Analysis of the Drosophila melanogaster anti-ovarian response to honey bee queen mandibular pheromone.

Queen mandibular pheromone (QMP) is a potent reproductive signal to which honey bee workers respond by suppressing their ovaries and adopting alloparental roles within the colony. This anti-ovarian effect of QMP on workers can, surprisingly, be induced in other insects, including fruit flies, in which females exposed to synthetic QMP develop smaller ovaries with fewer eggs. In this study, we use the Drosophila melanogaster model to identify the components of synthetic QMP required for the anti-ovarian effect. We found that virgin females respond strongly to 9-oxo-2-decenoic acid and 10-hydroxy-2-decenoic acid (10HDA), suggesting that the decenoic acid components of QMP are essential for the anti-ovarian response. Further, a nuclear factor of activated T-cells reporter system revealed neurones expressing the olfactory receptors Or-56a, Or-49b and Or-98a are activated by QMP in the antenna. In addition, we used olfactory receptor GAL4 drivers and a neuronal activator (a neuronal activating bacterial sodium channel) to test whether the candidate neurones are potential labelled lines for a decenoic acid response. We identified Or-49b as a potential candidate receiver of the 10HDA signal. Finally, the anti-ovarian response to synthetic QMP is not mediated by decreasing the titre of the reproductive hormones ecdysone and juvenile hormone.

Increased abundance of frost mRNA during recovery from cold stress is not essential for cold tolerance in adult Drosophila melanogaster.

Frost (Fst) is a candidate gene associated with the response to cold in Drosophila melanogaster because Fst mRNA accumulation increases during recovery from low temperature exposure. We investigated the contribution of Fst expression to chill-coma recovery time, acute cold tolerance and rapid cold hardening (RCH) in adult D. melanogaster by knocking down Fst mRNA expression using GAL4/UAS-mediated RNA interference. In this experiment, four UAS-Fst and one tubulin-GAL4 lines were used. We predicted that if Fst is essential for cold tolerance phenotypes, flies with low Fst mRNA levels should be less cold tolerant than flies with normal levels of cold-induced Fst mRNA. Cold-induced Fst abundance and recovery time from chill-coma were not negatively correlated in male or female flies. Survival of 2 h exposures to sub-zero temperatures in Fst knockdown lines was not lower than that in a control line. Moreover, a low temperature pretreatment increased survival of severe cold exposure in flies regardless of Fst abundance level during recovery from cold stress, suggesting that Fst expression is not essential for RCH. Thus, cold-induced Fst accumulation is not essential for cold tolerance measured as chill-coma recovery time, survival to acute cold stress and RCH response in adult D. melanogaster.

The structure of the homeodomain and its functional implications.

The three-dimensional structure of the homeodomain, as determined by nuclear magnetic resonance spectroscopy, reveals the presence of a helix-turn-helix motif, similar to the one found in prokaryotic gene regulatory proteins. Isolated homeodomains bind with high affinity to specific DNA sequences. Thus, the structure-function relationship is highly conserved in evolution.

Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae.

A differential hybridization screen of a genomic yeast DNA library previously identified 14 genes of Saccharomyces cerevisiae that are expressed preferentially during sporulation. Three of these sporulation-specific genes, SPS1, SPS2, and SPS3, have been shown to be closely linked. A mutational analysis has demonstrated that expression of the SPS1 gene, but not the SPS2 gene, is essential for the completion of sporulation. A diploid MATa/MAT alpha strain homozygous for a disruption of the SPS1 gene failed to form asci when subjected to sporulation conditions. The 3' end of the transcript encoded by the SPS1 gene was found to map only 185 base pairs from the 5' end of the SPS2 gene. The SPS1-SPS2 intergenic region was shown to contain all of the regulatory sequences necessary for the sporulation-specific activation of the SPS2 gene as assessed by expression of a translational SPS2-lacZ fusion gene present on a replicating, centromere-containing plasmid. The fusion gene was found to be expressed at the same time during sporulation as the chromosomal wild-type SPS2 gene.

Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae.

We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

Isolation of DNA sequences preferentially expressed during sporulation in Saccharomyces cerevisiae.

