Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation.

Bonus, a Drosophila TIF1 homolog, is a nuclear receptor cofactor required for viability, molting, and numerous morphological events. Here we establish a role for Bonus in the modulation of chromatin structure. We show that weak loss-of-function alleles of bonus have a more deleterious effect on males than on females. This male-enhanced lethality is not due to a defect in dosage compensation or somatic sex differentiation, but to the presence of the Y chromosome. Additionally, we show that bonus acts as both an enhancer and a suppressor of position-effect variegation. By immunostaining, we demonstrate that Bonus is associated with both interphase and prophase chromosomes and through chromatin immunoprecipitation show that two of these sites correspond to the histone gene cluster and the Stellate locus.

Enhancer of garnet/deltaAP-3 is a cryptic allele of the white gene and identifies the intracellular transport system for the white protein.

The white gene encodes an ABC-type transmembrane transporter that has a role in normal eye pigment deposition. In addition, overexpression in Drosophila leads to homosexual male courtship. Its human homologue has been implicated in cholesterol transport in macrophages and in mood disorders in human males. The garnet gene is a member of a group of other Drosophila eye colour genes that have been shown, or proposed, to function in intracellular protein transport. Recent molecular analysis indicates that it encodes the delta subunit of the AP-3 adaptin complex involved in vesicle transport from the trans-Golgi network to lysosomes and related organelles, such as pigment granules. This identification revealed a novel role for intracellular vesicular transport in Drosophila pigmentation. To further analyze this intracellular transport system, we examined the genetic interactions between garnet and a second site enhancer mutation, enhancer of garnet (e(g)). We show here that e(g) is a cryptic allele of the white gene. The white-garnet interaction is highly sensitive to the levels of both gene products but also shows some allele specificity for the white gene. The additive effect on pigmentation and the predicted protein products of these genes suggest that the garnet/AP-3 transport system ensures the correct intracellular localization of the white gene product. This model is further supported by the observation of homosexual male courtship behavior in garnet mutants, similar to that seen in flies overexpressing, and presumably mis-sorting, the white gene product. The w(e(g)) allele also enhances mutations in the subset of other eye-color genes with phenotypes similar to garnet. This observation supports a role for these genes in intracellular transport and leads to a model whereby incorrect sorting of the white gene product can explain the pigmentation phenotypes of an entire group of eye-color genes.

Expression analysis of a modified factor X in stably transformed insect cell lines.

A modified Factor X protein was combined with a cellulose-binding domain tag and expressed in insect cell lines. The protein, CBDFX, was expressed and secreted into the medium. Stable, transformed Hi5 and Sf9 insect cell lines were generated and tested for production of secreted CBDFX. The highest Sf9 and Hi5 CBDFX-producing cell lines were scaled up to 2-liter fermentors to evaluate production of this recombinant protein. Secreted protein production levels reached 4 mg/liter for the stable, transformed Hi5 cell line and 18 mg/liter for the stable, transformed Sf9 cell line. The protein was properly processed as determined by amino terminal sequencing and bound well to the cellulose substrate Avicel. In addition the activated recombinant CBDFX(a) was capable of recognizing and efficiently processing a Factor X cleavage site.

HMG-D and histone H1 interplay during chromatin assembly and early embryogenesis.

HMG-D is an abundant chromosomal protein associated with condensed chromatin during the first nuclear cleavage cycles of the developing Drosophila embryo. We previously suggested that HMG-D might substitute for the linker histone H1 in the preblastoderm embryo and that this substitution might result in the characteristic less compacted chromatin. We have now studied the association of HMG-D with chromatin using a cell-free system for chromatin reconstitution derived from Drosophila embryos. Association of HMG-D with chromatin, like that of histone H1, increases the nucleosome spacing indicative of binding to the linker DNA between nucleosomes. HMG-D interacts with DNA during the early phases of nucleosome assembly but is gradually displaced as chromatin matures. By contrast, purified chromatin can be loaded with stoichiometric amounts of HMG-D, and this can be displaced upon addition of histone H1. A direct physical interaction between HMG-D and histone H1 was observed in a Far Western analysis. The competitive nature of this interaction is reminiscent of the apparent replacement of HMG-D by H1 during mid-blastula transition. These data are consistent with the hypothesis that HMG-D functions as a specialized linker protein prior to appearance of histone H1.

Identification and analysis of Lydia, a LTR retrotransposon from Lymantria dispar.

Degenerative PCR primers to conserved amino acid motifs were used to identify an LTR retrotransposon from Lymantria dispar. The isolated retrotransposon, Lydia, is 6655 base pairs (bp) in length and contains perfect 300 bp terminal repeats. The identified gag and pol related ORFs have a high degree of similarity to the corresponding regions of the retrotransposon Ted from Trichoplusia ni, although several reading frameshifts and missense mutations are evident. The high degree of similarity between Lydia and Ted LTRs lends support for a family of lepidopteran retrotransposons. Southern blot analysis of individuals from two geographically distinct gypsy moth populations demonstrates that Lydia is found in both populations and the position of this element within the genome of these isolated populations is variable.

