Genetic mosaic analysis of decapentaplegic and wingless gene function in the Drosophila leg

 Genetically mosaic flies were constructed which lack a functional decapentaplegic (dpp) or wingless (wg) gene in portions of their leg epidermis, and the leg cuticle was examined for defects. Although dpp has previously been shown to be transcribed both ventrally and dorsally, virtually the only dpp-null clones that affect leg anatomy are those which reside dorsally. Conversely, wg-null clones only cause leg defects when they reside ventrally – a result that was expected, given that wg is only expressed ventrally. Both findings are consistent with models of leg development in which the future tip of the leg is specified by an interaction between dpp and wg at the center of the leg disc. Null clones can cause mirror-image cuticular duplications confined to individual leg segments. Double-ventral, mirror-image patterns are observed with dpp-null clones, and double-dorsal patterns with wg-null clones. Clones that are doubly mutant (null for both dpp and wg) manifest reduced frequencies for both types of duplications. Duplications can include cells from surrounding non-mutant territory. Such nonautonomy implies that both dpp and wg are involved in positional signaling, not merely in the maintenance of cellular identities. However, neither gene product appears to function as a morphogen for the entire leg disc, since the effects of each gene’s null clones are restricted to a discrete part of the circumference. Interestingly, the circumferential domains where dpp and wg are needed are complementary to one another. Received: 25 March 1996 / Accepted: 13 June 1996     

Accuracy of bristle placement on a leg segment in Drosophila melanogaster

Bristle positions in two rows of bristles on the basitarsus of the second leg of the fruitfly Drosophila melanogaster were analyzed in order to determine the accuracy of bristle placement within these rows. Within each row the positions of the two terminal bristles were found to be approximately equally variable, and positional variability was found to increase toward the middle of each row. Rows having fewer bristles manifested more positional variability in their midsection. These results are interpreted in terms of a possible bristle spacing mechanism involving repulsive forces between mobile bristle cells.     

Mutational analysis of the structure and function of opioid receptors

The cloning of the opioid receptors allows the investigation of receptor domains involved in the peptidic and nonpeptidic ligand interaction and activation of the opioid receptors. Receptor chimera studies and mutational analysis of the primary sequences of the opioid receptors have provided insights into the structural domains required for the ligand recognition and receptor activation. In the current review, we examine the current reports on the possible involvement of extracellular domains and transmembrane domains in the high-affinity binding of peptidic and nonpeptidic ligands to the opioid receptor. The structural requirement for the receptors' selectivity toward different ligands is discussed. The receptor domains involved in the activation and subsequent cellular regulation of the receptors' activities as determined by mutational analysis will also be discussed. Finally, the validity of the conclusions based on single amino acid mutations is examined.     

Mutational analysis of the Drosophila DNA repair and recombination gene mei-9

Drosophila mei-9 is essential for several DNA repair and recombination pathways, including nucleotide excision repair (NER), interstrand crosslink repair, and meiotic recombination. To better understand the role of MEI-9 in these processes, we characterized 10 unique mutant alleles of mei-9. These include a P-element insertion that disrupts repair functions but not the meiotic function; three nonsense mutations, one of which has nearly wild-type levels of protein; three missense mutations, one of which disrupts the meiotic function but not repair functions; two small in-frame deletions; and one frameshift.     

Mutational analysis of morphogenesis of the maize embryo

Morphogenesis of the maize embryo is controlled by many genes. A group of 51 embryo-specific (emb) mutations representing at least 45 independent mutation events and many different gene loci have been isolated from active Robertson's Mutator stocks. The authors have reported previously that the embryo phenotype of 27 of these mutations, characterized by examining mature embryos in fresh dissection. The maximal development capacity of the 24 emb mutations are reported here which have not been reported previously. All result in retarded embryos that are morphologically abnormal. Three of the mutants are blocked during the first phase of morphogenesis, the period in which the basal-apical asymmetry is established and the embryo is regionalized into suspensor and embryo proper. Nineteen mutants are blocked during the second phase, the period in which radial asymmetry appears, the embryonic axis is established at a different angle than the original basal-apical axis of the zygote and the vegetative organ primordia of the adult plant make their first appearance. Two mutants are blocked or altered during the third phase, the period in which vegetative structures are elaborated. Some of the mutants affected in the first phase of morphogenesis may have defective mechanisms for establishing basal-apical asymmetry, including possibly the asymmetric distribution of morphogenic determinants. Similarly, some of the mutants affected in the second phase may be altered in the mechanisms establishing radial asymmetry and the origin of the meristems. Mutations of the first type may act as early as the first cell division when the zygote undergoes a transverse division, while mutations of the second type are likely to act during the proembryo and transition stages. Both types include mutations affecting embryo pattern formation. Mutations affecting the third phase of morphogenesis may identify genes regulating reiterative morphogenic processes of vegetative plant development and events of embryo maturation. This group of 24 mutations is like that reported previously in representing genes that are crucial to embryo morphogenesis.     

The function of PS integrins in Drosophila wing morphogenesis.

