Boundary and insulator elements in chromosomes.

Recent progress in understanding boundary and insulator elements has concentrated on the identification of their protein components. BEAF-32 is a protein present in the scs' element of Drosophila that is also localized to most interband regions and puffs of polytene chromosomes, suggesting a role in the organization of structural chromosomal domains. The suppressor of Hairy-wing and modifier of mdg4 proteins have been characterized as components of the gypsy insulator. The latter seems to play a crucial role in conferring on the insulator its ability to unidirectionally affect enhancer function.

Interactions of retrotransposons with the host genome: the case of the gypsy element of Drosophila.

Insertion of the gypsy retrotransposon into various Drosophila genes results in mutant phenotypes that can be altered by second site mutations in a variety of modifier loci. One of these loci is the suppressor of Hairy-wing, which encodes a DNA-binding protein that binds to specific sequences of the gypsy element to regulate its expression. Interactions between the su(Hw) protein and transcription factors responsible for expression of the mutant genes are the primary cause of gypsy-induced phenotypes. Gypsy also appears to mediate effects in trans between copies of a gene located on homologous chromosomes. This interchromosomal communication allows transcriptional enhancers located in one chromosome to interact with their target promoter located on the other homolog.

Interactions among the gypsy transposable element and the yellow and the suppressor of hairy-wing loci in Drosophila melanogaster.

We cloned and characterized the yellow locus of Drosophila melanogaster. We also studied its transcription pattern in the suppressible allele y2, which is caused by the insertion of the transposable element gypsy, and the effect of mutations at the unlinked suppressor of Hairy-wing locus on the transcription of yellow RNAs. The gypsy element is transcribed in a temporal fashion that correlates with the pattern of expression of the yellow locus. We propose that the mutational effect of the gypsy element is due to developmentally specific transcriptional interference on yellow transcription. Mutations at the su(Hw) locus reverse this effect by altering the quantitative expression of gypsy.

Mutations at the suppressor of forked locus increase the accumulation of gypsy-encoded transcripts in Drosophila melanogaster.

We studied the effect of mutations at the suppressor of forked [su(f)] locus in Drosophila melanogaster on the accumulation of transcripts encoded by the gypsy transposable element. Mutations at this locus do not affect the pattern of developmental expression of gypsy, but they cause an increase in the total amount of gypsy RNA present at different stages of development as compared with wild-type or su(f)/+ flies. These results suggest that the su(f)-encoded products acts as a negative regulator of gypsy expression.

Correct temperature induction and developmental regulation of a cloned heat shock gene transformed into the Drosophila germ line.

We have constructed a size variant of the Drosophila hsp28 gene by deleting 207 base pairs of the protein coding region, beginning 33 base pairs downstream of the ATG protein initiation codon. After transformation of Drosophila melanogaster rosy (ry506) flies with this altered gene, using the P transposable element system, it was found that the transformed gene was regulated correctly both after temperature elevation and during the development of the flies. Levels of the variant mRNA were as high as those of the endogenous hsp28 during all patterns of expression, and the variant mRNA appeared in all cases to be processed correctly and to be as stable as the endogenous mRNA. Nevertheless, the chromosomal locus of the transformed gene did not puff after heat shock, suggesting that normal transcription of the gene does not require puffing of the locus. The deleted hsp28 gene retained the reading frame of the endogenous one, and a protein of the expected molecular weight of 18,500 was made after heat shock at levels comparable to those of the endogenous hsp28.

The Drosophila melanogaster gypsy transposable element encodes putative gene products homologous to retroviral proteins.

