The suppressor of forked locus in Drosophila melanogaster: genetic and molecular analyses

The suppressor of forked, su(f) locus is one of a class of loci in Drosophila whose mutant alleles are trans-acting allele-specific modifiers of transposable element-insertion mutations at other loci. Mutations of su(f) suppress gypsy insert alleles of forked and enhance the copia insert allele white apricot. Our investigations of su(f) include genetic and molecular analyses of 19 alleles to determine the numbers and types of genetic functions present at the locus. Our results suggest the su(f) locus contains multiple genetic functions. There are two distinct modifier functions and two vital functions. One modifier function is specific for enhancement and the other for suppression. One vital function is required for normal ecdysterone production in the third larval instar, the other is not. We present a restriction map of the su(f) genomic region and the results of an RFLP analysis of several su(f) alleles.     

Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.     

The recessive phenotype of forked can be uncovered by heat shock in Drosophila

Heat shock uncovers the recessive forked phenotype when heterozygotes between f^36a and wild-type are heated during sensitive periods in pupal development. We call the phenocopy of a mutant in such a heterozygote a heterocopy. The heterocopy in f^36a/+ is virtually identical to the mutant phenotype; however, bristles on different parts of the body are affected during different sensitive periods. We discuss the hypothesis that the heat shock acts by affecting expression of the wild-type gene product corresponding to the mutant gene. The sensitive period for heterocopy induction in a specific tissue is proposed to correspond to the normal time of gene expression for the forked gene product in a particular tissue.     

Nucleotide variation at the runt locus in Drosophila melanogaster and Drosophila simulans.

Intra- and interspecific nucleotide variation for the major developmental gene runt in Drosophila was studied in D. melanogaster and D. simulans. The 1.5-kb protein-coding region and the 0.4-kb intron of the runt gene were sequenced for 11 alleles in each species. The D. melanogaster alleles originated from east Africa. Estimated parameters of intraspecific variation in D. melanogaster (exons: theta = 0.018, pi = 0.018; intron: theta = 0.014, pi = 0.014) and D. simulans (exons: theta = 0.007, pi = 0.005; intron: theta = 0.008, pi = 0.005) were below average for other X-linked genes, while divergence between species (exons: D = 0.094; intron: D = 0.069) fell within the normal range for both silent and replacement changes. This estimate for runt, along with published values for three other genes in regions of normal recombination, show east African D. melanogaster to be roughly twice as polymorphic as D. simulans. The majority of nucleotide variation, silent and replacement, in both species was found to be selectively neutral using various statistical tests (HKA, McDonald-Kreitman, Tajima, and Fu and Li tests). Monte Carlo simulations of the coalescent process significantly rejected a Wright-Fisher model with respect to an amino acid polymorphism and the distribution of polymorphic sites among the D. simulans lines. This indicated an old lineage and may reflect ancestral population substructuring in D. simulans.     

Expression of the 53 kD forked protein rescues F-actin bundle formation and mutant bristle phenotypes in Drosophila.

forked mutations affect bristle development in Drosophila pupae, resulting in short, thick, gnarled bristles in the adult. The forked proteins are components of 200-300-microm-long actin fiber bundles that are present transiently during pupal development [Petersen et al., 1994: Genetics 136:173-182]. These bundles are composed of segments of 3-10 microm long, and forked protein is localized along the actin fiber bundle segments and accumulates at the junctions connecting them longitudinally. In the forked mutants, f(36a) and f(hd), F-actin bundles are greatly reduced in number and size, and bundle segmentation is absent. The p-element, P[w(+), falter] contains a 5.3-kb fragment of the forked gene that encodes the 53-kD forked protein [Lankenau et al., 1996: Mol Cell Biol 16:3535-3544]. Expression of only the 53-kD forked protein is sufficient to rescue the actin bundle and bristle phenotypes of f(36a) and f(hd) mutant flies. The 5.3-kb forked sequence, although smaller than the 13-kb region previously shown to rescue forked mutants [Petersen et al., 1994: Genetics 136:173-182], does contain the core forked sequence that encodes actin binding and bundling domains in cultured mammalian cells [Grieshaber and Petersen, 1999: J Cell Sci 112:2203-2211]. These data show that the 53-kD forked protein is sufficient for normal bristle development and that the domains shown previously to be important for actin bundling in cell culture may be all that are required for normal actin bundle formation in developing Drosophila bristles.     

