The Genetics of Dopa Decarboxylase and a-Methyl Dopa Sensitivity in Drosophila melanogaster

Dopa decarboxylase (DDC) in the Diptera is an enzyme involvedin sclerotinization of the cuticle in the epidermis and theproduction of neurogenic amines in the central nervous system.Its appearance in the epidermis at pupariation is induced bythe molting hormone ecdysone. The dietary administration ofthe analog inhibitor -methyl dopa (a MD) was used to isolateresistant and hypersensitive mutants. Two of three dominantresistant strains isolated increase DDC activity 35-70%. Forboth the increase is due to mutations between rdo (53) and pr(54.5) on the left arm of the 2nd chromosome (2L). The veryhighly resistant strain which does not affect DDC in any wayis located at 54.0 on 2L. Twelve dominant, l(2)amd^H —MD hypersensitive alleles located immediately to the right ofhk (53.9) on 2L have been recovered. All are recessive lethalsand exhibit some intracistronic complementation, and none ofthem, not even heteroallelic heterozygotes, affect DDC in anyway. The.recovery and analysis of 16 overlapping deficienciespermitted the localization of a DDC dosage effect to bands 37B10-C7on 2L; a region which includes the l(2)amd locus. Subsequentlyeight DDC deficient lethal alleles were recovered in this elevenband region which as heterozygotes reduce activities to 28–53% of controls. Some heteroallelic heterozygotes exhibit intracistroniccomplementation; most with viabilities 5% and with a mutantphenotype probably derived from inadequately sclerotinized cuticle.These Ddc alleles are within 0.004 Map Units to the right ofl(2)amd. None as Ddc/CyO heterozygotes are sensitive to -MD,and complementation occurs between the Ddc alleles and the l(2)amdalleles both on the basis of viability and DDC activity. Althoughthe protein product mutated by the l(2)amd alleles has not yetbeen identified, it seems likely that the two groups of mutantsare functionally related. Finally, the Ddc structural mutantsreduce DDC activity in the central nervous system as well asthe epidermis.     

Molecular Mapping of a Gene Cluster Flanking the Drosophila Dopa Decarboxylase Gene

Nine lethal complementation groups flanking the Drosophila Dopa decarboxylase (Ddc) gene, have been localized within 100 kb of cloned chromosomal DNA. Six of these complementation groups are within 23 kb of DNA, and all ten complementation groups, including Ddc, lie within 78-82 kb of DNA. The potential significance of this unusually high gene density is discussed.     

Tyrosine Decarboxylase and Dopa Decarboxylase in Drosophila virilis Under Normal Conditions and Heat Stress: Genetic and Physiological Aspects

The activity of tyrosine decarboxylase (TDC) and dopa decarboxylase (DDC) was studied in adults of two lines of Drosophila virilis,contrasting in their reaction to stress conditions. Differences were found in the activity of both enzymes between individuals of the examined lines. Genetic analysis of these differences was made. Each of the two enzymes was found to be controlled by a single gene or, possibly, by a block of closely linked genes. The gene responsible for TDC activity is located on one of the autosomes (excluding chromosome II). DDC activity in D. virilisis regulated by a gene located, apparently, on chromosome II. Adults of the line responding to stress by a stress reaction (r-line) were shown to react to a short-term heat stress (38°C, 60 min) by a decrease in TDC activity. TDC activity in flies of the line incapable of the stress reaction (nr-line) did not alter in such conditions. DDC activity of adults of both lines was found to be unchangeable under stress conditions.     

Evidence for Regulatory Variants of the Dopa Decarboxylase and Alpha-Methyldopa Hypersensitive Loci in Drosophila

