Maxwell J. Scott

Sexual Development in Lucilia cuprina (Diptera, Calliphoridae) Is Controlled by the Transformer Gene

Insects use an amazing variety of genetic systems to control sexual development. A Y-linked male determining gene (M) controls sex in the Australian sheep blowfly Lucilia cuprina, an important pest insect. In this study, we isolated the L. cuprina transformer (Lctra) and transformer2 (Lctra2) genes, which are potential targets of M. The LCTRA and LCTRA2 proteins are significantly more similar to homologs from tephritid insects than Drosophila. The Lctra transcript is alternatively spliced such that only females make a full-length protein and the presence of six TRA/TRA2 binding sites in the female first intron suggest that Lctra splicing is autoregulated as in tephritids. LCTRA is essential for female development as RNAi knockdown of Lctra mRNA leads to the development of male genitalia in XX adults. Analysis of Lctra expression during development shows that early and midstage male and female embryos express the female form of Lctra and males express only the male form by the first instar larval stage. Our results suggest that an autoregulatory loop sustains female development and that expression of M inhibits Lctra autoregulation, switching its splicing to the male form. The conservation of tra function and regulation in a Calliphorid insect shows that this sex determination system is not confined to Tephritidae. Isolation of these genes is an important step toward the development of a strain of L. cuprina suitable for a genetic control program.

MSL1 plays a central role in assembly of the MSL complex, essential for dosage compensation in Drosophila

In male Drosophila, histone H4 acetylated at Lys16 is enriched on the X chromosome, and most X‐linked genes are transcribed at a higher rate than in females (thus achieving dosage compensation). Five proteins, collectively called the MSLs, are required for dosage compensation and male viability. Here we show that one of these proteins, MSL1, interacts with three others, MSL2, MSL3 and MOF. The latter is a putative histone acetyl transferase. Overexpression of either the N‐ or C‐terminal domain of MSL1 has dominant‐negative effects, i.e. causes male‐specific lethality. The lethality due to expression of the N‐terminal domain is reduced if msl2 is co‐overexpressed. MSL2 co‐purifies over a FLAG affinity column with the tagged region of MSL1, and both MSL3 and MOF co‐purify with the FLAG‐tagged MSL1 C‐terminal domain. Furthermore, the MSL1 C‐terminal domain binds specifically to a GST–MOF fusion protein and co‐immunoprecipitates with HA‐tagged MSL3. The MSL1 C‐terminal domain shows similarity to a region of mouse CBP, a transcription co‐activator. We conclude that a main role of MSL1 is to serve as the backbone for assembly of the MSL complex.

Germ-line transformation of the Australian sheep blowfly Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina, is the most important economic insect pest for the sheep industries in Australia and New Zealand. piggyBac‐mediated germ‐line transformation of L. cuprina was achieved with a helper plasmid that had the Drosophila melanogaster hsp70 promoter controlling expression of the transposase and a piggyBac vector with an EGFP marker gene. Two transformant lines were obtained, at a frequency of approximately 1–2% per fertile G0. One of these lines has a single copy of the transgene, the other most likely has four copies. This is the first report of germ‐line transformation of L. cuprina and is an important step towards the generation of engineered strains that would be suitable for male‐only release eradication/suppression programmes.

The Amino-Terminal Region of Drosophila MSL1 Contains Basic, Glycine-Rich, and Leucine Zipper-Like Motifs That Promote X Chromosome Binding, Self-Association, and MSL2 Binding, Respectively

ABSTRACT In Drosophila melanogaster, X chromosome dosage compensation is achieved by doubling the transcription of most X-linked genes. The male-specific lethal (MSL) complex is required for this process and binds to hundreds of sites on the male X chromosome. The MSL1 protein is essential for X chromosome binding and serves as a central scaffold for MSL complex assembly. We find that the amino-terminal region of MSL1 binds to hundreds of sites on the X chromosome in normal males but only to approximately 30 high-affinity sites in the absence of endogenous MSL1. Binding to the high-affinity sites requires a basic motif at the amino terminus that is conserved among Drosophila species. X chromosome binding also requires a conserved leucine zipper-like motif that binds to MSL2. A glycine-rich motif between the basic and leucine-zipper-like motifs mediates MSL1 self-association in vitro and binding of the amino-terminal region of MSL1 to the MSL complex assembled on the male X chromosome. We propose that the basic region may mediate DNA binding and that the glycine-rich region may promote the association of MSL complexes to closely adjacent sites on the X chromosome.