We determined the complete nucleotide sequence of the gypsy element present at the forked locus of Drosophila melanogaster in the f1 allele. The gypsy element shares more homology with vertebrate retroviruses than with the copia element of D. melanogaster or the Ty element of Saccharomyces cerevisiae, both in overall organization and at the DNA sequence level. This transposable element is 7,469 base pairs long and encodes three putative protein products. The long terminal repeats are 482 nucleotides long and contain transcription initiation and termination signals; sequences homologous to the polypurine tract and tRNA primer binding site of retroviruses are located adjacent to the long terminal repeats. The central region of the element contains three different open reading frames. The second one encodes a putative protein which shows extensive amino acid homology to retroviral proteins, including gag-specific protease, reverse transcriptase, and DNA endonuclease.

The Drosophila tom retrotransposon encodes an envelope protein.

The tom transposable element of Drosophila ananassae is mobilized with high frequency in the germ line of females from the ca; px strain, and its insertion results in mutations that show almost exclusively dominant eye phenotypes. tom is a long terminal repeat-containing retrotransposon that encodes three different open reading frames (ORFs). It is expressed in the nurse cells during oogenesis, in the central and peripheral nervous systems during embryonic development, and in the imaginal discs of the larva. tom RNA accumulates in the germarium of ovaries from ca; px females but not in the parental inactive strain, suggesting that this altered pattern of tom expression might be the cause of the high rate of mobilization of this retrotransposon. The specificity of tom-induced eye phenotypes can be explained by the presence of regulatory sequences responsible for expression of tom in the eye imaginal discs of third-instar larvae. These sequences might cause overexpression of adjacent genes affected by tom-induced mutations, resulting in the death of undifferentiated cells located anterior to the morphogenetic furrow. In addition to the full-length RNA, tom is also transcribed into a spliced subgenomic transcript that encodes a protein resulting from the fusion between the amino-terminal region of the first (gag) and the third ORFs. The protein encoded by this RNA shows structural characteristics such as a signal peptide, glycosylation sites, endopeptidase cleavage site, and fusion peptide that are typical of the envelope proteins of retroviruses. Antibodies against tom ORF3 recognize two different proteins present in female ovaries, suggesting that tom might be able to form infective viral particles that could play a role in the horizontal transmission of this retrotransposon.

The RNA polymerase II 15-kilodalton subunit is essential for viability in Drosophila melanogaster.

A small, divergently transcribed gene is located 500 bp upstream of the suppressor of Hairy-wing locus of Drosophila melanogaster. Sequencing of a full-length cDNA clone of the predominant 850-nucleotide transcript reveals that this gene encodes a 15,100-Da protein with high homology to a subunit of RNA polymerase II. The RpII15 protein is 46% identical to the RPB9 protein of Saccharomyces cerevisiae, one of the smallest subunits of RNA polymerase II from that species. Among those identical residues are four pairs of cysteines whose spacing is suggestive of two metal-binding "finger" domains. The gene is expressed at all developmental stages and in all tissues. Two deletions within the RpII15 gene are multiphasic lethal deletions, with accumulation of dead animals commencing at the second larval instar. Ovary transplantation experiments indicate that survival of mutant animals to this stage is due to the persistence of maternal gene product throughout embryogenesis and early larval development. The RpII15 gene product is thus necessary for viability of D. melanogaster.

The ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene react very differently to perturbations of DNA sequence.

The effect of various types of DNA sequence alterations on the activity of the ovarian, ecdysterone, and heat-shock-responsive promoters of the Drosophila melanogaster hsp27 gene was studied by P element-mediated germ line transformation. Regions of DNA required for proper expression of the gene under these different conditions were identified. Wild-type levels of transcription during oogenesis are dependent on two elements respectively located within a 64-base-pair (bp) fragment in the transcribed untranslated region and between -227 and -958 bp upstream of the transcription start site. This ovarian expression is particularly sensitive to both chromosomal position effects and an increased distance between the distal upstream promoter element and the TATAA homology. The ecdysterone-mediated expression during metamorphosis is dependent on a 145-bp domain including the TATAA box and additional upstream sequences that augment transcription by two- to five-fold. Finally, sequences necessary for heat shock expression are located much further upstream from hsp27 than those previously found for hsp70, although basal expression was correlated with the presence of more proximal heat shock consensus sequences.