Mutational analysis of a histone deacetylase in Drosophila melanogaster: missense mutations suppress gene silencing associated with position effect variegation.

For many years it has been noted that there is a correlation between acetylation of histones and an increase in transcriptional activity. One prediction, based on this correlation, is that hypomorphic or null mutations in histone deacetylase genes should lead to increased levels of histone acetylation and result in increased levels of transcription. It was therefore surprising when it was reported, in both yeast and fruit flies, that mutations that reduced or eliminated a histone deacetylase resulted in transcriptional silencing of genes subject to telomeric and heterochromatic position effect variegation (PEV). Here we report the first mutational analysis of a histone deacetylase in a multicellular eukaryote by examining six new mutations in HDAC1 of Drosophila melanogaster. We observed a suite of phenotypes accompanying the mutations consistent with the notion that HDAC1 acts as a global transcriptional regulator. However, in contrast to recent findings, here we report that specific missense mutations in the structural gene of HDAC1 suppress the silencing of genes subject to PEV. We propose that the missense mutations reported here are acting as antimorphic mutations that "poison" the deacetylase complex and propose a model that accounts for the various phenotypes associated with lesions in the deacetylase locus.

Analysis of an insect neuropeptide, Schistocerca gregaria ion transport peptide (ITP), expressed in insect cell systems.

We have produced an active form of Schistocerca gregaria ion transport peptide (ITP) in an insect cell expression system. Transformed Drosophila Kc1 cells secreted a form of ITP into the cell culture medium that was proteolytically cleaved correctly at the amino (N)-terminus. Concentrated culture supernatant from transformed Kc1 and Hi5 cells had high biological activity when tested on isolated locust ilea. Conversely, ITP expressed by baculovirus-infected Sf9 cells was larger in size and had decreased specific activity compared to ITP produced by Kc1 cells due to incorrect cleavage of the peptide at the N-terminus in the baculovirus system. This demonstrates how processing of the secreted foreign protein (ITP) expressed under the late polyhedrin promoter is compromised in a baculovirus-infected cell. Transient transformation of Kc1 cells results in supernatants containing two forms of ITP; one form (A) co-elutes with synthetic ITP and the other form (B) has reduced electrophoretic mobility. In contrast, in stably transformed Kc1 cell supernatant, ITP is expressed in a single form, which has the same electrophoretic mobility and specific biological activity as form A produced by transiently transformed Kc1 cells. Arch.

Genomic imprinting and position-effect variegation in Drosophila melanogaster.

Genomic imprinting is a phenomenon in which the expression of a gene or chromosomal region depends on the sex of the individual transmitting it. The term imprinting was first coined to describe parent-specific chromosome behavior in the dipteran insect Sciara and has since been described in many organisms, including other insects, plants, fish, and mammals. In this article we describe a mini-X chromosome in Drosophila melanogaster that shows genomic imprinting of at least three closely linked genes. The imprinting of these genes is observed as mosaic silencing when the genes are transmitted by the male parent, in contrast to essentially wild-type expression when the same genes are maternally transmitted. We show that the imprint is due to the sex of the parent rather than to a conventional maternal effect, differential mitotic instability of the mini-X chromosome, or an allele-specific effect. Finally, we have examined the effects of classical modifiers of position-effect variegation on the maintenance and the establishment of the imprint. Factors that modify position-effect variegation alter the somatic expression but not the establishment of the imprint. This suggests that chromatin structure is important in maintenance of the imprint, but a separate mechanism may be responsible for its initiation.

Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines.

The ability of several lepidopteran and dipteran insect cell lines to express human melanotransferrin (p97), a glycosyl phosphatidylinositol (GPI)-anchored, iron-binding sialoglycoprotein, was assessed. Spodoptera frugiperda-derived (Sf9) cell lines, transformed with the p97 gene under control of a baculovirus immediate-early promoter, were able to constitutively express the protein and correctly attach it to the outer cell membrane via a GPI anchor as demonstrated by PI-PLC treatment. In contrast, stable constitutive expression could not be demonstrated with cell lines derived from either Drosophila melanogaster (Kc1 or SL2) or Lymantria dispar (Ld652Y) despite the observation that p97 could be detected in transient expression assays. This may indicate that the long-term expression and accumulation of p97 is inhibitory to Drosophila cells, possibly due to improper localization of the protein and resultant competition for cellular iron. In stably transformed Sf9 cells, p97 was expressed on the cell at a maximal level of 0.18 microg/10(6) cells and was secreted at a maximal rate of 9.03 ng/10(6) cells/h. This level was comparable to the amount expressed with the baculovirus system (0.37 microg/10(6) cells and 31.2 ng/10(6) cells/h) and transformed CHO cells (0.88 microg/10(6) cells and 7.8 ng/10(6) cells/h). Deletion of the GPI cleavage/attachment site resulted in an eightfold increase in the secretion rate of p97, when compared to the intact construct suggesting that the rate-limiting step involves processing of the GPI anchor.