A differential hybridization screen has been used to identify genes cloned from the yeast Saccharomyces cerevisiae that are expressed preferentially during sporulation. Duplicate copies of a partial Sau3A yeast DNA library prepared in the vector pBR322 were hybridized with radioactive cDNA probes representing the mRNA populations of sporulating a alpha cells and asporogenous alpha alpha cells at various times after transfer to sporulation medium. Thirty-eight clones showed an enhanced hybridization signal with the a alpha sporulation probe relative to the alpha alpha control cDNA probe. A comparison of the array of fragments produced by restriction endonuclease digestion of these plasmids suggested that 15 different sequences had been cloned. An RNA blot analysis using these cloned DNAs to probe RNAs purified from aa, a alpha, and alpha alpha cells harvested either during vegetative growth or at 10 h after transfer to sporulation medium indicated that 14 different sporulation-specific genes had been identified. Transcripts complementary to these genes are present only in a alpha cells after transfer to sporulation medium. Three of these clones contain two sporulation-specific genes. Three genes have been identified that are expressed in all cell types during vegetative growth and only in a alpha cells in sporulation medium.

Genetic characterization of the homeodomain-independent activity of the Drosophila fushi tarazu gene product.

The gene product of fushi tarazu (FTZ) has a homeodomain (HD)-independent activity. Ectopic expression of a FTZ protein that lacks half the HD in embryos results in the anti-ftz phenotype. We have characterized this FTZ HD-independent activity further. Ectopic expression of the HD-independent FTZ activity, in the absence of FTZ activity expressed from the endogenous ftz gene, was sufficient to result in the anti-ftz phenotype. Since the anti-ftz phenotype is a first instar larvae composed nearly entirely of FTZ-dependent cuticular structures derived from the even-numbered parasegments, this result suggests that expression of the HD-independent FTZ activity is sufficient to establish FTZ-dependent cuticle. Activation of FTZ-dependent Engrailed (EN) expression and activation of the ftz enhancer were HD-independent. The ftz enhancer element, AE-1, was activated by the HD-independent FTZ activity; however, the ftz enhancer element, AE-BS2CCC, which is the same as AE-1 except for the inactivation of two FTZ HD DNA-binding sites, was not. Activation of the ftz enhancer by ectopic expression of FTZ activity was effective only during gastrulation and germ band extension. In the discussion, we propose an explanation for these results.

Analysis in Drosophila melanogaster of the interaction between sex combs reduced and extradenticle activity in the determination of tarsus and arista identity.

Sex Combs Reduced (SCR) activity is proposed to be required cell nonautonomously for determination of tarsus identity, and Extradenticle (EXD) activity is required cell autonomously for determination of arista identity. Using the ability of Proboscipedia to inhibit the SCR activity required for determination of tarsus identity, we found that loss-of-EXD activity is epistatic to loss-of-SCR activity in tarsus vs. arista determination. This suggests that in the sequence leading to arista determination SCR activity is OFF while EXD activity is ON, and in the sequence leading to tarsus determination SCR activity is ON, which turns EXD activity OFF. Immunolocalization of EXD in early third-instar larval imaginal discs reveals that EXD is localized in the nuclei of antennal imaginal disc cells and localized in the cytoplasm of distal imaginal leg disc cells. We propose that EXD localized to the nucleus suppresses tarsus determination and activates arista determination. We further propose that in the mesodermal adepithelial cells of the leg imaginal discs, SCR is required for the synthesis of a tarsus-inducer that when secreted acts on the ectoderm cells inhibiting nuclear accumulation of EXD, such that tarsus determination is no longer suppressed and arista determination is no longer activated.

Analysis of murine HOXA-2 activity in Drosophila melanogaster.

