Gene Dmel\Ddc

Structural gene for dopa decarboxylase [DDC, 3-4-dihydroxy-L-phenylalanine-carboxylase (EC 4.1.28)] which catalyzes the decarboxylation of dopa to dopamine (Lunan and Mitchell, 1969, Arch. Biochem. Biophys. 132: 450-56) and 5-hydroxytryptophan to serotonin (5-hydroxytryptamine) but not tyrosine to tyramine (Livingstone and Tempel, 1983, Nature 303: 67-70). Native DDC isolated from mature larvae is a homodimer with subunit molecular weight 54 kd (Clark, Pass, Venkataraman, and Hodgetts, 1978, Mol. Gen. Genet 162: 287-97). Distinct DDC isoforms are generated in the CNS and hypoderm by alternate splicing of the Ddc primary transcript; the CNS isoform differs by the addition of 35 amino acids at the amino terminus (Morgan, Johnson, and Hirsh, 1986, EMBO J. 5: 3335-42). The predicted subunit molecular weights of these are 57.1 and 53.4 kd, respectively. DDC requires pyridoxal-5-phosphate for activity and is strongly inhibited by heavy-metal ions and the sulfhydryl reagent, N-ethylmaleimide. Initial velocity constants determined by Black and Smarrelli (1986, Biochim. Biophys. Acta 870: 31-40). The dopamine produced by DDC is necessary to effect sclerotization of the cuticle, being further metabolized both to N-acetyldopamine and N-β-alanyldopamine, which after oxidation to their respective quinones, crosslink cuticular proteins. Thus in adults and white prepupae more than 90% of the DDC activity is located in the epidermis (Lunan and Mitchell, 1969; Scholnick, Morgan, and Hirsh, 1983, Cell 34: 37-45). Some DDC activity (~5%) is found in the central nervous system of white prepupae and adults where it produces the neurotransmitters dopamine and serotonin [Wright, 1977, Amer. Zool. 17: 707-21; Livingstone and Tempel, 1983; White and Valles, 1985, Molecular Basis of Neural Development (Edelman, Gell, and Cowan (eds.). John Wiley and Sons, N.Y., pp 547-63]. The limited amounts found in the ovaries (Wright, Steward, Bentley and Adler, 1981, Dev. Genet. 2: 223-35) and proventriculus (Wright and Wright, Proc. Int. Congr. Genet., 15th, 1978, Part I, p. 615) are localized in associated neural ganglia (Konrad and Marsh, 1987, Dev. Biol. 122: 172-85). Five peaks of DDC activity evident during development: at the end of embryogenesis, the two larval molts, pupariation, and eclosion (Marsh and Wright, 1980, Dev. Biol. 80: 379-87; Kraminsky, Clark, Estelle, Gietz, Sage, O'Conner, and Hodgetts, 1980, Proc. Nat. Acad. Sci. USA 77: 4175-79). The largest peak, which occurs at pupariation, is induced by a coincident ecdysone peak of the molting larvae (Marsh and Wright, 1980) and has been shown to be attributable to a rapid increase in translatable DDC mRNA following administration of 20-0H-ecdysone (Kraminsky et al., 1980). Ecdysone induces Ddc expression in the mature larval epidermis within two to four hrs (Karminsky, et al., 1980; Clark, Doctor, Fristrom, and Hodgetts, 1986, Dev. Biol. 114: 141-50). Since cycloheximide addition is sufficient to largely abolish this induction, it appears that this response is an indirect action of ecdysone. A different response of Ddc to ecdysone occurs in cultured imaginal discs; Ddc induction occurs only subsequent to withdrawal of the hormone (Clark et al., 1986). Most mutations in Ddc are homozygous or hemizygous lethal. The effective lethal phases of the first eight lethal alleles, Ddcn1-Ddcn8, were almost identical. As hemizygotes over Df(2L)TW130 almost all mortality is late embryonic with actively moving larvae, exhibiting unpigmented cephalopharyngeal apparatuses and denticle belts, unable to hatch. When homozygous there is a fairly uniform shift in effective lethal phases with mean mortalities from all eight alleles in the cross of Ddcn/CyO x Ddcn/cn bw being 13.6% embryonic, 14.1% larval, and 4.8% pupal (Wright and Wright, 1978). Many larvae hemizygous for lethal alleles, or homozygous deficient for Ddc, when mechanically released from the egg membranes, continue development to the 3rd larval instar and to the pharate adult stage. Genotypes which produce individuals with drastically reduced DDC activities (~0.5-5% of wild type) exhibit an "escaper" phenotype characterized by incomplete pigmentation and sclerotization of the cuticle; developmental time can be prolonged for as many as four or five days; puparia are easily scored showing melanization at each end of the greenish-gray pupa case; adults often die or get stuck in the food within 24 hr of eclosion; macrochaetae may be very thin, long, and straw-colored or colorless; the whole body remains light, i.e., doesn't take on its normal pigmentation; abdominal markings are apparent but do not darken; upon aging a few hours wing axillae become melanized similar to the phenotype of sp, leg joints also become melanized perhaps due to the phenoloxidase wound reaction brought on by ruptures of weakened