The Drosophila micropia retrotransposon encodes a testis-specific antisense RNA complementary to reverse transcriptase.

The micropia transposable element of Drosophila hydei is a long terminal repeat-containing retrotransposon present in both the autosomes and the Y chromosome. micropia expression gives rise to a complex set of sense and antisense RNAs transcribed primarily during spermatogenesis. The most abundant sense RNAs constitute an assortment of heterogeneous high-molecular-weight transcripts expressed as constituents of the Y-chromosomal lampbrush loops of primary spermatocytes. In addition, micropia encodes a full-length RNA that extends between the two long terminal repeats of the element. The major 1.0-kb antisense RNA characterized is complementary to the reverse transcriptase and RNase H coding regions of micropia. It is expressed from a testis-specific promoter during the primary spermatocyte stages and is detectable until spermatid elongation stages. Sequence comparison of this promoter with the 5' region of other testis-specific genes allows the conception of a conserved sequence that is responsible for this pattern of expression. A 284-bp fragment containing this sequence is able to drive testis-specific expression of the Escherichia coli lacZ gene in Drosophila melanogaster. This sequence is conserved in the micropia elements present in other Drosophila species that also encode an antisense RNA. The evolutionary conservation of micropia antisense RNA expression and the sequences responsible for its testis-specific transcription suggests a role for this antisense RNA in the control of germ line expression of the full-length transcript or transposon-encoded proteins.

Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.

P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.

Ulysses transposable element of Drosophila shows high structural similarities to functional domains of retroviruses.

We have determined the DNA structure of the Ulysses transposable element of Drosophila virilis and found that this transposon is 10,653 bp and is flanked by two unusually large direct repeats 2136 bp long. Ulysses shows the characteristic organization of LTR-containing retrotransposons, with matrix and capsid protein domains encoded in the first open reading frame. In addition, Ulysses contains protease, reverse transcriptase, RNase H and integrase domains encoded in the second open reading frame. Ulysses lacks a third open reading frame present in some retrotransposons that could encode an env-like protein. A dendrogram analysis based on multiple alignments of the protease, reverse transcriptase, RNase H, integrase and tRNA primer binding site of all known Drosophila LTR-containing retrotransposon sequences establishes a phylogenetic relationship of Ulysses to other retrotransposons and suggests that Ulysses belongs to a new family of this type of elements.

Protein determinants of insertional specificity for the Drosophila gypsy retrovirus.

The gypsy retrovirus invades the germ line of Drosophila females, inserting with a high frequency into the ovo locus. Gypsy insertion sites in ovo are clustered within a region in the promoter of the ovo gene that contains multiple binding sites for the OvoA and OvoB proteins. We found that a 1.3-kb DNA fragment containing this region is able to confer gypsy insertional specificity independent of its genomic location. The frequency of gypsy insertions into the ovo gene is significantly lower in wild-type females than in ovoD1 females. In addition, gypsy insertions in ovoD1 females occur during most stages of germ-line development whereas insertions in wild-type females occur only in late stages. This pattern of temporally specific insertions, as well as the higher frequency of insertion in ovoD1 females, correlates with the presence of the OvoA or OvoD1 proteins. The results suggest that gypsy insertional specificity might be determined by the binding of the OvoA repressor isoform to the promoter region of the gene.

The gypsy insulator of Drosophila affects chromatin structure in a directional manner.

