Developmental control of transduced dopa decarboxylase genes in D. melanogaster

Summary Seventeen new euchromatic integration sites of the dopa-decarboxylase gene (Ddc) have been generated using p-mediated transduction. The developmental expression of the integrated genes was examined by monitoring the embryonic induction of dopa decarboxylase enzyme activity (DDC) and by monitoring the developmental pattern of DDC activity from late third instar to eclosion. The majority of inserts are regulated correctly within about 30% of controls. Several cases of multiple insertion events were recovered and these show correspondingly elevated levels of activity and are regulated normally. The pattern of expression of one insert (15C) falls outside the normal range. Multiple copies of transduced Ddc genes are used to test for effects of elevated gene dose on levels of expression. One insert on the X chromosome shows little or no dosage compensation. Possible reasons for the differences between the regulation of transduced genes in Drosophila and the regulation of transformed genes in mammalian systems are discussed.     

Functional analysis of a naturally occurring variant dopa decarboxylase gene in Drosophila melanogaster using P element mediated germ line transformation

Summary Expression of the gene which encodes dopa decarboxylase in Drosophila melanogaster (Ddc) is temporally controlled. The variant strain Ddc ⁺⁴ shows an altered pattern of enzyme activity during development compared with the standard Canton S laboratory strain. An examination of the DNA sequences which might control the expression of the variant gene was undertaken by reintroducing a cloned genomic fragment containing Ddc ⁺⁴ into Drosophila via P element mediated genetic transformation. The analogous fragment from the Canton S strain was reintroduced as a control. Despite a generally reduced expression in one transformed line, all the reintegated Ddc alleles revealed temporal patterns of Ddc expression characteristic of the strain from which the transforming DNA had originally been derived. Thus, we conclude that the essential information of the variant Ddc ⁺⁴ phenotype was included on a fragment which extended 2.9 kb upstream of the cap site for Ddc mRNA and 0.9 kb downstream of the poly(A) addition site.     

Ddcde1, a Mutant Differentially Affecting Both Stage and Tissue Specific Expression of Dopa Decarboxylase in Drosophila

The isolation and characterization of a unique Dopa decarboxylase (Ddc) mutant in Drosophila melanogaster is reported. This mutant, DdcDE1, exhibits stage- and tissue-specific altered Ddc expression. Homozygous DdcDE1 embryos, central nervous systems (CNSs) at pupariation and newly eclosed adult epidermis all have approximately 5% as much specific dopa decarboxylase (DDC) activity as the pr control stock in which DdcDE1 was induced. In contrast, the DdcDE1 epidermis at pupariation has roughly 50% as much DDC activity as controls, a 10-fold increase over the relative activity detected in other tissues and stages. Although the adult cuticle lacks proper pigmentation as expected in flies with low DDC activity (less than or equal to 5%), the bristles unexpectedly have wild-type black pigmentation. This implies that the bristle forming cells have more DDC activity than the rest of the adult epidermis. This variegated phenotype, black bristles and pale cuticle, plus the fact that DdcDE1 was originally isolated in a reciprocal translocation between proximal X heterochromatin and the euchromatic left arm of the second chromosome, 42 bands from the Ddc locus, suggested that the mutant might be an example of position-effect variegation. All tests for position-effect variegation, including persistence of the mutant phenotype when DdcDE1 was removed from the translocation, were negative. At pupariation DDC cross-reacting material (CRM) levels are similar in DdcDE1 and wild-type controls, but in newly eclosed adults CRM levels are approximately 35% of wild-type controls. This suggests that DDC produced by DdcDE1 adults has less activity per DDC molecule than the DDC produced at pupariation by DdcDE1. If the DDC enzyme produced by DdcDE1 at adult eclosion had full DDC activity (35% DDC CRM = 35% DDC activity) then no mutant phenotype would be exhibited by DdcDE1 since flies with as little as 10% activity have a wild-type phenotype. DDC thermolability assays clearly demonstrate that DDC from DdcDE1 is more thermolabile than control DDC at both pupariation and adult eclosion. Furthermore, DDC from adults in both DdcDE1 and the pr control is more thermolabile than DDC from white prepupae. Mixing experiments indicate the difference in DDC thermolability between pr white prepupae and pr adults is not due to a difference in the white prepupal and adult supernatants. This suggests that in wild-type different isoforms of DDC are produced either by differences in post-translational modification or as a result of a different primary amino acid sequence.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Genetics of Dopa Decarboxylase in DROSOPHILA MELANOGASTER . II. Isolation and Characterization of Dopa-Decarboxylase-Deficient Mutants and Their Relationship to the α-Methyl-Dopa-Hypersensitive Mutants

