J. Peter Gergen

The isolation and characterization of Drosophila yolk protein genes

We have isolated recombinant DNA clones that contain the genomic sequences coding for the three most abundant proteins in Drosophila eggs, the yolk proteins (YP1, YP2 and YP3). The identity of these cloned genes was established by a two-step procedure. We used the genes to isolate complementary mRNA from total Drosophila RNA; we than showed the in vitro translation products of the isolated mRNAs to be the yolk proteins by comparing their protease digestion products to those of yolk proteins isolated from eggs. An examination of these isolated genes and of their DNA complements in the Drosophila genome showed that each of the three coordinately expressed yolk proteins is encoded by a different single-copy gene. Three genes were cloned; each has a different pattern of restriction endonuclease sites and each appears to be homologous to a different yolk protein mRNA. Southern transfer blots demonstrated that there is only one copy of each gene in the Drosophila genome. Our structural studies have shown that these three genes are not adjacent. In situ hybridization to polytene chromosomes demonstrated, in fact, that the YP3 gene is approximately 1000 kb from the closely spaced YP1 and YP2 genes. Thus, if the coordinate synthesis of the yolk proteins is due to transcriptional control, there must be coordinate control of initiation at two distant sites.

DNA-binding by Ig-fold proteins.

Recent structures of the Runx1–CBFβ heterodimer bound to DNA brings to six the number of characterized eukaryotic transcription factor families that use an immunoglobulin (Ig) fold to bind to DNA. Variation in the loop regions accounts for the diversity of DNA sequences recognized by Ig-fold proteins, but there are recurring themes in the interactions made by specific loop regions and how these interactions are regulated.

The localized requirements for a gene affecting segmentation in Drosophila: Analysis of larvae mosaic for runt☆

The runt gene is required in a developing Drosophila embryo for proper segmentation. Mutant embryos fail to hatch but secrete a larval cuticle in which pattern defects are apparent. In runt embryos, there are pattern deletions spaced at two segment intervals along the antero-posterior axis of the animal. The deleted regions are replaced by mirror-image duplications of the remaining regions. This paper investigates the localized requirements for runt+ activity by analyzing the segmentation patterns in larval genetic mosaics. This analysis is aided by the faintoid and shavenbaby mutations which affect larval cuticle morphology without affecting segmentation. These two mutations serve as markers of the regions of larval cuticle secreted by genetically runt cells. The analysis of the runt mosaic patterns indicates the effects of the gene on segmentation are primarily cell autonomous. This includes both the pattern deletions and the associated mirror-image duplications. This indicates the mirror-image duplications are not due to regeneration but result from a more direct effect of runt on patterning in the embryo. The mosaic patterns also reveal other aspects of the process of pattern formation in the larval epidermis. Based on these results a model is presented for the generation of the larval pattern.

Dosage requirements for runt in the segmentation of Drosophila embryos

The runt gene is required in a Drosophila embryo for normal segmentation. We investigate this requirement by analyzing runt mutations of varying strength and by manipulating wild-type gene dosage. Elimination of runt causes periodic deletions in the segmentation pattern which are spaced at two segment intervals along the antero-posterior axis. The pattern deletions produced by partial loss of function mutations and by halving the normal wild-type gene dosage reveal a gradation in the requirement for runt, with the centers of the affected regions being most sensitive to deletion. Significantly, increased runt+ dosage causes an anti-runt phenotype consisting of periodic pattern deletions that are out of phase with those caused by runt mutations.

A novel functional activator of the Drosophila JAK/STAT pathway, unpaired2, is revealed by an in vivo reporter of pathway activation

Striking similarities continue to emerge between the mammalian and Drosophila JAK/STAT signaling pathway. However, until now there has not been the ability to monitor global pathway activity during development. We have generated a transgenic animal with a JAK/STAT responsive reporter gene that can be used to monitor pathway activation in whole Drosophila embryos. Expression of the lacZ reporter regulated by STAT92E binding sites can be detected throughout embryogenesis, and is responsive to the Janus Kinase hopscotch and the ligand upd. The system has enabled us to identify the effect of a predicted gene related to upd, designated upd2, whose expression initiates during germ band extension. The stimulatory effect of upd2 on the JAK/STAT reporter can also be demonstrated in Drosophila tissue culture cells. This reporter system will benefit future investigations of JAK/STAT signaling modulators both in whole animals and tissue culture.

