which for several reasons is an underestimate, is 7.05 x 10(-4) exchanges per generation.

The physiological effects of dumpy mutations on de-novo pyrimidine synthesis in Drosophila melanogaster

kjaer, T. 1987. The physiological effects of dumpy mutations on de-novo pyrimidine synthesis in Drosophila rnelanogaster. —Hereditas 107:21S-217. Lund, Sweden. ISSN 0018–0661. Received February 9, 1987 The activity of three de-novo pyrimidine synthesis enzymes, aspartate transcarbamylase, orotate phos-phoribosyltransferase and orotidine-5′-phosphate decarboxylase, is investigated by in vitro enzyme assays. The results show an elevated activity in all three enzymes in homozygous viable strains, whereas most of the homozygous lethal strains have a lower activity than the wild type. This phenomenon is discussed with relevance to the composition of the cuticle. The activity of the same enzymes is also assayed in strains known to be defective in pyrimidine anabolism, and in a deficiency covering the entire dumpy gene. All results accumulate to support the theory that dp might have some regulatory effect on the pyrimidine synthesis which might be caused by an alteration in the chitin synthesis.

Concerted Evolution Within the Drosophila dumpy Gene

We have determined by reverse Southern analysis and direct sequence comparisons that most of the dumpy gene has evolved in the dipteran and other insect orders by purifying selection acting on amino acid replacements. One region, however, is evolving rapidly due to unequal crossing over and/or gene conversion. This region, called "PIGSFEAST," or PF, encodes in D. melanogaster 30-47 repeats of 102 amino acids rich in serines, threonines, and prolines. We show that the processes of concerted evolution have been operating on all species of Drosophila examined to date, but that an adjacent region has expanded in Anopheles gambiae, Aedes aegypti, and Tribolium castaneum, while the PF repeats are reduced in size and number. In addition, processes of concerted evolution have radically altered the codon usage patterns in D. melanogaster, D. pseudoobscura, and D. virilis compared with the rest of the dumpy gene. We show also that the dumpy gene is expressed on the inner surface of the micropyle of the mature oocyte and propose that, as in the abalone system, concerted evolution may be involved in adaptive changes affecting Dumpy's possible role in sperm-egg recognition.

Drosophila dumpy is a gigantic extracellular protein required to maintain tension at epidermal-cuticle attachment sites.

Growth and morphogenesis during development depend both on patterning genes, which assign positional information, and on genes that regulate mechanical forces. The dumpy gene of the fruit fly Drosophila melanogaster is an example of the latter class, with mutant phenotypes affecting size and shape of the limbs, thoracic cuticle, trachea and mouthparts. The genetically complex dumpy locus was found to span over 100 kb and encode a gigantic 2.5 MDa extracellular matrix protein. Dumpy represents an extreme form of modular protein evolution, containing 308 epidermal growth factor (EGF) modules, interspersed with a new module class, DPY, and terminating in a crosslinking zona pellucida domain and membrane anchor sequence. We determined the three-dimensional structure of the DPY module by nuclear magnetic resonance (NMR) spectroscopy and found that it forms a disulphide-stabilised beta sheet motif, capable of linking end-to-end with EGF modules to form a fibre. Consistent with its cuticle phenotypes, dumpy is expressed at several sites of cuticle-epidermal cell attachment, including the trachea and the muscle tendon cells, which mediate anchorage of the muscles to the cuticle. The dumpy gene encodes a gigantic extracellular molecule that we predict to be a membrane-anchored fibre of almost a micrometer in length. Insertion and crosslinking of this fibre within the cuticle may provide a strong anchor for the underlying tissue, allowing it to maintain mechanical tension at sites under stress. This would explain its contribution to tissue morphogenesis through its regulation of mechanical properties.

NOVEL NEMATODE AMBER SUPPRESSORS

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.-Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).-Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.

Rare pseudoallelic crossover between two phenotypically identical alleles at a restricted sublocus of dumpy in Drosophila melanogaster.

THE dumpy locus has been extensively mapped1–4. Many of the sites within the dumpy gene have phenotypic specificity ; that is, mutants of similar phenotypes map at the same restricted part of the region. A series of mutants which show an ov phenotype (oblique wings and thoracic vortices) were mapped to the right of lv1 and to the left of v2 (ref. 4). The ov mutants are viable as homozygotes and in combination with each other, except for ovh, which is a facultative lethal in the homozygote but which is viable with the other ov alleles. The ovh allele also shows a more extreme phenotype, and it is distinguishable in combination with other ov mutants by a more extreme phenotype. A crossover between ov1 and ovh was obtained by Southin5. The work reported here was carried out using other ov alleles of indistinguishable phenotype (ov1 and ov52b) in an attempt to establish critically the fine structure of the dumpy locus by obtaining crossovers between those alleles.

COMPARATIVE MUTAGENESIS OF THE DUMPY LOCUS IN DROSOPHILA MELANOGASTER. I. X-RAY TREATMENT OF MATURE SPERM–FREQUENCY AND DISTRIBUTION

Evidence is presented to support the view that the majority of x-ray- induced mutations at the dumpy locus are gene mutations not associated with multibreakage events. Nonvariegated mosaicism at the dumpy locus is usually free of detectable breakage events. Complete mutations (those affecting the entire body) form a mixed class of breakage events and gene mutations. The mutation frequency of the dumpy gene was not altered when sperm bearing a pre-existing dumpy mutation were used for irradiation. Gross mutation detected at the clot locus is one tenth that of the dumpy locus. Criteria are evaluated for determining the true gene mutation frequencies at both loci. The results of the x-ray analysis of the dumpy locus support a theory of mutagenesis proposed by Muller, Carlson, and Schalet (1961). (auth)