cuticle; flies walk on tibias rather than tarsi, but leg movements appear to be coordinated (Wright, Bewley, and Sherald, 1976). Genotypes that produce flies exhibiting the "escaper" phenotype include heteroallelic intragenic complementing heterozygotes with less than 5% of the expected number of survivors (Wright, Bewley, and Sherald, 1976), hemizygotes of the ts allele Ddcts2 raised continuously at 22 or 25, or homozygotes for Ddcts1 or Ddcts2 exposed to the restrictive temperature 30 for 24- or 48-hour pulses at the end of the pupal stage (Wright). Ddc temperature-sensitive mutants have been reported to show reduced learning after a three-day period at the restrictive temperature (Tempel, Livingstone, and Quinn, 1984, Proc Nat. Acad. Sci. USA 81: 3577-81). However, these results cannot presently be reproduced by other investigators (see Tully, 1987, Trends in Neurosci. 10: 330-35; Hirsh, 1989, Dev. Genet. 10: 232-38). It is possible that this lack of reproducibility is due to the accumulation of genetic modifiers. In homozygous deficient larvae normally-serotonin-containing neurons lack immunologically detectable serotonin but display normal levels of uptake of exogenously supplied serotonin (Valles and White, J. Neurosci. 6: 1482-91). Further studies of these Ddc- larvae, on which catecholamine histofluorescence studies were performed, revealed novel neuronal subsets lighting up, which become fluorogenic earlier than the wild-type-like neurons in the mutant CNS (Budnik, Martin-Morris, and White, 1986, J. Neurosci. 6: 1482-91). Certain serotonin-containing nerve fibers in developing larvae are still able to reach their normal targets in Ddc- animals (which therefore are intrinsically serotonin-minus), but there is anomalous extra branching associated with the incoming fibers (Budnik, Wu, and White, 1989, J. Neurosci. 9: 2866-77). Ddc mosaics generated by crossing a transduced Ddc+ insert into R(1)wvC (Gailey, Bordne, Valles, Hall, and White, 1987, Genetics 115: 305-11). Such adult mosaics used to reveal no absolute requirement of DDC in any particular portion of epidermis or CNS, but there was low recovery of gynandromorphs with large Ddc- patches. Larval mosaics show that DDC-positive neurons always contain serotonin, but some serotonin-positive cells (which were near DDC+) have no detectable enzyme protein; hence, the serotonin phenotype can be nonautonomous (Valles and White, 1990). In addition to the naturally occurring alleles, DdcRE, DdcRS, and Ddc+4, which are described separately, three surveys of natural populations for Ddc variants have been reported. Estelle and Hodgetts (1984, Mol. Gen. Genet. 195: 434-41) measured DDC levels in 109 strains isogenic for second chromosomes isolated independently by Bewley (1978, Biochem. Genet. 16: 769-75) from collections at Raleigh, NC, Bloomington, IN., and Webster Groves, MO. (WGM). Two (WGM) strains (including Ddc+4) had increased activities and two had reduced activities when compared with a Canton-S control. Marsh and Wright report DDC activities from twelve different wild-type strains maintained in laboratories for many years. Relative to Oregon-R (DdcC) females, they ranged from a low of 68% for Urbana males to a high for Canton-S females (180%) and males (130%) with most strains with activities between Oregon-R and Canton-S. Aquadro, Jennings, Bland, Laurie-Ahlberg, and Langley (1984, Genetics 107: s3) surveyed forty-six second chromosome lines isolated from five natural populations for restriction fragment variations in the 80kb region surrounding Ddc and for adult DDC activity. No consistent pattern of association between level of DDC activity and restriction site haplotype was apparent although the lines showed a two-fold variation in DDC activity. Two lines with 5kb and 1.5kb inserts within an intron and at the 5' end of Ddc showed normal adult DDC activities. The temperature-sensitive periods causing lethality for Ddcts2 homozygotes are primarily during embryogenesis and late in the third larval instar. Heat shocks, 30 for 24 or 48 hr, during metamorphosis do not increase lethality significantly but produce adults with the extreme "escaper" phenotype. DDC in extracts from adult Ddcts1 and Ddcts2 homozygotes is significantly more thermolabile than that from wild-type controls. DDC from Ddcts1/+ heterozygotes is much less labile showing a biphasic inactivation curve. Ddcts2/+ DDC is no more thermolabile than wild-type DDC (Wright, unpublished data). Genotypes with reduced levels of DDC activity, e.g. Ddcn5/Ddcn8 and Ddcn1/Ddcn8 with less than 4% DDC activity, are not more sensitive to dietary alpha methyl dopa nor are genotypes with increased levels of DDC activity more resistant (Marsh and Wright, 1986, Genetics 112: 249-65). In fact, the reverse may be true: reduced DDC, more resistant; increased DDC, more sensitive.

