Genetic elements near the structural gene modulate the level of dopa decarboxylase during Drosophila development

Summary Measurement of dopa decarboxylase (DDC) levels in 109 strains of Drosophila melanogaster isogenic for second chromosomes isolated independently from natural populations was undertaken. One of the most extreme variants, designated Ddc ⁺⁴, was shown to have about 20% more DDC activity at adult eclosion than a standard laboratory strain used for comparison. The DDC overproduction was shown to segregate with the second chromosome and was mapped to a position within 0.15 map units of the DDC structural gene. The variant was shown to be an underproducer of DDC activity at pupariation and the genetic element responsible for this trait mapped in an identical fashion to that causing overproduction. The temporal phenotype described above was observed in the epidermis but DDC activity levels in neural tissue were normal. Examination of CRM levels at pupariation and eclosion revealed that altered DDC protein levels were responsible for the variant DDC activity levels. Electrophoretic analysis under both denaturing and non-denaturing conditions indicated that the DDC molecules in Ddc ⁺⁴ and the laboratory strain were indistinguishable. These results suggest that alterations in a genetic element (or elements) lying in close proximity to the structural gene are responsible for the complex temporal phenotype of DDC activity exhibited in the variant Ddc ⁺⁴.Abbreviations CRM cross-reacting material - DDC dopa decarboxylase - PTU phenylthiourea     

Regulation of dopa decarboxylase gene expression in the larval epidermis of the tobacco hornworm by 20-hydroxyecdysone and juvenile hormone

Dopa decarboxylase (DDC) which converts dopa to dopamine is important for cuticular melanization and sclerotization in insects. An antibody to Drosophila DDC was found to precipitate both DDC activity and a 49-kDa polypeptide synthesized by the epidermis of molting Manduca larvae. Using the Drosophila DDC gene, we isolated the Manduca DDC gene which on hybrid selection produced a 49-kDa translation product precipitable by the Drosophila DDC antibody. The 3.1-kb DDC mRNA appeared 12 hr after head capsule slippage (HCS) and reached maximal levels 7 hr later. Peak expression was twofold higher in melanizing allatectomized larvae and could be depressed to normal levels by application of 0.1 micrograms juvenile hormone I at HCS. Infusion of 1 microgram/hr 20-hydroxyecdysone (20-HE) for 18 hr beginning 2 hr after HCS or addition of 1 microgram/ml 20-HE to the culture medium for 24 hr prevented the normal increase in DDC mRNA. When Day 2 fourth instar epidermis was explanted before the molting ecdysteroid rise and cultured with 1-3 micrograms/ml 20-HE for 17 hr and then for 24 hr in hormone-free medium, DDC expression was three- to fourfold higher than that in epidermis cultured in the absence of hormone. Twelve or more hours of incubation with 20-HE was required for an increase in DDC mRNA, but continuous exposure to 20-HE prevented the increase. In all cultures an initial rapid increase in DDC mRNA was observed which decayed with time in vitro and apparently was associated with the wound response. Thus, ecdysteroid during a larval molt is necessary to program the later expression of DDC, but the subsequent decline of the ecdysteroid is required for this expression to occur.     

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.     

Human dopa decarboxylase: localization to human chromosome 7p11 and characterization of hepatic cDNAs.

We have cloned full-length DDC cDNAs from a human hepatoma cDNA library [DDC; dopa decarboxylase; aromatic-L-amino acid decarboxylase, EC 4.1.1.28]. The protein encoded by hepatoma cells is the same as that encoded by adrenal chromaffin derived pheochromocytoma cells, despite reported differences in biochemical properties. We have confirmed the location of the DDC gene to chromosome 7 using a new panel of somatic cell hybrids, and we have localized the gene to band p11 on chromosome 7 by fluorescent in situ hybridization. The human gene retains 65% amino acid identity with Drosophila DDC (Accession No. X04426) and considerable structural similarity with other enzymes (F.R. Jackson, 1990, J. Mol. Evol. 31:325-329, and references therein).     

Reduced cellular immune competence of a temperature-sensitive dopa decarboxylase mutant strain of Drosophila melanogaster against the parasite Leptopilina boulardi.

1. The melanotic encapsulation response made by larvae of a temperature-sensitive dopa decarboxylase (DDC) mutant strain of Drosophila against the parasitic wasp Leptopilina was severely compromised in hosts with reduced levels of DDC. 2. Dopa and 5,6-dihydroxyindole (DHI) were two hemolymph components identified in hosts exhibiting a melanotic encapsulation response. 3. This is the first study to implicate DDC in insect cellular immune responses, and to provide chemical evidence that the pigment formed during such responses is eumelanin derived from tyrosine.     

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.     

Immuno-cross-reactivity of histidine and dopa decarboxylases

Immuno-cross-reactivity between histidine decarboxylase (HDC) and dopa decarboxylase (DDC) was investigated. By comparing the cDNA sequences of rat HDC with rat and guinea-pig DDCs, we found a region that may possibly be related to the cross-reactivity of anti-rat HDC antibody with guinea-pig DDC. The peptide encoded by this region was synthesized and anti-peptide antibody was prepared. We also purified HDC and DDC homogeniously from fetal rat liver and guinea-pig liver, respectively. On immunoblotting, anti-peptide antibody recognized both rat HDC and guinea-pig DDC. Anti-HDC polyclonal antibody which also recognizes both enzymes detected only rat HDC when it was absorbed by the peptide. This result indicates that this region is responsible for the immuno-cross-reactivity of anti-rat HDC antibody with guinea-pig DDC.     

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.     

A kinetic analysis of Drosophila melanogaster dopa decarboxylase

The kinetic mechanism of dopa decarboxylase (3,4-dihydroxy-L-phenylalanine carboxy-lyase, EC 4.1.1.28) was investigated in Drosophila melanogaster. Based on initial velocity and product inhibition studies, an ordered reaction is proposed for dopa decarboxylase. This kinetic mechanism is interpreted in the context of measured enzyme activities and the catecholamine pools in Drosophila. The 1(2)amd gene is immediately adjacent to the gene coding for dopa decarboxylase (Ddc) and determines hypersensitivity to alpha-methyldopa in Drosophila. Dopa decarboxylase does not decarboxylate alpha-methyldopa and hence does not generate a toxic product capable of inhibiting 1(2)amd gene function. We propose that the 1(2)amd gene is involved with an unknown catecholamine pathway involving dopa but not dopamine.     

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)