We have analyzed two variants of Drosophila melanogaster (RS and RE) which lead to the dual phenotype of elevated DDC activity and increased resistance to dietary alpha-methyldopa relative to Oregon-R controls. Both phenotypes show tight genetic linkage to the dopa decarboxylase, Ddc, and l(2)amd genes (i.e., less than 0.05 cM distant). We find that low (Oregon-R), medium (RS) and high (RE and Canton-S) levels of DDC activity seen at both pupariation and eclosion in these strains are completely accounted for by differences in accumulation of DDC protein as measured by immunoprecipitation. Genetic reconstruction experiments in which Ddc+ and amd+ gene doses are varied show that increasing DDC activity does not lead to a measurable increase in resistance to dietary alpha-methyldopa. This suggests that the increased resistance to dietary alpha-methyldopa is not the result of increased DDC activity but, rather, results from increased l(2)amd+ activity. Both cytogenetic and molecular analyses indicate that these overproduction variants are not the result of small duplications of the Ddc and amd genes, nor are they associated with small (greater than or equal to 100 bp) insertions or deletions. Measurements of DDC activity in wild-type strains of Drosophila reveal a unimodal distribution of activity levels with the Canton-S and RE strains at the high end of the scale, the Oregon-R control at the low end and RS near the modal value. We conclude that accumulated changes in a genetic element (or elements) in close proximity to the Ddc+ and amd+ genes lead to the coordinated changes in the expression of the Ddc and amd genes in these strains.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Retrotransposon-Induced Ectopic Expression of Cut Causes the Om(1a) Mutant in Drosophila Ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The γ-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.     

The Response of Dopa Decarboxylase Activity to Variations in Gene Dosage in Drosophila: A Possible Location of the Structural Gene

A location of the structural gene(s) for dopa decarboxylase (EC 4.1.1.26) is proposed on the basis of enzyme determinations in a set of duplication-bearing aneuploids, which revealed only one dosage-sensitive region in the Drosophila genome. This region lies between 36EF and 37D on the left arm of chromosome 2.     

Morewright (Mwr), a New Meiotic Mutant of Drosophila Melanogaster Affecting Nonexchange Chromosome Segregation

A new meiotic mutation, morewright (mwr) was identified by screening for new mutations that act as dominant enhancers of the dosage-sensitive Drosophila melanogaster female meiotic mutant, nod(DTW). mwr is a recessive meiotic mutant, specifically impairing the segregation of nonexchange chromosomes. Cytological evidence suggests that the meitoic defect in mwr/mwr females is in homologue recognition because the chromosomes appear to be misaligned on an intact spindle. The mwr mutation was recovered during a screen of random P-element insertions on a chromosome with a single insertion located at 50C. The P-element insertion is a recessive female-sterile mutation. While excision of the P element from the mwr-bearing chromosome partially relieves the female sterility, the excisions retain the dominant nod(DTW)-enhancing activity. The mwr meiotic phenotype maps very close to the female-sterile P insertion. Thus the mwr locus appears to encode a function required for partner recognition in meiosis, although its relationship to the neighboring female-sterile mutation remains to be elucidated.     

Ornithine Decarboxylase Activity in Brain Regulated by a Specific Macromolecul, the Antizyme

Mouse brain ornithine decarboxylase activity is about 70-fold higher at the time of birth compared with that of adult mice. Enzyme activity declines rapidly after birth and reaches the adult level by 3 weeks. Immunoreactive enzyme concentration parallels very closely the decrease of enzyme activity during the first postnatal week, remaining constant thereafter. The content of brain antizyme, the macromolecular inhibitor to ornithine decarboxylase, in turn is very low during the first 7 days and starts then to increase and at the age of 3 weeks it is about six times the level of that in newborn mice. This may explain the decrease in enzyme activity during brain maturation, and suggests the regulation of polyamine biosynthesis by an antizyme-mediated mechanism in adult brain.     

Molecular Cloning, Water Channel Activity and Tissue Specific Expression of Two Isoforms of Radish Vacuolar Aquaporin1

A major membrane intrinsic protein (VM23) in vacuoles of radish(Raphanus) tap root was investigated. The cDNAs for two isoformsof VM23,      

Inhibition of Multiplication in Acanthamoeba castellanii by Specific Inhibitors of Ornithine Decarboxylase1

Proliferation of Acanthamoeba castellanii (Neff strain) in either a broth medium or a defined medium was arrested by α-monofluoromethyldehydroornithine (Δ-MFMOme), α-difluoromethylornithine (DFMO), and (R, R')-δ-methyl-α-acetylenic putrescine (MAP), three specific inhibitors of ornithine decarboxylase. Although all three inhibited the ameba enzyme, Δ-MFMOme was the most effective inhibitor of multiplication. Growth inhibition was reversed by the addition of polyamines. The inhibitors did not induce differentiation by themselves although DFMO caused encystment when supplemented with CaC1₂ or MgSO₄.