A genetic and molecular characterization of the garnet gene of Drosophila melanogaster.

The garnet gene was one of the first genes to be identified in Drosophila melanogaster. Mutations in the garnet gene affect both of the biochemically distinct types of pigments in the eye and disrupt pigmentation of other organs. As an initial step in the analysis of this gene, we have analyzed the pigmentation defects in several of the garnet alleles. We have also cloned the gene and examined its expression in various tissues and at different stages of development. The garnet gene is expressed throughout development and in all tissues examined. Structurally related sequences can be detected in a variety of other eukaryotes. The predicted protein sequence of the garnet product resembles clathrin and nonclathrin adaptin proteins and is highly similar to the delta subunit of the newly isolated mammalian AP-3 adaptin complex, which is associated with the trans-Golgi network and endosomes. This suggests that garnet encodes a protein that acts in the intracellular sorting and trafficking of vesicles from the trans-Golgi network to endosomes, and related specialized organelles such as the pigment granule. This finding provides an explanation for the phenotype of garnet mutations and predicts that other Drosophila eye-colour genes will be a rich resource for the genetic dissection of intracellular vesicle transport.

Suppressors of position-effect variegation in Drosophila melanogaster affect expression of the heterochromatic gene light in the absence of a chromosome rearrangement.

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.

A series of broad host range shuttle vectors for constitutive and inducible expression of heterologous proteins in insect cell lines.

A series of shuttle vectors have been constructed that allow expression of heterologous proteins in either dipteran or lepidopteran insect cell lines. Constitutive expression in a broad range of host cells is mediated by the Orgyia pseudotsugata multicapsid nucleopolyhedrosis virus (OpMNPV) immediate-early 2 (ie2) promoter. Alternatively, if inducible expression is required, for example to express cytotoxic proteins, a vector has been constructed that uses the Drosophila metallothionein (Mtn) promoter for metal-inducible protein expression in dipteran cell lines. A chimeric synthetic bacterial-OpMNPV ie promoter-Zeocin resistance gene cassette has been included to facilitate cloning in E. coli as well as the generation of stably transformed insect cell lines. The utility of the system is demonstrated by the constitutive and inducible expression of the highly processed glycosylphosphatidylinositol-anchored glycoprotein, human melanotransferrin, in transformed insect cell lines.

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

Molecular characterization of a retrotransposon in Drosophila melanogaster, nomad, and its relationship to other retrovirus-like mobile elements.

The nomad element was identified as a retrovirus-like transposon in Drosophila melanogaster. DNA sequence analysis showed that the nomad element contains three long ORFs that are similar to the gag, pol and env genes of retrovirus- and the copia-like elements of D. melanogaster. The nomad element terminates with 519-bp terminal repeats, each of which contains eukaryotic consensus transcription initiation and termination signals. nomad elements are located at approximately 10-15 sites within the euchromatic arms of the genome and at the chromocenter, as shown by in situ hybridization. The host DNA sequence TANA was found to be duplicated on each side of the nomad element and appears to be a preferential target site for insertion of nomad elements. Analysis of the zinc finger motif in the pol gene product of retrotransposons known to have target site preference suggests involvement of the integrase subunit in target site selection for those retrotransposons that display insert site specificity. A comparison of the predicted amino acid sequence of the pol-like genes of several known retrotransposons was made and the phylogenetic relationship between nomad and other retrovirus-like mobile elements was determined. It was clear from this conceptual protein analysis and from analysis of their structural characteristics that retrotransposons of the gypsy class can be generally classified as members of one of two distinct groups. The phylogenetic relationships of these groups are also discussed.

Unique gene organization: alternative splicing in Drosophila produces two structurally unrelated proteins.

The Ub80 gene in eukaryotes produces a ubiquitin fusion protein in which ubiquitin is fused in frame to a tail protein (Redman and Rechsteiner, 1988; Finley et al., 1989; Barrio et al., 1994). The tail protein is incorporated into the ribosome, and ubiquitin is thought to act as a chaperone. The DUb80 gene of Drosophila melanogaster was cloned by Barrio et al. (1994) and contains a 5'-untranslated exon, followed by a large intron and then the first coding exon. We report that the large intron of DUb80 contains an open reading frame, which produces a 259-aa protein (IP259) that is conserved in eukaryotes from yeast to mammals. Transcription of the DUb80 and IP259 mRNAs begins at the same start sites. However, alternate splicing of the primary transcript produces two structurally unrelated proteins. This is the second reported instance of two structurally unrelated proteins being produced via alternate splicing, suggesting that this form of genomic organization may be more common than previously thought.