Chromatin insulators are thought to regulate gene expression by establishing higher-order domains of chromatin organization, although the specific mechanisms by which these sequences affect enhancer-promoter interactions are not well understood. Here we show that the gypsy insulator of Drosophila can affect chromatin structure. The insulator itself contains several DNase I hypersensitive sites whose occurrence is dependent on the binding of the Suppressor of Hairy-wing [Su(Hw)] protein. The presence of the insulator in the 5' region of the yellow gene increases the accessibility of the DNA to nucleases in the promoter-proximal, but not the promoter-distal, region. This increase in accessibility is not due to alterations in the primary chromatin fiber, because the number and position of the nucleosomes appears to be the same in the presence or absence of the insulator. Binding of the Su(Hw) protein to insulator DNA is not sufficient to induce changes in chromatin accessibility, and two domains of this protein, presumed to be involved in interactions with other insulator components, are essential for this effect. The presence of Modifier of mdg4 [Mod(mdg4)] protein, a second component of the gypsy insulator, is required to induce these alterations in chromatin accessibility. The results suggest that the gypsy insulator affects chromatin structure and offer insights into the mechanisms by which insulators affect enhancer-promoter interactions.

Characterization of functional domains of the su(Hw) protein that mediate the silencing effect of mod(mdg4) mutations.

The suppressor of Hairy-wing [su(Hw)] protein represses enhancer function in a unidirectional fashion: enhancers segregated from the promoter by the su(Hw) binding region are rendered inactive. whereas those in the same domain are unaffected. In the case of the gypsy-induced y2 allele, the repressive effect of su(Hw) is rendered bidirectional in mod(mdg4) mutant flies, and all enhancers of the affected gene become inactive. This silencing of enhancer elements might be due to exposure of specific domains of su(Hw) when the mod(mdg4) protein is absent. Two of three regions of su(Hw) that are located adjacent to the leucine zipper motif and are conserved across Drosophila species are necessary for both the unidirectional and bidirectional repression of transcription by su(Hw). In contrast, two acidic domains that are dispensable for the unidirectional repression of enhancer elements are critical for the bidirectional silencing of enhancer activity observed in mutants lacking functional mod(mdg4) protein.

The suppressor of Hairy-wing protein regulates the tissue-specific expression of the Drosophila gypsy retrotransposon.

The gypsy retrotransposon of Drosophila melanogaster causes mutations that show temporal and tissue-specific phenotypes. These mutant phenotypes can be reversed by mutations in su(Hw), a gene that also regulates the transcription of the gypsy element. Gypsy encodes a full-length 7.0-kb RNA that is expressed in the salivary gland precursors and fat body of the embryo, imaginal discs and fat body of larvae, and fat body and ovaries of adult females. The su(Hw)-binding region inserted upstream of the promoter of a lacZ reporter gene can induce beta-galactosidase expression in a subset of the embryonic and larval tissues where gypsy is normally transcribed. This expression is dependent on the presence of a functional su(Hw) product, suggesting that this protein is a positive activator of gypsy transcription. Flies transformed with a construct in which the 5' LTR and leader sequences of gypsy are fused to lacZ show beta-galactosidase expression in all tissues where gypsy is normally expressed, indicating that sequences other than the su(Hw)-binding site are required for proper spatial and temporal expression of gypsy. Mutations in the zinc fingers of su(Hw) affect its ability to bind DNA and to induce transcription of the lacZ reporter gene. Two other structural domains of su(Hw) also play an important role in transcriptional regulation of gypsy. Deletion of the amino-terminal acidic domain results in the loss of lacZ expression in larval fat body and adult ovaries, whereas mutations in the leucine zipper region result in an increase of lacZ expression in larval fat body and a decrease in adult ovaries. These effects might be the result of interactions of su(Hw) with activator and repressor proteins through the acidic and leucine zipper domains to produce the final pattern of tissue-specific expression of gypsy.

Effects of transposable elements on the expression of the forked gene of Drosophila melanogaster.