The murine HOXA-2 protein shares amino acid sequence similarity with Drosophila Proboscipedia (PB). In this paper, we test whether HOXA-2 and PB are functionally equivalent in Drosophila. In Drosophila, PB inhibits SCR activity required for larval T1 beard formation and adult tarsus formation and is required for maxillary palp and proboscis formation. HOXA-2 expressed from a heat-shock promoter weakly suppressed SCR activity required for T1 beard formation. But interestingly neither PB nor HOXA-2 expressed from a heat-shock promoter suppressed murine HOXA-5 activity, the murine SCR homologue, from inducing ectopic T1 beards in T2 and T3, indicating that HOXA-5 does not interact with PB. HOXA-2 activity expressed from the Tubulin alpha 1 promoter modified the pb null phenotype resulting in a proboscis-to-arista transformation, indicating that HOXA-2 was able to suppress SCR activity required for tarsus formation. However, HOXA-2 expressed from a Tubulin alpha 1 promoter was unable to direct maxillary palp determination when either ectopically expressed in the antenna or in the maxillary palp primordia of a pb null mutant. HOXA-2 was also unable to rescue pseudotrachea formation in a pb null mutant. These results indicate that the only activity that PB and HOXA-2 weakly share is the inhibition of SCR activity, and that murine HOXA-5 and Drosophila SCR do not share inhibition by PB activity.

Analysis of Drosophila proboscipedia mutant alleles.

Proboscipedia (PB) is a HOX protein required for adult maxillary palp and proboscis formation. To identify domains of PB important for function, 21 pb point mutant alleles were sequenced. Twelve pb alleles had DNA sequence changes that encode an altered PB protein product. The DNA sequence changes of these 12 alleles fell into 2 categories: missense alleles that effect the PB homeodomain (HD), and nonsense or frameshift alleles that result in C-terminal truncations of the PB protein. The phenotypic analysis of the pb homeobox missense alleles suggests that the PB HD is required for maxillary palp and proboscis development and pb - Sex combs reduced (Scr) genetic interaction. The phenotypic analysis of the pb nonsense or frameshift alleles suggests that the C-terminus is an important region required for maxillary palp and proboscis development and pb-Scr genetic interaction. PB and SCR do not interact directly with one another in a co-immunoprecipitation assay and in a yeast two-hybrid analysis, which suggests the pb-Scr genetic interaction is not mediated by a direct interaction between PB and SCR.

Genetic characterization of the role of the two HOX proteins, Proboscipedia and Sex Combs Reduced, in determination of adult antennal, tarsal, maxillary palp and proboscis identities in Drosophila melanogaster.

Both Proboscipedia (PB) and Sex Combs Reduced (SCR) activities are required for determination of proboscis identity. Here we show that simultaneous removal of PB and SCR activity results in a proboscis-to-antenna transformation. Dominant negative PB molecules inhibit the activity of SCR indicating that PB and SCR interact in a multimeric protein complex in determination of proboscis identity. These data suggest that the expression pattern of PB and SCR and the ability of PB and SCR to interact in a multimeric complex control the determination of four adult structures. The absence of PB and SCR expression leads to antennal identity; expression of only PB leads to maxillary palp identity; expression of only SCR leads to tarsus identity; and expression of both PB and SCR, which results in the formation of a PB-SCR-containing complex, leads to proboscis identity. However, the PB-SCR interaction is not detectable in vitro and is not detectable genetically in the head region during embryogenesis, indicating the PB-SCR interaction may be regulated and indirect. This regulation may also explain why ectopic expression of SCR(Q50K) and SCR do not result in the expected transformation of the maxillary palp to an antennae and proboscis, respectively. Previous analysis of the requirements of SCR activity for adult pattern formation has shown that ectopic expression of SCR results in an antenna-to-tarsus transformation, but removal of SCR activity in a clone of cells does not result in a tarsus-to-arista transformation. Here we show in five independent assays the reason for this apparent contradictory requirement of SCR activity in tarsus determination. SCR activity is required cell nonautonomously for tarsus determination. Specifically, we propose that SCR activity is required in the mesodermal adepithelial cells of all leg imaginal discs at late second/early third instar larval stage for the synthesis of a mesoderm-specific, tarsus-inducing, signaling factor, which after secretion from the adepithelial cells acts on the overlaying ectodermal cells determining tarsus identity. This study characterizes a combinatorial interaction between two HOX proteins; a mechanism that may have a major role in patterning the anterior-posterior axis of other animals.