Localized requirements for gene activity in segmentation of Drosophila embryos: analysis of armadillo, fused, giant and unpaired mutations in mosaic embryos

SummaryIn order to investigate the localized requirements for the activity of genes that are required in a Drosophila embryo for segmentation, we have analyzed the patterns in genetic mosaics. In this paper, we describe our results with four different X-chromosome linked segmentation loci: armadillo, fused, giant and unpaired. For each locus, we first describe in detail the cuticle phenotype of mutant embryos. We then describe the segmentation patterns in embryos mosaic for these mutations, in each case utilizing the shavenbaby (svb) larval cuticle marker mutation to identify the regions of pattern made by genetically mutant cells. For all four loci, we can identify embryos containing large regions of both mutant and wildtype pattern. In these mosaics the regions of mutant pattern are marked with svb and the genetically wildtype (svb+) cells make wildtype pattern. The interpretations of the patterns in embryos mosaic for fused and unpaired are complicated by the variability of the phenotypes. However, after taking these complications into account, our principal conclusion is that the requirement for embryonic gene activity seems to be primarily cell autonomous. Based on the descriptions of the mutant phenotypes of these four loci and the analysis of the mosaics, we speculate on the possible roles these genes play in the process of segmentation.

Distinct in vivo requirements for establishment versus maintenance of transcriptional repression

Low-level ectopic expression of the Runt transcription factor blocks activation of the Drosophila melanogaster segmentation gene engrailed (en) in odd-numbered parasegments and is associated with a lethal phenotype. Here we show, by using a genetic screen for maternal factors that contribute in a dose-dependent fashion to Runt-mediated repression, that there are two distinct steps in the repression of en by Runt. The initial establishment of repression is sensitive to the dosage of the zinc-finger transcription factor Tramtrack. By contrast, the co-repressor proteins Groucho and dCtBP, and the histone deacetylase Rpd3, do not affect establishment but instead maintain repression after the blastoderm stage. The distinction between establishment and maintenance is confirmed by experiments with Runt derivatives that are impaired specifically for either co-repressor interaction or DNA binding. Other transcription factors can also establish repression in Rpd3-deficient embryos, which indicates that the distinction between establishment and maintenance may be a general feature of eukaryotic transcriptional repression.

Regulation of phototransduction responsiveness and retinal degeneration by a phospholipase D–generated signaling lipid

Drosophila melanogaster phototransduction proceeds via a phospholipase C (PLC)–triggered cascade of phosphatidylinositol (PI) lipid modifications, many steps of which remain undefined. We describe the involvement of the lipid phosphatidic acid and the enzyme that generates it, phospholipase D (Pld), in this process. Pld null flies exhibit decreased light sensitivity as well as a heightened susceptibility to retinal degeneration. Pld overexpression rescues flies lacking PLC from light-induced, metarhodopsin-mediated degeneration and restores visual signaling in flies lacking the PI transfer protein, which is a key player in the replenishment of the PI 4,5-bisphosphate (PIP2) substrate used by PLC to transduce light stimuli into neurological signals. Altogether, these findings suggest that Pld facilitates phototransduction by maintaining adequate levels of PIP2 and by protecting the visual system from metarhodopsin-induced, low light degeneration.

Ftz modulates Runt-dependent activation and repression of segment-polarity gene transcription.

A crucial step in generating the segmented body plan in Drosophila is establishing stripes of expression of several key segment-polarity genes, one stripe for each parasegment, in the blastoderm stage embryo. It is well established that these patterns are generated in response to regulation by the transcription factors encoded by the pair-rule segmentation genes. However, the full set of positional cues that drive expression in either the odd- or even-numbered parasegments has not been defined for any of the segment-polarity genes. Among the complications for dissecting the pair-rule to segment-polarity transition are the regulatory interactions between the different pair-rule genes. We have used an ectopic expression system that allows for quantitative manipulation of expression levels to probe the role of the primary pair-rule transcription factor Runt in segment-polarity gene regulation. These experiments identify sloppy paired 1 (slp1) as a gene that is activated and repressed by Runt in a simple combinatorial parasegment-dependent manner. The combination of Runt and Odd-paired (Opa) is both necessary and sufficient for slp1 activation in all somatic blastoderm nuclei that do not express the Fushi tarazu (Ftz) transcription factor. By contrast, the specific combination of Runt + Ftz is sufficient for slp1 repression in all blastoderm nuclei. We furthermore find that Ftz modulates the Runt-dependent regulation of the segment-polarity genes wingless (wg) and engrailed (en). However, in the case of en the combination of Runt + Ftz gives activation. The contrasting responses of different downstream targets to Runt in the presence or absence of Ftz is thus central to the combinatorial logic of the pair-rule to segment-polarity transition. The unique and simple rules for slp1 regulation make this an attractive target for dissecting the molecular mechanisms of Runt-dependent regulation.

Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster

Background Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. Principal Findings In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of ∼74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative “catalytic-inactive” mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. Conclusion Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.