The products of the forked gene are involved in the formation and/or maintenance of a temporary fibrillar structure within the developing bristle rudiment of Drosophila melanogaster. Mutations in the forked locus alter this structure and result in aberrant development of macrochaetae, microchaetae and trichomes. The locus has been characterized at the molecular level by walking, mutant characterization and transcript analysis. Expression of the six forked transcripts is temporally restricted to mid-late pupal development. At this time, RNAs of 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kilobases (kb) are detected by Northern analysis. The coding region of these RNAs has been found to be within a 21-kb stretch of genomic DNA. The amino terminus of the proteins encoded by the 5.4- and 5.6-kb forked transcripts contain tandem copies of ankyrin-like repeats that may play an important role in the function of forked-encoded products. The profile of forked RNA expression is altered in seven spontaneous mutations characterized during this study. Three forked mutations induced by the insertion of the gypsy retrotransposon contain a copy of this element inserted into an intron of the gene. In these mutants, the 5.6-, 5.4- and 2.5-kb forked mRNAs are truncated via recognition of the polyadenylation site in the 5' long terminal repeat of the gypsy retrotransposon. These results help explain the role of the forked gene in fly development and further our understanding of the role of transposable elements in mutagenesis.

The lawc gene is a new member of the trithorax-group that affects the function of the gypsy insulator of Drosophila.

Mutations in the lawc gene result in a pleiotropic phenotype that includes homeotic transformation of the arista into leg. lawc mutations enhance the phenotype of trx-G mutations and suppress the phenotype of Pc mutations. Mutations in lawc affect homeotic gene transcription, causing ectopic expression of Antennapedia in the eye-antenna imaginal disc. These results suggest that lawc is a new member of the trithorax family. The lawc gene behaves as an enhancer of position-effect variegation and interacts genetically with mod(mdg4), which is a component of the gypsy insulator. In addition, mutations in the lawc gene cause alterations in the punctated distribution of mod(mdg4) protein within the nucleus. These results suggest that the lawc protein is involved in regulating the higher-order organization of chromatin.

Dominant effects of suppressor of Hairy-wing mutations on gypsy-induced alleles of forked and cut in Drosophila melanogaster.

Mutations induced by the gypsy retrotransposon in the forked (f) and cut (ct) loci render their expression under the control of the suppressor of Hairy-wing [su(Hw)] gene. This action is usually recessive, but su(Hw) acts as a dominant on the alleles fk, ctk and ctMRpN30. Molecular analysis of the gypsy element present in fk indicates that this allele is caused by the insertion of a modified gypsy in which the region normally containing twelve copies of the octamer-like repeat that interacts with the su(Hw) product is altered. Analysis of the gypsy element responsible for the ctk and ctMRpN30 mutations also reveals a correlation between the dominant action of su(Hw) and disruption of the octamer region. We propose that these disruptions alter the affinity and interaction of su(Hw) protein with gypsy DNA, thereby sensitizing the mutant phenotype to fluctuations in su(Hw) product.

forked proteins are components of fiber bundles present in developing bristles of Drosophila melanogaster.

The forked (f) gene of Drosophila melanogaster encodes six different transcripts 6.4, 5.6, 5.4, 2.5, 1.9, and 1.1 kb long. These transcripts arise by the use of alternative promoters. A polyclonal antibody raised against a domain common to all of the forked-encoded products has been used to identify forked proteins on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and in Drosophila pupal tissues. The antibody stains fiber bundles present in bristle cells for about 15 hr during normal pupal development. Electron microscopy shows that these fibers are present from 40 to 53 hr in bristles of wild-type flies but are absent in the null f36a mutant. The forked protein(s) thus appear to be an essential part of the bristle fibers. The phenotype of the f36a mutation can be rescued by a 13-kb fragment of the forked locus containing the coding regions for the 2.5, 1.9, and 1.1-kb transcripts, suggesting that the proteins encoded by the three large forked RNAs are dispensable during bristle development. Increasing the copy number of a P[w+,f+] construct containing the 13-kb fragment induces a hypermorphic bristle phenotype whose severity correlates with the number of copies of P[w+,f+] present. These results indicate that alterations in the ratios among the forked proteins, or between forked products and other components of the fiber, result in abnormal assembly of the fibrillar cytoplasmic structures necessary for bristle morphogenesis.