Integrins are found on many cell types during the development of most organisms. In Drosophila their functions can be analysed genetically. An analysis of lethal mutations in a PS integrin gene showed that the integrins were required for muscle attachment and for certain cell sheet migrations during embryogenesis. In this paper we use viable mutations in integrin component genes to look at integrin function in the later stages of development of one adult structure, the wing. We show that two known viable mutations, one which has its primary effect on the fly's escape response, the other on wing morphogenesis, are mutations in the beta and PS2alpha subunits, respectively, of the PS integrins. The mutation non-jumper (mys(mj42)) in the beta subunit leads to wasting of the thoracic jump muscles. Flies in which the dosage of this allele is reduced (and no wildtype copy is present) show defects also in wing morphogenesis. The two surfaces of the wing fail to connect properly, resulting in 'blistering' of the wing and the formation of extra crossveins. The mutation in the gene for the PS2alpha integrin subunit, inflated, also leads to a failure in wing surface apposition and consequent wing blistering. When the two mutations are combined, the mutant phenotype is greatly enhanced. Thus, one of the roles of the PS integrins in late Drosophila development is to ensure the correct apposition and patterning of the wing epithelia.     

Variance component analysis of bristle characters in local populations of Drosophila melanogaster.

The genetic variabilities of sternopleural and abdominal bristle numbers existing in local natural populations were assessed. Using second chromosome lines of Drosophila melanogaster extracted from three natural populations in Japan (the Ishigakijima, Ogasawara and Aomori populations), experiments were conducted to estimate the components of genetic variances, additive and dominance variances. The following results were obtained: For both sternopleural and abdominal bristle numbers, the additive genetic variances (sigma 2A) were much larger than the dominance variances (sigma 2D) especially in the southern populations. For example, in the Ishigakijima population, for females sternopleural bristle numbers of the inversion-free chromosome group, the additive and dominance variances were estimated to be 1.255 +/- 0.2034 and 0.0552 +/- 0.0180, respectively. The magnitudes of the estimates of additive genetic variances were nearly equal from north to south. By comparing the additive genetic variances of the inversion-free chromosome group with those of the In(2L)t-carrying chromosome group, it was inferred that sufficient number of generations to achieve the equilibrium state has not passed since the introduction of a single or a small number of the In(2L)t-carrying chromosomes to the Ishigakijima population.     

The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart

In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also Gellon et al., 1997), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heat tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages.     

Arrangement of bristles as a function of bristle number on a leg segment inDrosophila melanogaster

Summary The arrangement of bristles on a leg segment of the fruitflyDrosophila melanogaster was studied in various mutants that have abnormal numbers of bristles on this segment. Eighteen mutations at six different genetic loci were analyzed, plus five double or triple mutant combinations. Recessive mutations at theachaete-scute locus were found to affect distinct groups of bristles:achaete mutations remove mechanosensory bristles, whereasscute mutations remove mainly chemosensory bristles. Mechanosensory bristles remain uniformly spaced along the longitudinal axis unless their number decreases below a certain threshold, suggesting that spacing is controlled by cell interactions that cannot function when bristle cells are too far apart. Above a certain threshold, bristle spacing and alignment both become irregular, perhaps due to excessive force from these same interactions. Chemosensory bristles occupy definite positions that are virtually unaffected by removal of individual bristles from the array. Extra chemosensory bristles develop only near the six normal sites. At two of the six sites the multiple bristles tend to exhibit uniform longitudinal spacing — a property confined to mechanosensory bristles in wild-type flies. To explain the various mutant phenotypes the following scheme is proposed, with different mutations directly or indirectly affecting each step: (1) spots and stripes are demarcated within the pattern area, (2) one bristle cell normally arises within each spot, multiple bristle cells within each stripe, (3) incipient bristle cells inhibit neighboring cells from becoming bristle cells, and (4) the bristle cells within each stripe become aligned to form rows and then repel one another to generate uniform spacing.     

Mutational analysis of X-linked adrenoleukodystrophy gene

X-linked adrenoleukodystrophy (ALD) is an inherited peroxisomal disorder characterized by progressive neurological dysfunction, occasionally associated with adrenal insufficiency. The clinical thenotypes of ALD are quite variable, and include childhood ALD, adult-onset ALD, adrenomyeloneuropathy, and Addison's disease only. Although the causative gene for ALD has been identified, the physiological role of the gene product remains to be clarified. Despite many mutations having been identified in patients with these clinical phenotypes, the genotype-phenotype correlations have not been clarified. The authors investigated genotype-phenotype correlatons in ALD by analyses on 29 unrelated Japanese patients with ALD and by a review of the literature. All the phenotypes were associated with mutations leading to protein truncation, as well as those resulting in subtle amino acid changes. Furthermore, there were no differences in phenotypic expression among the natures of the subtle amino acid changes. All these data indicate that no obvious correlations exist between the phenotypes of ALD patients and their geneotypes, suggesting that other genetic or environmental factors may also be involved in determining phenotypic expression in ALD.     

Mutational analysis of residue roles in AraC function

The previously isolated hemiplegic, induction-negative, repression-positive mutants, H80R and Y82C, were found to be defective in the binding of arabinose. Randomization of other residues close to arabinose in the three-dimensional structure of AraC or that make strong interactions with arabinose yielded induction-negative, repression-positive mutants. The induction and repression properties of mutants obtained by randomizing individual residues of the N-terminal arm of AraC allowed identification of the domain with which that residue very likely makes its predominant interactions. Residues 8-14 of the arm appear to make their predominant interaction with the DNA-binding domain. Although the side-chain of residue 15 interacts directly with arabinose bound to the N-terminal dimerization domain, the properties of mutant F15L indicate that this mutation increases the affinity of the arm for the DNA-binding domain.