P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F.

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover approximately 25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven "super-contigs" that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the alphagamma element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.

Position-effect variegation in Drosophila depends on dose of the gene encoding the E2F transcriptional activator and cell cycle regulator.

A dominant mutation due to the insertion of a P-element at 93E on the third chromosome of Drosophila melanogaster enhances position-effect variegation. The corresponding gene was cloned by transposon tagging and the sequence of the transcript revealed that it corresponds to the gene encoding the transcriptional activator and cell cycle regulator dE2F. The transposon-tagged allele is homozygous viable, and the insertion of the transposon in an intron correlates with a strong reduction in the amount of transcript. A homozygous lethal null allele was found to behave as a strong enhancer when heterozygous. Overexpression of the gene in transgenic flies has the opposite effect of suppressing variegation. A link is established here, and discussed, between the dose of a transcriptional activator, which controls the cell cycle, and epigenetic silencing of chromosomal domains in Drosophila.

Identification of two bone morphogenetic protein type I receptors in Drosophila and evidence that Brk25D is a decapentaplegic receptor.

Drosophila sequences at chromosomal positions 25D (Brk25D) and 43E (Brk43E) are similar to the TGF beta type I receptor serine/threonine kinases and are expressed broadly during embryogenesis. Brk25D binds dpp protein and bone morphogenetic protein 2 with high affinity. Mutations affecting Brk25D map to the gene thick veins and block the expression of two decapentaplegic-responsive (dpp-responsive) genes, dpp and labial, in the embryonic midgut. Defects in Brk25D receptor function combined with reduced expression of dpp ligand produce mutant phenotypes in the embryo and adult. Brk43E is the product of the gene saxophone, which also interacts with dpp. We conclude that dpp signaling in vivo is mediated by at least two receptors, Brk25D and Brk43E.

P transposon-induced dominant enhancer mutations of position-effect variegation in Drosophila melanogaster.

P transposon induced modifier mutations of position-effect variegation (PEV) were isolated with the help of hybrid dysgenic crosses (pi 2 strain) and after transposition of the mutator elements pUChsneory+ and P[lArB]. Enhancer mutations were found with a ten times higher frequency than suppressors. The 19 pUChsneory(+)- and 15 P[lArB]-induced enhancer mutations can be used for cloning of genomic sequences at the insertion sites of the mutator elements via plasmid rescue. Together with a large sample of X-ray-induced (48) and spontaneous (93) enhancer mutations a basic genetic analysis of this group of modifier genes was performed. On the basis of complementation and mapping data we estimate the number of enhancer genes at about 30 in the third chromosome and between 50 and 60 for the whole autosome complement. Therefore, enhancer of PEV loci are found in the Drosophila genome as frequently as suppressor genes. Many of the enhancer mutations display paternal effects consistent with the hypothesis that some of these mutations can induce genomic imprinting. First studies on the developmentally regulated gene expression of PEV enhancer genes were performed by beta-galactosidase staining in P[lArB] induced mutations.

The fatty acid constitution and ordering state of membranes in dominant temperature-sensitive lethal mutation and wild-type Drosophila melanogaster larvae.

The ordering state and changes in fatty acid composition of microsomal (MS) and mitochondrial (MC) membranes of two dominant temperature-sensitive (DTS) lethal mutations and the wild-type Oregon-R strain larvae of Drosophila melanogaster have been studied at 18 and 29 degrees C and after temperature-shift experiments. The membranes of wild-type larvae have a stable ordering state, with "S" values between 0.6 (18 degrees C) and 0.5 (29 degrees C) in both membranes which remained unchanged in shift experiments, although the ratios of saturated/unsaturated fatty acids were changed as expected. The strongly DTS mutation 1(2) 10DTS forms very rigid membranes at the restrictive temperature (29 degrees C) which cannot be normalized after shift down, while shift up or development at the permissive temperature results in normal ordering state. This mutant is less able to adjust MS and MC fatty acid composition in response to the growth temperature than the wild type. The less temperature-sensitive 1(2)2DTS allele occupies an intermediate state between Oregon-R and 1(2)10DTS in both respects. We assume and the genetical data suggest that the DTS mutant gene product is in competition with the wild-type product, resulting in a membrane structure which is not able to accommodate to the restrictive temperature.

Isolation and characterization of dominant female sterile mutations of Drosophila melanogaster. II. Mutations on the second chromosome.