Of 84 lethals isolated over the dopa decarboxylase (DDC) deficiency Df(2L)50, 8 have been identified as DDC-deficient alleles on the basis of their effect on DDC activity when heterozygous over the CyO balancer chromosome with activities ranging from 28% to 53% of controls. Some of the Ddc-deficient alleles exhibit intracistronic complementation. Most of the complementing pairs of alleles are much reduced in viability, e.g. < 5% of expected, and express a common syndrome of mutant phenes which can reasonably be inferred to derive from inadequately sclerotinized cuticle. Individuals heterozygous for the noncomplementing allele, Ddcⁿ⁷, over the 12-band DDC deficiency, Df(2L)130, die at the end of embryogenesis as unhatched larvae with unpigmented mouth parts.

The Ddc alleles and the l(2)amd α-methyl dopa (αMD) hypersensitive alleles are both located within the 11 band region 37B10-C7. The l(2)amd locus is immediately to the right of hk(2–53.9).Ddc has been mapped within 0.004 Map Units to the right of l(2)amd with a maximum estimated recombination frequency of 0.01%. None of the Ddc/CyO strains are sensitive to the dietary administration of α-methyl dopa (αMD), and complementation occurs between the Ddc deficient alleles and the l(2)amd alleles both on the basis of viability and DDC activity. No effect on DDC by the amd alleles has been found to date. Even in the complementing heterozygote, amd^H1/amd ^H89, the level of activity, thermostability, and in vitro αMD inhibition of DDC remains unaffected. Although no biochemical phene has yet been established for the αMD hypersensitive amd alleles, it seems likely that the two groups of mutants are functionally related.

     

The genetics of dopa decarboxylase in Drosophila melanogaster

Summary Data on 46 mutations in the structural gene, Ddc. for dopa decarboxylase and 33 mutations in the methyl dopa hypersensitive gene, 1(2)amd, in Drosophila melanogaster are presented including information on their isolation, their effects on DDC activity, and their sensitivity to dietary methyl dopa. Intragenic complementation of both loci is documented, the effects of heteroallelic complementing heterozygosity on DDC activity, in vitro thermolability of DDC, and on temperature sensitive viability are presented. Data are marshalled to support rejection of the hypothesis that Ddc mutations and 1(2)amd, mutations are lesions in a single gene.     

Dopa decarboxylase from Drosophila melanogaster

Summary Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to -methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.     

The genetics of dopa decarboxylase in Drosophila melanogaster

Of 204 mutations located in the 8–12 band Df(2L)130 region, 37B9-C1,2;37D1-2, 199 have been assigned to twelve lethal genes and one visible gene (hook). The 13 genes are not evenly distributed. Twelve, (possibly all thirteen) are in the seven band region 37B10-C4 giving a gene-to-band ratio of almost two. Only one gene, 1(2)37Cf, may be in the four band region 37C5-7, and none are localized in band 37D1. In situ hybridization places the dopa decarboxylase structural gene, Ddc, in or very close to band 37C1,2 (Hirsh and Davidson, 1981). The methyl dopa hypersensitive gene, 1(2) amd, is 0.002 map units distal to Ddc. Df(2L)VA17, 37C1,2; 37F5-38A1 may actually break in the 37C1,2 singlet. It places six genes, hook, 1(2)amd, and four lethal genes, in a maximum of five bands, 37B10, 11, 12, 13 and perhaps part of the 37C1,2 singlet and localizes six genes, Ddc plus five lethal genes, in a maximum of three bands; probably part of the 37C1,2 singlet plus bands, C3, and C4. Wild type activity of five of twelve lethal genes is necessary for female fertility. — Band 37C5 puffs at the time of pupariation; Puff Stages 8–10. Twelve of eighteen alleles of 1(2)37Cf havs been examined as heterozygotes over CyO and none affect the appearance of a homozygous 37C5 puff. — Of the 204 mutations considered here only one Ddc ^p1, affects the function of more than one gene. It eliminates Ddc ⁺ and l(2) 37Ca ⁺ function and at 30 ° C reduces l(2)37Ce ⁺ function. It is not a deficiency but could be a polar mutant.Prof. Beermann's co-authors are very pleased to dedicate this paper to him in honor of his sixtieth birthday and in recognition of his seminal, most significant, extensive, and authoritive contributions on the functional organization of chromosomes     