DDC activity and resistance to dietary alpha methyl dopa in the normal range for Oregon-R derived stocks. This strain was put through the same genetic manipulations as DdcRE and DdcRS so it could serve as a valid control for those strains.

Hemizygous adults (9% of expected eclose) exhibit an extreme "escaper" phenotype (see Ddc above) except macrochaetae are normally pigmented suggesting that DdcDE1 is differentially active in the epidermis vis-a-vis the bristle-forming cells. Pupa cases of DdcDE1 homo- and hemizygotes are wild type. DDC activity in newly eclosed adult DdcDE1 homozygotes is 4.4 _ 0.2% and in hemizygotes is 0.6 _ 0.1% of wild-type controls. Homozygous late embryos have 4.8 _ 2.3% activity. In striking contrast homozygous white prepupae have 46.5 _ 2.8% DDC activity. However, central nervous systems dissected from these DdcDE1 homozygous white prepupae show a tissue specific difference having 4.8 _ 2.3% DDC activity compared to wild-type CNS. Specific DDC activity in DdcDE1 homozygotes ranges significantly more than two times DDC levels in DdcDE1/Df(2L)TW130 hemizygotes. DDC from DdcDE1 homozygotes, crawling third instar larvae and adults, is less thermostable in vitro in comparison to controls. Late DdcDE1/DdcDE1 embryos (16-20 hr) have no detectable mature 2.0 kb Ddc RNA and have reduced levels of the 2.3 kb RNA. The precise reason for the differential expression has yet to be established but is not due to position effect variegation (Bishop and Wright, 1987, Genetics 115: 477-91). DdcDE1 phenotype rescued by a 7.5kb transformant of Ddc+ DNA.

Some hemizygous adults exhibit the incomplete sclerotization "escaper" phenotype. Not temperature sensitive: hemizygotes being equally viable at 18, 25, and 30 (40-56% of expected). DDC from Ddclo1 hemizygotes is not more thermolabile in vitro than that from wild type. Heterozygous Ddclo1/CyO have about 77% wild type specific DDC activity and Ddclo1 homozygotes have 15-30% activity.

Dual phenotype of elevated DDC activity and increased resistance to dietary alpha methyl dopa relative to Oregon-R derived controls (DdcC). Specific DDC activity of newly eclosed adults 158% and DDC crossreacting material (CRM) 156% of the DdcC control. LD50 for alpha methyl dopa is ~0.4 mM vs. ~0.2 mM for the DdcC control. Gene dosage studies with Ddc+ and l(2)amd+ demonstrate that increased resistance to alpha methyl dopa is not the result of increased DDC activity. Thus, the dual phenotype is inferred to arise from a coordinated increase in Ddc+ activity and l(2)amd+ activity produced either by accumulated changes in a genetic element (or elements) in the close proximity to the Ddc and amd genes.

Dual phenotype of elevated DDC activity and increased resistance to dietary and methyl dopa relative to Oregon-R derived controls (DdcC). Specific DDC activity of newly eclosed adults 141% and DDC crossreacting material (CRM) 137% of the DdcC control. Interpretation of phenotype identical to that for DdcRE.

No visible phenotype: Ddc+4 overproduces DDC activity at embryonic hatching, the second to third instar molt, and at adult eclosion relative to a Canton-S control: 141%, 150%, and 118% respectively; in contrast, underproduces DDC at pupariation: 50%. These temporal differences are found in epidermis but not in neural tissues where DDC activities are normal. DDC CRM at pupariation and adult eclosion are 49% and 140% respectively of Canton-S CRM. No difference was found in the electrophoretic mobility of non-denatured and denatured DDC molecules. DDC mRNA is 140%, 52%, and 148% of Canton-S at embryonic hatching, pupariation, and adult eclosion respectively indicating that the temporal phenotype is reflected in mRNA levels.