Twenty-four, second chromosome, dominant female sterile (Fs) mutations in Drosophila are described. Fs(2) were isolated at a frequency of approximately 1 per 1000 EMS-treated chromosomes screened. In comparison the isolation of frequency for second chromosome zygotic recessive lethal mutations was approximately 550 per 1000. Complementation analysis of the Fs(2) revertants showed that the 24 Fs(2) mutations identify 13-15 loci, calculated to be about 65-75% of the second chromosome genes EMS mutable to dominant female sterility. Two of the Fs(2) mutations are useful tools for the dominant female sterile technique: Fs(2)1 for induction and detection of germ-line clones and Fs(2)Ugra for follicle cell clones. Several of the Fs(2) mutations bring about novel mutant phenotypes. Seven of them alter egg shape, whereas the others arrest development primarily at two stages: around fertilization by five Fs(2) and during cleavage divisions [by Fs(2) in three loci]. The remaining that allow development to the larval stage of differentiation include four new dorsal alleles and one dominant torso allele. Analysis of germ-line chimeras revealed that with two exceptions all the Fs(2) mutations are germ-line dependent. The Fs(2) mutations were mapped mainly on the basis of mitotic recombination induced in the female germ-line cells of adult females. That most of the Fs(2) may be gain-of-function mutations is indicated by the unusual behavior of the Fs+ germ-line clones and also by the fact that 90% of the could be induced to revert.

The genetics of position-effect variegation modifying loci in Drosophila melanogaster.

The dose dependent effects of position-effect variegation (PEV) modifying genes were studied in chromosome arms 2L, 2R and 3R. Four groups of PEV modifying genes can be distinguished: haplo-abnormal suppressor and enhancer loci with or without a triplo-effect. Using duplications four triplo-abnormal suppressor and four triplo-abnormal enhancer functions were localized. In two cases we proved that these functions correspond to a converse haplo-abnormal one. Altogether 43 modifier loci were identified. Most of these loci proved not to display significant triplo-effects (35). The group of haplo-abnormal loci with a triplo-effect may represent genes which play an important role in heterochromatin packaging.

Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila.

Lethal mutations at the fat locus in Drosophila cause imaginal discs to continue to grow by cell proliferation far beyond their normal final size. During a greatly extended larval period, the overgrowing imaginal discs develop additional folds and lobes, but retain a single-layered epithelial structure. In the wing disc, the additional lobes originate in the proximal fold area, and in the extra tissue the cells are less columnar than normal. Mutant disc cells lack zonulae adherents as well as associated microtubules and microfilaments, and they show an abnormal distribution and reduced density of gap junctions. The effect on growth is disc-autonomous as shown by transplantation experiments. The overgrown imaginal discs retain the ability to differentiate adult cuticular structures, as shown by metamorphosis of discs after transplantation into wild-type larval hosts and by the ability of some mutant animals to develop to the pharate adult stage. The structures differentiated by mutant discs show many abnormalities including ingrowths, outgrowths, separated cuticular vesicles, and areas of reversed bristle polarity; some of these abnormalities suggest that the mutations interfere with cell adhesion as well as the control of cell proliferation. The fat locus is located in cytogenetic interval 24D5.6-7, and 18 alleles are known including spontaneous, chemically induced, X-ray-induced, and dysgenic mutations.

Characterization of Fs(2)1, a germ-line dependent dominant female sterile mutation of Drosophila.

Fs(2)1 is a germ-line dependent dominant female sterile mutation of Drosophila melanogaster. Fs(2)1 heterozygous females deposit very few abnormal eggs (collapsed, with malformed chorion). The degeneration of egg primorida starts around the end of egg maturation. Mitotic recombination mapping locates Fs(2)1 in a distal region of the left arm of the 2nd chromosome. Fs(2)1 is a good tool for studying germ-line functions (by the dominant female sterile technique) because the frequency of germ-line mosaicism exceeds 20% upon irradiation of adult females. Salivary gland polytene chromosomes of Fs(2)1 and the revertant heterozygous larvae appear normal.

Cytogenetic analysis of variegation suppressors and a dominant temperature-sensitive lethal in region 23-26 of chromosome 2L in Drosophila melanogaster.