The suppressor of forked mutation in Drosophila melanogaster: Interactions with the lozenge gene

An interaction between the lozenge gene and the suppressor of forked gene of Drosophila melanogaster has been investigated both spectrophotometrically and electrophoretically. The nature of this interaction is such that certain lozenge alleles appear to be phenotypically suppressed while others are enhanced or unaffected, and the results reported demonstrate that the effect can clearly be observed at the biochemical level. Earlier observations have suggested that the suppressor of forked gene codes for a ribosomal protein, and this hypothesis is discussed.These studies were supported by USPHS Grants GM-18485 and GM-20361 to P. D. S. P. D. S. is a recipient of USPHS Research Career Development Award GM-70758.     

Intragenic recombination at the white locus of Drosophila hydei.

A fine-structure analysis of the white locus in Drosophila hydei was carried out by means of allele recombination. Four mutants, derived from wild type, mapped at three subloci. These are possibly homologous to the main subloci 2, 3, and 4 of D. melanogaster. Three secondary mutants, derived from the primary wiv allele, were located in the proximal part of the gene. One of them appeared as a homoallele of the original wiv, whereas the remaining two are better explained either as double mutants or as mutants which facilitate irregular exchange. Intragenic recombination at the white locus seems to be more frequent in D. hydei than in D. melanogaster. The comparatively high incidence is probably a general characteristic, common to intragenic and intergenic recombination in D. hydei.     

Cloning of heat-shock locus 93D from Drosophila melanogaster.

Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.     

The involvement of the Notch locus in Drosophila oogenesis.

The Notch gene in Drosophila encodes a transmembrane protein with homology to EGF that, in a variety of tissues, appears to mediate cell interactions necessary for cell fate choices. Here we demonstrate that oogenesis and spermatogenesis depend on Notch. We examine the phenotypes of the temperature-sensitive Notch allele, Nts1, and, using a monoclonal antibody, determine the cellular and subcellular distribution of Notch protein during oogenesis. We show that Nts1 is associated with a missense mutation in the extracellular, EGF homologous region of Notch and that at non-permissive temperatures oogenesis is blocked and the subcellular distribution of the protein is altered. In wild-type ovaries, Notch protein is found on the apical surface of somatically derived follicle cells, while in the germline-derived cells the protein is not polarized. These findings are discussed in view of the hypothesis that Notch acts as a multifunctional receptor to mediate developmentally important cell interactions.     

A novel trans-regulatory locus in Drosophila

A new trans-regulatory gene, leg-arista-wing complex (lawc), was earlier identified in a system of prolonged genetic instability induced by transpositions of structurally different mobile genetic elements, the P element and MDG Stalker. Mutations of this gene alter expression of many loci, including the achaete and scute proneural genes and the cut locus coding for a homeobox-containing protein. A product of lawc is involved in regulating expression of white. This role is played in cooperation with the product of Zeste which provides for a contact between the white promoter and enhancer. A possible role of the new locus in organogenesis, neurogenesis, and embryonic development is discussed on the basis of experimental findings.     

Nucleotide polymorphism at the xanthine dehydrogenase locus in Drosophila pseudoobscura.

Sequential polyacrylamide electrophoresis has revealed 20 allozymes of xanthine dehydrogenase (XDH) in Drosophila pseudoobscura. DNA sequence determination of seven isolates of the Xdh locus that represent six allozyme classes are presented here. Of the 5,456 sites examined, 180 are polymorphic, with 27 polymorphisms occurring at nonsynonymous, or replacement, sites. An average of nine amino acids differ between XDH allozyme classes, with 85% of the polymorphic amino acids singly represented. The level and pattern of variation observed at Xdh argue that the effective population size of the species is quite large--i.e., on the order of 2 x 10(6)--and that the populations sampled are quite ancient. In addition, as judged by two statistical tests, the levels of nucleotide polymorphism observed at Xdh are compatible with predictions from the neutral theory of molecular evolution.     

Euchromatic inversions causing mosaic gene expression in Drosophila hydei: A study of forked and Zebra

In D. hydei two new mutants, In(1)f³ and IN(5)Z, show obvious mosaic gene expression. Their phenotypic expression is susceptible to the breeding temperature and to the addition of a supernumerary Y chromosome to the chromosome set. In this respect the mutants resemble standard cases of position-effect variegation based on the action of heterochromatin. However, since neither centromeric nor sex chromosomal heterochromatin apparently are involved, the mutations point to a new type of variegation provoked by euchromatic sections. The mosaic patterns of these mutants, in particular those of In(1)f³, will be described.