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
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
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.