The yellow condition in Drosophila melanogaster. A biological structuralist approach to the study of phenocopies

A phenocopy, as defined by Goldschmidt, is an environmentally induced form which resembles a genetic mutant. They have a site of induction at a number of developmental levels and are not only produced by interfering with gene gene expression. Phenocopies and the corresponding genetic mutant, are also not necessarily generated through equivalent developmental pathways. The yellow phenocopy of Drosophila melanogaster, where both cuticle colour and mating behaviour resembles that of the yellow mutant, serves to illustrate the above points. It is suggested that if the term phenocopy is to be used the term should correspond to the generative processes and hence transformations which mutant and phenocopy share, rather than purely on phenotype. Finally, phenocopies are discussed in relation to structuralist biology, showing how they are instructive in describing generative processes, sensitive periods in development and differing modes of inheritance in such a research programme.     

Genotoxicity testing of five herbicides in the Drosophila wing spot test

Four triazine herbicides: amitrole, metribuzin, prometryn and terbutryn, and the bipyridal compound diquat dibromide have been evaluated for genotoxicity in the wing somatic mutation and recombination test of Drosophila melanogaster, following standard procedures. Third-instar larvae trans-heterozygous for the third chromosome recessive markers multiple wing hairs (mwh) and flare-3 (flr(3)) were chronically fed with different concentrations of the test compounds. Feeding ended with pupation of the surviving larvae. Genetic changes induced in somatic cells of the wing's imaginal discs lead to the formation of mutant clones on the wing blade. Point mutation, chromosome breakage and mitotic recombination produce single spots; while twin spots are produced only by mitotic recombination. Exposure to 0.5 mM and 1 mM of amitrole clearly increased the frequency of small single, large single and total spots. Terbutryn, at the concentration of 5 mM, induced a slight increase in the frequency of small single and total spots, but this result could be false positive. The other three herbicides tested did not show any genotoxic effect. When heterozygous larvae for mwh and the multiple inverted TM3 balancer chromosomes were treated, significant increases in the frequency of mutant spots were only detected for amitrole. The observed spot frequencies were lower than those found in mwh/flr(3)50%) of the total spot induction was due to mitotic recombination.     

Genetic effects of mitotic poisons and their modification by heat-shock in strains of Drosophila melanogaster having defective adaptive response]

Teratogenic effect of two mitotic poisons, griseofulvin and colchicine, was confirmed. A similar effect of another antimitotic agent, vinblastin, was demonstrated. The teratogenic effect of these poisons is expressed as a reduction of ommatidia in adult flies when the drug is fed to larvae. The highest frequency of phenocopies was recorded in temperature- and mutagen-sensitive strains. The mutagenic activity of vinblastin and griseofulvin was confirmed by the wing-spot test (somatic mutation and recombination test, SMART) in Drosophila melanogaster. In addition, this test demonstrated mutagenic activity of colchicine. All of the mitotic poisons induced small single spots but did not increase frequency of twin spots mwh/flr. Spot frequency was significantly higher in mutagen-sensitive mutants having defective excision repair. Heat shock (45-min exposure at 37 degrees C) decreased the frequency of phenocopies induced by the mitotic poisons. When third-instar larvae were subjected to heat shock prior to drug administration, the frequency of mutant cell clones was significantly reduced. These results indicate participation of heat-shock proteins in the protection of microtubules in actively proliferating cells of D. melanogaster.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila

We have found that the actin and microtubule cytoskeletons have overlapping, but distinct roles in the morphogenesis of epidermal hairs during Drosophila wing development. The function of both the actin and microtubule cytoskeletons appears to be required for the growth of wing hairs, as treatment of cultured pupal wings with either cytochalasin D or vinblastine was able to slow prehair extension. At higher doses a complete blockage of hair development was seen. The microtubule cytoskeleton is also required for localizing prehair initiation to the distalmost part of the cell. Disruption of the microtubule cytoskeleton resulted in the development of multiple prehairs along the apical cell periphery. The multiple hair cells were a phenocopy of mutations in the inturned group of tissue polarity genes, which are downstream targets of the frizzled signaling/signal transduction pathway. The actin cytoskeleton also plays a role in maintaining prehair integrity during prehair development as treatment of pupal wings with cytochalasin D, which inhibits actin polymerization, led to branched prehairs. This is a phenocopy of mutations in crinkled, and suggests mutations that cause branched hairs will be in genes that encode products that interact with the actin cytoskeleton.     

Effects of heat shock on protein processing and turnover in developing Drosophila wings

Developmental defects called phenocopies can be induced by heating Drosophila melanogaster pupae at specific developmental stages. The induction of the defects is thought to be a result of interference with gene expression at some level (Petersen and Mitchell, Dev Biol 1987; 121:335-341, 1987). Here we look at protein turnover in developing 52-hour wings and at the effect of heat on the proteolytic processing of three proteins that normally turn over rapidly. The effect of the heat treatment itself on the turnover of each protein is different. However, all of the proteins appear to be stabilized at 25 degrees C during recovery from severe heat shocks.     

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.     

Gradients of differentiation in wild-type and bithorax mutants of Drosophila

We present evidence to show that differentiation in wing cells to produce hairs is synchronous over the distal 90% of the wing surface (approximately 28,000 cells). In spite of this synchrony within such a large area a temporal gradient exists between zones (in general anterior to posterior) on the animal surface with rather sharp boundaries in between. In order to evaluate the basis for the gradient we studied two mutants which carry different combinations of the genes of the bithorax complex. These were examined with respect to the temporal aspects of sensitivity to heat shock induction of the multihair phenocopy on wings and the time of initiation of the program of protein synthesis that is related to hair formation. Results show that the gradient observed is based on predetermined properties within specific areas of tissue rather than on the position of the cells in the animal.     

