Ross B. Hodgetts

Ecdysone titers during postembryonic development of Drosophila melanogaster

Abstract The level of ecdysone in Drosophila melanogaster was determined by a radioimmune assay in organisms selected between the second larval instar and maturity. Maxima in the titer of the hormone were observed at puparium formation and 38 hr later, just prior to the secretion of the adult cuticle. The level of ecdysone was very low in adults of either sex. However, adult females had significantly more ecdysone per organism than did males. The magnitude of this difference could be correlated with ovarian development, suggesting a possible role for ecdysone in ovarian maturation in this organism.

Effects of reforestation methods on genetic diversity of lodgepole pine: an assessment using microsatellite and randomly amplified polymorphic DNA markers

Abstract We examined the effects of different methods of forest regeneration on the genetic diversity of lodgepole pine (Pinus contorta var ‘latifolia’) using two different DNA-based molecular markers [randomly amplified polymorphic DNA (RAPDs) and microsatellites or simple sequence repeats (SSRs)]. Genetic diversity was estimated for 30 individuals in each of four populations for the following three stand types: (1) mature lodgepole pine (>100 years); (2) 20- to 30-year-old harvested stands left for natural regeneration; (3) 20- to 30-year-old planted stands (4 stands of each type); and one group of 30 operationally produced seedlings. There was no significant effect of stand type on expected heterozygosity, although allelic richness and diversity were much higher for SSRs than for RAPDs. Expected heterozygosity ranged from 0.39 to 0.47 based on RAPDs and from 0.67 to 0.77 based on SSRs. The number of alleles per locus for SSRs ranged from 3 to 34 (mean 21.0), and there was a significant relationship between sequence repeat length and the number of alleles at a locus. Both marker types showed that over 94% of the variation was contained within the populations and that the naturally regenerated stands sampled had lower (not significant) expected heterozygosity than the planted or unharvested stands. The group of seedlings (assessed by RAPDs only) had expected heterozygosity and allele frequencies similar to those of the unharvested stands. Genetic distance measures were higher than obtained previously in the species using isozyme markers. There was no correlation between the two marker types for pair-wise genetic distances based on populations analyzed by both methods. Pair-wise genetic distance measures and an ordination of allele frequencies for both marker types showed little effect of geographic location or stand type on genetic similarity.

Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony

Abstract Microinjection of radioactive tyrosine, dopa, and dopamine into mature larvae of Drosophila revealed that the sclerotization pathway is similar but not identical to that in Calliphora : (a) tyrosine is converted to tyrosine-o-phosphate and not to dopa, and (b) the substrate N-acetyldopamine does not accumulate. Larvae of the mutant ebony appear to be similar to the wild type with respect to tyrosine, dopa, and dopamine utilization. About the time of eclosion, however, ebony has twice as much dopamine as normal. Some implications of this are discussed with reference to the mutant phenotype.

A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster

A neuropeptide hormone-signalling pathway controls events surrounding eclosion in Drosophila melanogaster. Ecdysis-triggering hormone, eclosion hormone and crustacean cardioactive peptide (CCAP) together control pre-eclosion and eclosion events, whereas bursicon, through its receptor rickets (RK), controls post-eclosion development. Cuticular tanning is a convenient visible marker of the temporally precise post-eclosion developmental progression, and we investigated how it is controlled by the ecdysis neuropeptide cascade. Together, two enzymes, tyrosine hydroxylase (TH, encoded by ple) and dopa decarboxylase (DDC, encoded by Ddc), produce the dopamine that is required for tanning. Levels of both the ple and Ddc transcripts begin to accumulate before eclosion, coincident with the onset of pigmentation of the pharate adult bristles and epidermis. Since DDC activity is high before the post-eclosion onset of tanning, a different factor must be regulated to switch on tanning. Transcriptional control of ple does not regulate the onset of tanning because ple transcript levels remain unchanged from 24 hours before to 12 hours after eclosion. TH protein present before eclosion is degraded, and no TH activity can be detected at eclosion. However, TH protein rapidly accumulates within an hour of eclosion and we provide evidence that CCAP controls this process. Furthermore, we show that TH is transiently activated during tanning by phosphorylation at Ser32, as a result of bursicon signalling. We conclude that the ecdysis hormone cascade acts as a regulatory switch to control the precise onset of tanning by both translational and activational control of TH.

A characterization of dopamine acetyltransferase in Drosophila melanogaster

Dopamine acetyltransferase isolated from Drosophila melanogaster has a mol. wt of 2.9 × 104 daltons and readily acetylates both dopamine and tyramine. The enzyme activity reaches maximum levels at pupariation and during the adult stage. Unlike dopa decarboxylase, which precedes it in the sclerotization pathway, dopamine acetyltransferase is not found in the epidermal cells at pupariation. We have postulated the enzyme is present in the oenocytes of both the mature larva and adult, and it is found also in the adult nervous system. The data in this study provide no support for the existence of two acetyltransferases, one functioning in sclerotization and the other in the inactivation of neurogenic amines, although the criteria were not sufficiently rigorous to exclude this possibility.

Biochemical characterization of mutants affecting the metabolism of β-alanine in Drosophila

Abstract Microinjection of 1- 14 C- β -alanine into immobile larvae at spiracle eversion confirmed that this substance is utilized during tanning of the pupal case in Drosophila melanogaster . Two mutants, ebony and black , which fail to tan the pupal case were shown to be defective in the metabolism of β -alanine. Pharate pupae of ebony are able to manufacture the compound but unable to utilize it. Pharate pupae of black are unable to manufacture the substance, but can utilize β -alanine if it is supplied to them during the larval stage. The variation in the concentration of β -alanine throughout development was determined and a comparison between ebony and normal was made. Beta-alanine is normally manufactured at puparium formation and adult eclosion. Pharate pupae and young adults of ebony have excess amounts of β -alanine, because of the apparent inability of this mutant to utilize the substance. However, mature ebony adults have normal levels of β -alanine. Therefore, it is improbable that the abnormalities observed in thevisual behaviour of this mutant can be ascribed to excess β -alanine.

The characterization of DINE-1, a short, interspersed repetitive element present on chromosome and in the centric heterochromatin of Drosophila melanogaster.

Abstract.The banded portion of chromosome 4 (the ”dot” chromosome) in Drosophila melanogaster displays some properties of β-heterochromatin, which is normally found within the centric domain of the chromosomes. The nature and distribution of repetitive elements on chromosome 4 could play a role in the establishment of this unusual chromatin configuration. We describe here one such element: a short, interspersed repetitive sequence named DINE-1. Determination of a consensus sequence for the element reveals that there are two conserved regions (A and B) separated by a highly variable spacer. The conserved sequences are ∼400 bp long but degenerate at both ends, opening the possibility that a yet-to-be-discovered mother element may be present in the genome. DINE-1 bears few of the properties of the mammalian short interspersed elements (SINEs) to which it bears a superficial resemblance in size. It does not appear to be the product of reverse transcription and lacks any polymerase III promoter consensus. The elements are not flanked by target site duplications and their termini lack direct or inverted repeats, suggesting that they themselves are not transposable. Our analysis of cosmid clones from chromosome 4, and elsewhere in the genome, revealed that the euchromatic locations of DINE-1 are almost exclusively confined to chromosome 4. In situ hybridization of a DINE-1 probe to polytene chromosomes confirmed the preferential distribution along 4, in addition to its presence in the centric heterochromatin. This unusual genomic distribution of bias toward chromosome 4 is also seen in the sibling species, D. simulans, whose dot chromosomes exhibit poorly resolved polytene bands and lack crossing over during meiosis like those of D. melanogaster. However, the dot chromosome of D. virilis, which exhibits a well-defined banded structure on polytene chromosomes and can cross over, has only a single, discrete site of DINE-1 element hybridization. The presence of DINE-1 within these regions suggests a role in the heterochromatic nature of chromosome 4 in D. melanogaster and supports the contention that repeats accumulate in regions of diminished crossing over.

Dopa decarboxylase from Drosophila melanogaster

SummaryDopa 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 α-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.

Differential responses of the dopa decarboxylase gene to 20-OH-ecdysone in Drosophila melanogaster☆

The dopa decarboxylase gene (Ddc) of Drosophila melanogaster responds to 20-OH-ecdysone in the mature larval epidermis and in imaginal discs (presumptive adult epidermis) in a tissue-specific manner. Exposure of the mature larval epidermis to 20-OH-ecdysone caused a rapid accumulation of DDC transcripts. In the absence of protein synthesis, transcript accumulation was substantially reduced suggesting an indirect hormonal effect on DDC transcription (and/or RNA turnover). By contrast, neither DDC activity induction nor transcript accumulation was detected in imaginal discs cultured in the continuous presence of the hormone. However, when discs were exposed to 20-OH-ecdysone and then cultured in its absence, DDC activity and DDC transcript levels started to increase 6 hr after hormone withdrawal. A Northern analysis failed to reveal any novel transcripts in discs making the utilization of an alternative promotor an unlikely explanation for the very different responses of the Ddc gene in the two epidermal tissues. The results demonstrate that the Ddc gene in the larval epidermis responds rapidly to an increase in hormone titer. In imaginal discs a fall in hormone titer is required before DDC transcripts accumulate.

A Member of the p38 Mitogen-Activated Protein Kinase Family Is Responsible for Transcriptional Induction of Dopa decarboxylase in the Epidermis of Drosophila melanogaster during the Innate Immune Response†

ABSTRACT Drosophila innate immunity is controlled primarily by the activation of IMD (immune deficiency) or Toll signaling leading to the production of antimicrobial peptides (AMPs). IMD signaling also activates the JUN N-terminal kinase (JNK) cascade, which is responsible for immune induction of non-antimicrobial peptide immune gene transcription though the transcription factor AP-1. Transcription of the Dopa decarboxylase (Ddc) gene is induced in response to gram-negative and gram-positive septic injury, but not aseptic wounding. Transcription is induced throughout the epidermis and not specifically at the site of infection. Ddc transcripts are detectible within 2 h and remain high for several hours following infection with either gram-negative or gram-positive bacteria. Using Ddc-green fluorescent protein (GFP) reporter gene constructs, we show that a conserved consensus AP-1 binding site upstream of the Ddc transcription start site is required for induction. However, neither the Toll, IMD, nor JNK pathway is involved. Rather, Ddc transcription depends on a previously uncharacterized member of the p38 mitogen-activated protein kinase family, p38c. We propose that the involvement of DDC in a new pathway involved in Drosophila immunity increases the levels of dopamine, which is metabolized to produce reactive quinones that exert an antimicrobial effect on invading bacteria.