Baculovirus immediate-early promoter-mediated expression of the Zeocin resistance gene for use as a dominant selectable marker in dipteran and lepidopteran insect cell lines.

The antibiotic Zeocin, a derivative of phleomycin, was evaluated for use as a selection system in both dipteran and lepidopteran insect cell lines. Growth of Drosophila cell lines, Kc1 and SL2, was inhibited at Zeocin concentrations of 50 and 75 microg/ml, respectively, while the Spodoptera cell line, Sf9, was inhibited at a concentration of 250 microg/ml Zeocin. The mammalian cytomegalovirus (CMV) and Simian virus 40 (SV40) early promoters did not function in these insect cell lines. Several baculovirus-derived immediate-early (IE) promoters from the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) and Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were used to drive expression of the Zeocin resistance gene (ble) in these cell lines. The resulting plasmid vectors enabled selection of Zeocin-resistant cell lines within 3-4 weeks. Gene amplification events in the presence of increasing Zeocin concentrations were not detected using Southern blot analysis. Furthermore, the function of the baculovirus IE promoters, as demonstrated by beta-galactosidase expression, was not detectable in a variety of mammalian cell lines tested. A cloning/shuttle vector, containing ten unique restriction sites, was constructed which allows for selection of Zeocin resistance in insect cell lines and in Escherichia coli.

Competition between different variegating rearrangements for limited heterochromatic factors in Drosophila melanogaster.

Position effect variegation (PEV) results from the juxtaposition of a euchromatic gene to heterochromatin. In its new position the gene is inactivated in some cells and not in others. This mosaic expression is consistent with variability in the spread of heterochromatin from cell to cell. As many components of heterochromatin are likely to be produced in limited amounts, the spread of heterochromatin into a normally euchromatic region should be accompanied by a concomitant loss or redistribution of the protein components from other heterochromatic regions. We have shown that this is the case by simultaneously monitoring variegation of a euchromatic and a heterochromatic gene associated with a single chromosome rearrangement. Secondly, if several heterochromatic regions of the genome share limited components of heterochromatin, then some variegating rearrangements should compete for these components. We have examined this hypothesis by testing files with combinations of two or more different variegating rearrangements. Of the nine combinations of pairs of variegating rearrangements we studied, seven showed nonreciprocal interactions. These results imply that many components of heterochromatin are both shared and present in limited amounts and that they can transfer between chromosomal sites. Consequently, even nonvariegation portions of the genome will be disrupted by re-allocation of heterochromatic proteins associated with PEV. These results have implications for models of PEV.

Identification of a 5' truncated non-LTR-retrotransposon, YAKPs1, from the variegated cutworm, Peridroma saucia, using PCR.

Retrotransposable elements encode for several polypeptides that contain a number of conserved amino acid motifs, especially in the region encoding reverse transcriptase. We have used these motifs to design primers for the PCR amplification of retrotransposon DNA. These primers have allowed us to isolate a retroposon, or LINE (long interspersed nuclear element), from the pest insect, Peridroma saucia. DNA sequence analysis of this element, YAKPs1, demonstrated a high degree of homology to a number of retroposons from Drosophila melanogaster, in particular the Fw and Doc elements with homologies of up to 69%. Determination of the complete sequence of the YAKPs1 element will enable a detailed analysis of its evolutionary relatedness to other elements as well as a greater insight into its mode of action.

Simple plaque hybridization method for the detection of differentially represented repetitive DNA.

A differential DNA hybridization method of detecting moderately repetitive strain-specific or species-specific DNA is described. Two Drosophila melanogaster strains, one with and one without transposable elements, were utilized as a model system to demonstrate the effectiveness of this procedure. A genomic library was constructed from flies of the pi 2 strain, which contains both P and hobo transposable elements. Duplicate plaque lifts of this library were probed with DNA from the same strain and with DNA from the Canton-S strain, which contains neither of these two families of transposable elements. Plaques that hybridized stronger to the genomic DNA that contained elements were noted, and then the filters were stripped and reprobed with P and hobo element DNA. Many of the differentially hybridizing plaques were shown to contain DNA homologous to one of the two known elements. This method should allow the isolation and cloning of any repetitive DNA present in one species or isolate, but absent or present in reduced copy number in another species or isolate. By analogy to the recent invasion of D. melanogaster by P elements, such differentially represented DNA is likely to represent recently invading transposable elements that are actively mobile.