DNA binding properties of the purified Antennapedia homeodomain.

The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed. Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively. Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study. The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones. The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared.

The interaction with DNA of wild-type and mutant fushi tarazu homeodomains.

The in vitro DNA binding properties of wild-type and mutant fushi tarazu homeodomains (ftz HD) have been analysed. The DNA binding properties of the ftz HD are very similar to those of the Antp HD. In interference experiments with mutant ftz HDs, close approaches between specific portions of the ftz HD peptide and specific regions of the binding site DNA were mapped. A methylation interference, G7 on the beta strand of BS2, is absent from the interference pattern with a mutant ftz HD [ftz (R43A) HD] in which the Arg43 at the second position of helix III (the recognition helix) is replaced by an Ala. This indicated that Arg43 of the ftz HD is in close proximity to the N7 of G7 of the beta strand of BS2 in the major groove. The methylation and ethylation interference patterns with the ftz (NTD) HD, in which the first six amino acids of the homeodomain were deleted, were extensively altered relative to the ftz HD patterns. Methylation of A11 and G12 of the alpha strand and ethylation of the phosphate of nucleotide A12 of the alpha strand no longer interfere with binding. This indicated that the first six amino acids of the homeodomain of ftz interact with A11 of the alpha strand in the minor groove, the phosphate of the nucleotide A13 on the alpha strand and G12 of the alpha strand in the adjacent major groove of BS2. In a binding study using a change of specificity mutation [ftz (Q50K) HD], in which the Gln50 at the ninth position of the third helix is exchanged for a Lys (as in the bicoid HD), and variant binding sites, we concluded that position 50 of the ftz HD and the ftz (Q50K) HD peptides interacts with base pairs at positions 6 and 7 of BS2. These three points of contact allowed us to propose a crude orientation of the ftz HD within the protein-DNA complex. We find that the ftz HD and the Antp HD peptides contact DNA in a similar way.

Homeodomain-independent activity of the fushi tarazu polypeptide in Drosophila embryos.

The Drosophila segmentation gene fushi tarazu (ftz) encodes a homeodomain-containing protein, ftz, that can act as a DNA-binding activator of transcription. In the developing embryo, ftz is expressed in seven stripes which correspond to the even-numbered parasegments. These parasegments are missing in ftz- embryos. When ftz is expressed throughout blastoderm embryos under the control of a heat-shock promoter, the odd-numbered parasegments are lost. This 'anti-ftz' phenotype has been attributed to autoactivation of the endogenous ftz gene by the ectopically expressed protein. Here we show that the same phenotype is induced by ectopic expression of a ftz polypeptide containing a deletion in the homeodomain. Thus, ftz can alter gene expression without binding directly to DNA.

Glucagon inhibits phosphatidylcholine biosynthesis via the CDP-choline and transmethylation pathways in cultured rat hepatocytes.

The short-term influence of insulin and glucagon on phosphatidylcholine biosynthesis in monolayer cultures of rat hepatocytes was investigated. Under conditions in which insulin (100 nM) stimulated [3H]acetate incorporation into fatty acid almost twofold, synthesis of phosphatidylcholine via CDP-choline and from phosphatidylethanolamine were unaffected. By contrast, glucagon (100 nM), even in the presence of insulin (100 nM), reduced the rate of phosphatidylcholine formation from [Me-3H]phosphocholine by approximately 25% (p less than 0.05) within 1 h. Similarly, [3H]phosphatidylethanolamine incorporation into phosphatidylcholine was inhibited in cells exposed to glucagon. Insulin and glucagon had little or no effect on [Me-3H]choline uptake by the hepatocytes. No changes in the activities of the phosphatidylcholine biosynthetic enzymes in cytosol and microsomes from glucagon-treated cells could be detected.

FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs.

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.

The nuclear receptor homologue Ftz-F1 and the homeodomain protein Ftz are mutually dependent cofactors.

Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz), which encodes a homeodomain protein (Ftz), is required zygotically for the formation of alternate segments in the developing embryo. Here we show that the orphan nuclear receptor alphaFtz-F1 (ref. 3), which is deposited in the egg during oogenesis, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.