Three suppressor loci for position-effect variegation, one dominant temperature-sensitive (DTS), three Minute genes, and two recessive visible mutants (ed, tkv) have been cytogenetically localized by using duplications and deficiencies in regions 23-25 of chromosome arm 2L of Drosophila melanogaster. Two of the suppressor loci studied proved to represent haplo-abnormal genes localized in regions 23A6-23F6 and 24E2-25A1, respectively. The third one is a strong triplo-abnormal suppressor mapping in 25F4-26B9 which affects white variegation in wm4h when present in three doses. The l(2)2DTS mutation, which belongs to a group of noncomplementing dominant temperature-sensitive mutations, is localized in the 25A4-B1 region. Furthermore, two Minute genes have been localized in region 24 that are included in Df(2L)M11 and can be separated employing translocation (Y;2)P8 (24E2-4): M(2)LS2 in 24D3-4-24E2-4, and M(2)z in 24E4-5-24F5-7. A third Minute gene (M(2)S1) is localized in 25C3-8-25C9-D1. The usefulness of the isolated chromosomal rearrangements for further genetic studies of region 23-26 is discussed.

Developmental analysis of fs(1)1867, an egg resorption mutation of Drosophila melanogaster.

The X-linked 1867+ gene seems to be a pleiotropic one. Mutation in this gene causes delay in development and abnormal bristle morphology. These phenotypes are expressed autonomously in genetic mosaics. There is no focus for the delay. The female sterility could be localized to the ovary (based on ovary transplantations). It seems that the 1867+ gene is expressed in the follicular cells at one of the last steps of oogenesis. This is suggested by the results of mosaic analysis based on mitotic recombination. Possible drawbacks of the mitotic recombination type of analyses are also discussed.

Behavioral mutants of Drosophila melanogaster. III. Isolation and mapping of mutations by direct visual observations of behavioral phenotypes.

Sex-linked behavioral mutants were induced in Drosophila melanogaster with ethyl methanesulfonate (EMS) and isolated by direct visual observation of abnormal phenotypes. The four behavioral phenotypes used were flight-reduction, hyperactivity, hypoactivity and stress-sensitivity, and are easily discernable in either single or small populations of mutant flies. In one screen, forty-two behavioral mutants were recovered from strains derived from 800 mutagen-treated X chromosomes In a second screen, 139 behavioral mutants were obtained from 2369 X chromosomes. The high rate at which behavioral mutants were recovered in the second screen, when compared to new visibles (28) and new temperature-sensitive lethals (124), suggests that the isolation of behavioral mutations on the autosomes of Drosophila and in the genomes of larger insects should be practical.

Abnormalities of ultrastructure and calcium distribution in the flight muscle of a flightless mutant of Drosophila melanogaster.

An X-linked mutant of Drosophila melanogaster was isolated which was completely unable to fly. The map position of the mutation is 43 +/- 0.1. Gynandromorph analysis indicated that the mutation is autonomously expressed in the flight muscle. Fate-map data show that the focus of the mutation lies close to the ventral midline of the blastoderm. Flightlessness appears to be the result of abnormalities of the thoracic musculature, including highly irregular arrangement of the fibrils, lack of the normal striation pattern and abnormal structure of the mitochondria. X-ray microanalysis (EDAX) demonstrates a pronounced difference in the distribution of calcium in mutant and wild-type flight muscle at the fine-structural level. We propose that an abnormal calcium distribution in the mutant may be associated with the ultrastructural abnormalities and ultimately responsible for the flightless phenotype.

The effect of dexamethasone on the karyotype of A9HT cells.

In the majority of glucocorticoid-resistant receptor containing variants of A9HT cells, the numbers of dot chromosomes was found to be significantly higher than in the parent cells. The higher number of dot chromosomes, however, did not always correlate with the resistance of L cells to dexamethasone. The formation of dot chromosomes was not the consequence of dexamethasone-induced chromosome breaks. Dot-chromosome formation could not be induced by dexamethasone in human diploid cells. It seems likely that dexamethasone can select for A9HT cells with higher number of dot chromosomes.

Isolation and characterization of X-linked lethal mutants affecting differentiation of the imaginal discs in Drosophila melanogaster.

Twenty-seven late larval or early pupal lethal mutations were isolated for the X-chromosome, some of which showed structural and/or functional deficiencies of the imaginal discs. The mutants were grouped according to the size and morphology of their discs as follows: 1. discs normal: 18 mutants. 2. discs small: 2 mutants. 3. discs degenerate: 4 mutants. 4. discless: 1 mutant. 5. discs heterogeneous: 2 mutants. Preliminary characterization of the mutants included a study of disc morphology, puparium formation and pupal molt, in vivo and in vitro evagination of the imaginal discs, autonomy of the mutation in the disc tissue (differentiation after transplantation and gynander mosaicism test). Possible relations between disc morphology and the former characteristics are discussed.