The genetics of dopa decarboxylase in Drosophila melanogaster. III. Effects of a temperature sensitive dopa decarboxylase deficient mutation on female fertility

The mutation Ddc^ts1 effects female sterility when homozygous, hemizygous, or heterozygous over a series of Ddc null alleles (Ddc^x) indicating that some aspect of Ddc gene function is necessary for female fertility. Ovary transplant experiments demonstrate that the female sterility phenotype is ovary autonomous. Two to 3% of the total DDC activity measurable in newly hatched females is localized in their previtellogenic ovaries. The degree to which females heterozygous for Ddc^ts1 over different Ddc null alleles are fertile at 22°C reflects a continuous spectrum of allelic complementation similar to that observed for the effects of these genotypes on viability at 30°C. Fertility of all the Ddc^ts1/Ddc^x females tested is significantly depressed at 30 vis-a-vis 22°C providing evidence that it is the DDC enzyme activity itself which is required for female fertility. Ddc^ts1/Ddc^ts1 homozygous and Ddc^ts1/Df hemizygous females are nonconditionally, completely sterile at 18, 20, 22, 25, and 30°C. Although all homo- and hemizygous females do lay some eggs, no evidence of embryogenesis or fertilization has ever been detected. The absolute, nonconditional sterility of Ddc^ts1 homo- and hemizygous females stands in stark contrast to the conventional temperature dependent effects of these same genotypes on viability and to the temperature sensitive effects of Ddc^ts1/Ddc^x heterozygous females on both fertility and viability. Reasons for these tissue-specific and genotypic differences are 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.     

Control of Dopa decarboxylase gene expression by the Broad-Complex during metamorphosis in Drosophila

The induction of the Dopa decarboxylase gene (Ddc) in the epidermis of Drosophila at pupariation is a receptor-mediated response to the steroid molting hormone, ecdysone. Activity is also dependent on the Broad-Complex (BR-C), an early ecdysone response gene that functions during metamorphosis. BR-C encodes a family of zinc-finger protein isoforms, BR-C(Z1-Z4). Genetic experiments have shown that the Z2 isoform is required for epidermal Ddc to reach maximum expression at pupariation. In this paper, we report that BR-C regulates Ddc expression at two different developmental stages through two different cis-acting regions. At pupariation, BR-C acts synergistically with the ecdysone receptor to up-regulate Ddc. DNase I foot printing has identified four binding sites of the predominant Z2 isoform within a distal regulatory element that is required for maximal Ddc activity. The sites share a conserved core sequence with a set of BR-C sites that had been mapped previously to within the first Ddc intron. Using variously deleted Ddc genomic regions to drive reporter gene expression in transgenic organisms, we show that the intronic binding sites are required for Ddc expression at eclosion. At both pupariation and eclosion, BR-C releases Ddc from an active silencing mechanism, operating through two distinct cis-acting regions of the Ddc genomic domain at these stages. Transgenes, bearing a Ddc fragment from which one of the cis-acting silencers has been deleted, exhibit beta-galactosidase reporter activity in the epidermal cells prior to the appearance of endogenous DDC. Our finding that BR-C is required for Ddc activation at eclosion is the first evidence to suggest that this important regulator of the early metamorphic events, also regulates target gene expression at the end of metamorphosis.     