Genotoxic evaluation of selective serotonin-reuptake inhibitors by use of the somatic mutation and recombination test in Drosophila melanogaster

This study evaluated different concentrations of selective serotonin-reuptake inhibitors (citalopram and sertraline) for genotoxicity by use of the somatic mutation and recombination test (SMART) in Drosophila melanogaster. Three-day-old larvae, trans-heterozygous for the multiple wing hairs (mwh) and flare (flr3) genes were treated with these two compounds. Two recessive markers were located on the left arm of chromosome 3, i.e. 'multiple wing hairs' (mwh) in map position 0.3 and 'flare-3' (flr3) at 38.8, while the centromere was located in position 47.7. SMART is based on the loss of heterozygosity, which may occur through various mechanisms, such as mitotic recombination, mutation, deletion, half-translocation, chromosome loss, and non-disjunction. Genetic changes occurring in somatic cells of the wing's imaginal discs, cause the formation of mutant clones on the wing blade. The results of this study show that citalopram had a genotoxic effect in the Drosophila SMART. Sertraline, however, did not show any genotoxic effect in balancer heterozygous wings. This study concluded that more information is needed to be certain regarding the mutagenic effects of sertraline.     

Mutagenicity of sumithion tested in Drosophila somatic and germ cells

Sumithion, a broad-spectrum insecticide, was tested for its mutagenicity in the Drosophila wing-spot test and sex-linked recessive lethal test. Strains carrying the recessive mutant markers mwh and flr3 in their third chromosomes, expressed phenotypically as multiple trichomes or thickened and misshapen wing hairs in the adult wings, were used in the wing-spot test. Larvae transheterozygous for these markers were exposed to the insecticide in instant food and the sex-linked recessive lethal test was performed by the standard technique using the Basc strain. The compound is mutagenic in the wing primordial cells and induces recombination at high doses. Further, the frequency of induction of sex-linked recessive lethals is significant only at high treatment doses.     

Differential gene expression in salt-tolerant rice mutant and its parental variety

The differential expressions of three genes rbcL, salT and rab!6 in response to ABA, NaCl, PEG and heat shock were investigated in seedlings of a salt-tolerant rice mutant 20 (mutant 20) and its parental variety Oryza sativa var. japonica 77-170(170). By Northern blot analysis it was found that ABA induced the expression of all three genes of rbcL, salT and rab16 in shoots and roots of both 170 and mutant 20 with the exceptions of rab16 in shoots of mutant 20 and rbcL in roots of 170. Lower concentrations of NaCl induced rbcL expression in shoots of mutant 20 but not 170. Higher concentrations of NaCl decreased rbcL expression but induced expressions of salT and rab16 in shoots of both 170 and mutant 20. PEG(15%) and 37℃ heat shock showed almost no effects on the expression of the three genes in mutant 20. However, they caused a decrease in rbcL expression and slight induction of the rab16 gene in 170, with salT expression unaffected. These results indicated that mutant 20 was relatively less responsiv     

Genetic control of galactokinase synthesis in Saccharomyces cerevisiae: evidence for constitutive expression of the positive regulatory gene gal4.

Temperature-sensitive (ts) mutants for the gal80 and gal4 genes of Saccharomyces cerevisiae were isolated and characterized. These mutants were classified into two categories; one showed thermolability (TL) and the other showed temperature-sensitive synthesis (TSS) of the respective products. Both the TL and TSS gal80 mutants are constitutive for galactokinase activity at 35 degrees C and, because they are derived from a dominant super-repressible GAL80s mutant, are uninducible at 25 degrees C. Both the TL and TSS gal4 mutants are galactose negative at 35 degrees C and galactose positive at 25 degrees C. None of the ts gal4 mutations affected the thermolability of galactokinase activity in cell extracts. Induction of galactokinase activity was studied with these mutants. The results indicate that the gal80 gene codes for a repressor and the gal4 gene codes for a positive factor indispensable for the expression of the structural genes or their products. However, striking evidence that the expression of the gal4 gene is constitutive and not under the control of gal80 was provided by a kinetic study with the TL gal4 mutant. The TL gal4 mutant pregrown in glycerol nutrient medium at 35 degrees C showed a prolonged lag period (35 min) in the induction of galactokinase activity at 25 degrees C, whereas the same mutant pregrown at 25 degrees C showed the same lag period as those observed in the wild-type strain and a revertant clone derived from the TL gal4 mutant (15 min).     

Bithorax phenocopy and pattern formation. I. Spatiotemporal characteristics of the phenocopy response

In four different lines of Drosophila melanogaster the highest frequencies of bithorax phenocopies result from ether treatment during the precellular blastoderm stages. The spatial characteristics of the phenocopy spots are dependent on the precise timing of phenocopy induction, and are not in accord with those predicted from the model of sequential determination of whole compartments put forward by Garcia-Bellido et al. in 1976. Ether treatment for 13 successive previous generations gave rise to changes in the spatiotemporal characteristics of the phenocopy response. Our results suggest the involvement of continuous diffusion-like processes in the prepatterning event which is affected by ether.