Developmental plasticity and the evolution of parasitism in an unusual nematode, Parastrongyloides trichosuri

BackgroundParasitism is an important life history strategy in many metazoan taxa. This is particularly true of the Phylum Nematoda, in which parasitism has evolved independently at least nine times. The apparent ease with which parasitism has evolved amongst nematodes may, in part, be due to a feature of nematode development acting as a pre-adaptation for the transition from a free-living to a parasitic life history. One candidate pre-adaptive feature for evolution in terrestrial nematodes is the dauer larva, a developmentally arrested morph formed in response to environmental signals.ResultsWe investigated the role of dauer development in the nematode, Parastrongyloides trichosuri, which has retained a complete free-living life cycle in addition to a life cycle as a mammalian gastrointestinal parasite. We show that the developmental switch between these life histories is sensitive to the same environmental cues as dauer arrest in free-living nematodes, including sensitivity to a chemical cue produced by the free-living stages. Furthermore, we show that genetic variation for the sensitivity of the cue(s) exists in natural populations of P. trichosuri, such that we derived inbred lines that were largely insensitive to the cue and other lines that were supersensitive to the cue.ConclusionsFor this parasitic clade, and perhaps more widely in the phylum, the evolution of parasitism co-opted the dauer switch of a free-living ancestor. This lends direct support to the hypothesis that the switch to developmental arrest in the dauer larva acted as a pre-adaptation for the evolution of parasitism, and suggests that the sensory transduction machinery downstream of the cue may have been similarly co-opted and modified.

Incorporation of the noncoding roX RNAs alters the chromatin-binding specificity of the Drosophila MSL1/MSL2 complex.

ABSTRACT The male-specific lethal (MSL) protein-RNA complex is required for X chromosome dosage compensation in Drosophila melanogaster. The MSL2 and MSL1 proteins form a complex and are essential for X chromosome binding. In addition, the MSL complex must integrate at least one of the noncoding roX RNAs for normal X chromosome binding. Here we find the amino-terminal RING finger domain of MSL2 binds as a complex with MSL1 to the heterochromatic chromocenter and a few sites on the chromosome arms. This binding required the same amino-terminal basic motif of MSL1 previously shown to be essential for binding to high-affinity sites on the X chromosome. While the RING finger domain of MSL2 is sufficient to increase the expression of roX1 in females, activation of roX2 requires motifs in the carboxyl-terminal domain. Binding to hundreds of sites on the X chromosome and efficient incorporation of the roX RNAs into the MSL complex require proline-rich and basic motifs in the carboxyl-terminal domain of MSL2. We suggest that incorporation of the roX RNAs into the MSL complex alters the binding specificity of the chromatin-binding module formed by the amino-terminal domains of MSL1 and MSL2.

A de novo transcriptome assembly of Lucilia sericata (Diptera: Calliphoridae) with predicted alternative splices, single nucleotide polymorphisms and transcript expression estimates.

The blow fly Lucilia sericata (Diptera: Calliphoridae) (Meigen) is a nonmodel organism with no reference genome that is associated with numerous areas of research spanning the ecological, evolutionary, medical, veterinary and forensic sciences. To facilitate scientific discovery in this species, the transcriptome was assembled from more than six billion bases of Illumina and twenty‐one million bases of 454 sequence derived from embryonic, larval, pupal, adult and larval salivary gland libraries. The assembly was carried out in a manner that enabled identification of putative single nucleotide polymorphisms (SNPs) and alternative splices, and that provided expression estimates for various life history stages and for salivary tissue. The assembled transcriptome was also used to identify transcribed transposable elements in L. sericata. The results of this study will enable blow fly biologists, dipterists and comparative genomicists to more rapidly develop and test molecular and genetic hypotheses, especially those regarding blow fly development and salivary gland biology.

Organization and expression of the Australian sheep blowfly (Lucilia cuprina) hsp23, hsp24, hsp70 and hsp83 genes.

In this study we report the isolation and characterization of a heat shock protein 70 (hsp70) gene, the hsp83 gene and two genes that encode small Hsps (Lchsp23 and Lchsp24) from the Australian sheep blowfly, Lucilia cuprina, a major agricultural pest. Phylogenetic analyses indicate that the LcHsp23 protein is the orthologue of Drosophila melanogaster Hsp23 and LcHsp24 is the orthologue of Sarcophaga crassipalpis Hsp23. Quantitative reverse‐transcriptase PCR analysis showed that the basal level of Lchsp83 RNA is relatively high at all developmental stages and only moderately induced by heat shock. In contrast, Lchsp70 transcripts are present at low levels and strongly induced by heat shock at all stages. The basal levels of expression and degrees of heat induction of the Lchsp23 and Lchsp24 transcripts were more variable across the different developmental stages. Putative heat shock factor binding sites were identified in the Lchsp24, Lchsp70 and Lchsp83 gene promoters. The isolation of these hsp gene promoters will facilitate constitutive or conditional expression of a gene of interest in transgenic Lucilia.

piggyBac-mediated transposition in Drosophila melanogaster: an evaluation of the use of constitutive promoters to control transposase gene expression.

Transgenic non‐Drosophilid insects have been made using insect transposable elements that have a broad host range such as the piggyBac element. However, the success rate is often low. Previous piggyBac helper plasmids have used either the piggyBac or the hsp70 promoter from Drosophila melanogaster to control expression of the transposase gene. Here we show that plasmids with the piggyBac transposase gene regulated by constitutive promoters can be effective ‘helpers’ for mediating transposition in D. melanogaster. We also present preliminary evidence on the use of an RNA helper that encodes the transposase. Our results suggest that for germ‐line transformation of non‐Drosophilid insects it may be advantageous to isolate a constitutive promoter from the species of interest to control transposase expression.

Structural and Biochemical Studies on the Chromo-barrel Domain of Male Specific Lethal 3 (MSL3) Reveal a Binding Preference for Mono- or Dimethyllysine 20 on Histone H4

We have determined the human male specific lethal 3 (hMSL3) chromo-barrel domain structure by x-ray crystallography to a resolution of 2.5 Å (r = 0.226, Rfree = 0.270). hMSL3 contains a canonical methyllysine binding pocket made up of residues Tyr-31, Phe-56, Trp-59, and Trp-63. A six-residue insertion between strands β1 and β2 of the hMSL3 chromo-barrel domain directs the side chain of Glu-21 into the methyllysine binding pocket where it hydrogen bonds to the NH group of a bound cyclohexylamino ethanesulfonate buffer molecule, likely mimicking interactions with a histone tail dimethyllysine residue. In vitro binding studies revealed that both the human and Drosophila MSL3 chromo-barrel domains bind preferentially to peptides representing the mono or dimethyl isoform of lysine 20 on the histone H4 N-terminal tail (H4K20Me1 or H4K20Me2). Mutation of Tyr-31 to Ala in the hMSL3 methyllysine-binding cage resulted in weaker in vitro binding to H4K20Me1. The same mutation in the msl3 gene compromised male survival in Drosophila. Combined mutation of Glu-21 and Pro-22 to Ala in hMSL3 resulted in slightly weaker in vitro binding to H4K20Me1, but the corresponding msl3 mutation had no effect on male survival in Drosophila. We propose MSL3 plays an important role in targeting the male specific lethal complex to chromatin in both humans and flies by binding to H4K20Me1. Binding studies on the related dMRG15 chromo-barrel domain revealed that MRG15 prefers binding to H4K20Me3.