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.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.     

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.     

Dopa decarboxylase activity in Aedes aegypti: A preadult profile and its subsequent correlation with ovarian development

Dopa decarboxylase activity was monitored throughout the entire life cycle of Aedes aegypti. Peaks of activity were detected at each larval molt, at the larval-pupal ecdysis, and at eclosion. The dopa decarboxylase activity in adults was high right after eclosion, but it then dropped rapidly and after 5 days very little activity was detectable. This activity, however, was persistent and remained essentially constant, albeit low, for up to 15 days of adult life. Throughout this part of the study no sex differences in enzymatic activity were observed.A dramatic increase in the level of dopa decarboxylase was noted after adult females were allowed to blood feed. Since a blood meal is necessary in order to initiate ovarian development in this species and since the rate of increase of enzymatic activity paralleled oocyte maturation a causal relationship was indicated. Specifically, we suggest that the dopa decarboxylase is incorporated into the eggs to be used later for subsequent sclerotization.Injection of the molting hormone β-ecdysone into non-blood fed females resulted in a marked stimulation of dopa decarboxylase activity. No such stimulation was observed in saline-injected adult females. The adult female enzymatic activity profile obtained with time after hormone injection was qualitatively the same as that seen after a blood meal. The possibility that ecdysone or an ecdysonelike hormone is necessary for normal ovarian development in Aedes aegypti is discussed.     

Developmental expression and spatial distribution of dopa decarboxylase in Drosophila

Regulation of the dopa decarboxylase gene of Drosophila has been studied at the genetic and molecular levels. Here we report a direct assay for the tissue and temporal regulation of Ddc. A dopa decarboxylase (DDC) peptide was obtained by bacterial expression of a portion of the DDC gene in a pUC plasmid. Antisera raised against this biologically purified DDC peptide react specifically with Drosophila DDC in histological preparations and protein blots. The levels of DDC cross-reacting material closely parallel the levels of enzyme activity observed during development, indicating that DDC is degraded during periods of declining activity. We find that DDC is expressed in only two tissues, namely, the epidermis and the nervous system of the larva and adult. Epidermal DDC was found within the epidermal cells and was not detected in the overlying cuticle. DDC-containing neurons were observed in the central as well as in the visceral nervous system. Paired and unpaired midline neurons in the ventral ganglia are arranged in a segmental pattern. A subset of the DDC-positive neurons appears to correlate with the serotonin-positive neurons suggesting that the others are producing only dopamine. We find that the DDC activity associated with the proventriculus and ovary is due to the presence of DDC in the stomatogastric and caudal system neurons specifically associated with those structures.     

Nucleotide variation at the dopa decarboxylase (<Emphasis Type="Italic">Ddc</Emphasis>) gene in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We studied nucleotide sequence variation at the gene coding for dopa decarboxylase (Ddc) in seven populations of Drosophila melanogaster. Strength and pattern of linkage disequilibrium are somewhat distinct in the extensively sampled Spanish and Raleigh populations. In the Spanish population, a few sites are in strong positive association, whereas a large number of sites in the Raleigh population are associated nonrandomly but the association is not strong. Linkage disequilibrium analysis shows presence of two groups of haplotypes in the populations, each of which is fairly diverged, suggesting epistasis or inversion polymorphism. There is evidence of two forms of natural selection acting on Ddc. The McDonald-Kreitman test indicates a deficit of fixed amino acid differences between D. melanogaster and D. simulans, which may be due to negative selection. An excess of derived alleles at high frequency, significant according to the H-test, is consistent with the effect of hitchhiking. The hitchhiking may have been caused by directional selection downstream of the locus studied, as suggested by a gradual decrease of the polymorphism-to-divergence ratio. Altogether, the Ddc locus exhibits a complicated pattern of variation apparently due to several evolutionary forces. Such a complex pattern may be a result of an unusually high density of functionally important genes.     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage