Suppression of heterochromatic gene variegation can be used to distinguish and characterize E(var) genes potentially important for chromosome structure in Drosophila melanogaster.

Hundreds of genic modifiers of position effect variegation (PEV) have been isolated in Drosophila melanogaster with a view to identifying genes important for chromosome structure. Here we propose a supplementary genetic screen to pinpoint candidate genes that are most likely to function in chromosome organization, within the enhancer of variegation [E(var)] class of modifiers. Our strategy takes advantage of the fact that variegating euchromatic and heterochromatic genes respond oppositely to changes in the dosage of heterochromatin proteins. Consequently, only when enhancement of euchromatic gene variegation results from increased formation of heterochromatin should suppression of heterochromatic gene variegation be observed. Mutations in four E(var) genes were tested for the ability to suppress variegation of multiple alleles of the heterochromatic light ( lt) gene in a variety of tissues and at several developmental stages. Mutations in E(var)3-4, E(var)3-5 and modifier of mdg4 [ mod(mdg4)] suppressed lt variegation. In contrast, a mutation in the Trithorax-like ( Trl) gene, which encodes GAGA factor, enhanced or had no effect on lt variegation, consistent with its known role in promoting transcription. These data show that suppression of lt variegation can be used as an assay to distinguish between members of the E(var) class of modifiers.

The teflon gene is required for maintenance of autosomal homolog pairing at meiosis I in male Drosophila melanogaster.

In recombination-proficient organisms, chiasmata appear to mediate associations between homologs at metaphase of meiosis I. It is less clear how homolog associations are maintained in organisms that lack recombination, such as male Drosophila. In lieu of chiasmata and synaptonemal complexes, there must be molecules that balance poleward forces exerted across homologous centromeres. Here we describe the genetic and cytological characterization of four EMS-induced mutations in teflon (tef), a gene involved in this process in Drosophila melanogaster. All four alleles are male specific and cause meiosis I-specific nondisjunction of the autosomes. They do not measurably perturb sex chromosome segregation, suggesting that there are differences in the genetic control of autosome and sex chromosome segregation in males. Meiotic transmission of univalent chromosomes is unaffected in tef mutants, implicating the tef product in a pairing-dependent process. The segregation of translocations between sex chromosomes and autosomes is altered in tef mutants in a manner that supports this hypothesis. Consistent with these genetic observations, cytological examination of meiotic chromosomes suggests a role of tef in regulating or mediating pairing of autosomal bivalents at meiosis I. We discuss implications of this finding in regard to the evolution of heteromorphic sex chromosomes and the mechanisms that ensure chromosome disjunction in the absence of recombination.

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.

Chromosome rearrangements induce both variegated and reduced, uniform expression of heterochromatic genes in a development-specific manner.

In Drosophila melanogaster, chromosome rearrangements that juxtapose euchromatin and heterochromatin can result in position effect variegation (PEV), the variable expression of heterochromatic and euchromatic genes in the vicinity of the novel breakpoint. We examined PEV of the heterochromatic light (lt) and concertina (cta) genes in order to investigate potential tissue or developmental differences in chromosome structure that might be informative for comparing the mechanisms of PEV of heterochromatic and euchromatic genes. We employed tissue pigmentation and in situ hybridization to RNA to assess expression of lt in individual cells of multiple tissues during development. Variegation of lt was induced in the adult eye, larval salivary glands and larval Malpighian tubules for each of three different chromosome rearrangements. The relative severity of the effect in these tissues was not tissue-specific but rather was characteristic of each rearrangement. Surprisingly, larval imaginal discs did not exhibit variegated lt expression. Instead, a uniform reduction of the lt transcript was observed, which correlated in magnitude with the degree of variegation. The same results were obtained for cta expression. These two distinct effects of rearrangements on heterochromatic gene expression correlated with the developmental stage of the tissue. These results have implications for models of heterochromatin formation and the nuclear organization of chromosomes during development and differentiation.

The paternal effect gene ms(3)sneaky is required for sperm activation and the initiation of embryogenesis in Drosophila melanogaster.

Although a large number of maternal factors are known to be essential for fertilization or the earliest stages of embryogenesis in Drosophila melanogaster, the role of paternally supplied products is not clearly understood. Paternal effect mutations provide a means to identify factors specifically required by the sperm after its entry into the egg. Here we describe the third strict paternal effect gene to be identified in Drosophila ms(3)sneaky(snky), which defines the earliest developmental arrest phenotype so far described. Characterization of two independently isolated snky mutations showed that they affected male fertility, but not viability or female fertility. Cytological analyses showed that spermatogenesis proceeded normally in snky males. However, the snky defect was evident after sperm entry into the egg; snky sperm did not undergo nuclear decondensation, form a functional male pronucleus, or initiate mitotic divisions in the egg. Immunolocalization of tubulin and Drosophila Centrosomin, a known centrosomal component, showed that snky-inseminated eggs failed to reconstitute a microtubule-organizing center. In addition, snky sperm chromatin retained the histochemical properties of mature sperm chromatin for several hours after sperm entry, showed reduced staining with membrane-impermeant nuclear dyes, and failed to replicate. We conclude that the snky+ product is required for the initial response of the sperm to cytoplasmic cues in the egg and for the subsequent initiation of embryogenesis in Drosophila. We suggest that all of the snky defects can be explained by the failure of the sperm plasma membrane to break down after entry into the egg.

Paternal effects in Drosophila: implications for mechanisms of early development.

The study of paternal effects on development provides a means to identify sperm-supplied products required for fertilization and the initiation of embryogenesis. This review describes paternal effects on animal development and discusses their implications for the role of the sperm in egg activation, centrosome activity, and biparental inheritance in different animal species. Paternal effects observed in Caenorhabditis elegans and in mammals are briefly reviewed. Emphasis is placed on paternal effects in Drosophila melanogaster. Genetic and cytologic evidence for paternal imprinting on chromosome behavior and gene expression in Drosophila are summarized. These effects are compared to chromosome imprinting that leads to paternal chromosome loss in sciarid and coccid insects and mammalian gametic imprinting that results in differential expression of paternal and maternal loci. The phenotypes caused by several early-acting maternal effect mutations identify specific maternal factors that affect the behavior of paternal components during fertilization and the early embryonic mitotic divisions. In addition, maternal effect defects suggest that two types of regulatory mechanisms coordinate parental components and synchronize their progression through mitosis. Some activities are coordinated by independent responses of parental components to shared regulatory factors, while others require communication between paternal and maternal components. Analyses of the paternal effects mutations sneaky, K81, paternal loss, and Horka have identified paternal products that play a role in mediating the initial response of the sperm to the egg cytoplasm, participation of the male pronucleus in the first mitosis, and stable inheritance of the paternal chromosomes in the early embryo.

Cis-effects of heterochromatin on heterochromatic and euchromatic gene activity in Drosophila melanogaster.

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light+ (lt+) gene and carried the euchromatic white+ (w+) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w+ gene. Heterochromatic genes differed in their response to deletions. The lt+ gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w+ phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.

Developmental genetical analysis and molecular cloning of the abnormal oocyte gene of Drosophila melanogaster.

Studies of the abnormal oocyte (abo) gene of Drosophila melanogaster have previously been limited to the analysis of a single mutant allele, abnormal oocyte1 (abo1). The abo1 mutation causes a maternal-effect lethality that can be partially rescued zygotically by the abo+ allele and by increasing the dosage of specific regions of heterochromatin denoted ABO. This report describes the properties of abo2, a new P-element-induced allele that allowed us to reexamine the nature of maternal-effect defect. Comparisons of the phenotype of progeny of abo1/abo1 and abo1/abo2 females show that the preblastoderm lethality previously described as a component of the abo mutant maternal effect results from a recessive fertilization defect associated with the abo1 chromosome. We demonstrate here that the abo-induced maternal effect lethality occurs predominately late in embryogenesis after cuticle deposition but before hatching. The phenocritical period for zygotic rescue by heterochromatin coincides with this period of late embryogenesis. We have used the abo2 mutation to map and molecularly clone the gene. We show that the abo gene is located in the 32C cytogenetic interval and identify the putative abo transcript from mRNA isolated from adult females. Using germline transformation, we show that a 9-kb genomic fragment to which the transcript maps, partially fulfills requirement for maternal and zygotic abo+ function.

Genetic characterization of ms (3) K81, a paternal effect gene of Drosophila melanogaster.

The vast majority of known male sterile mutants of Drosophila melanogaster fail to produce mature sperm or mate properly. The ms(3) K81(1) mutation is one of a rare class of male sterile mutations in which sterility is caused by developmental arrest after sperm entry into the egg. Previous studies showed that males homozygous for the K81(1) mutation produce progeny that arrest at either of two developmental stages. Most embryos arrest during early nuclear cycles, whereas the remainder are haploid embryos that arrest at a later stage. This description of the mutant phenotype was based on the analysis of a single allele isolated from a natural population. It was therefore unclear whether this unique paternal effect phenotype reflected the normal function of the gene. The genetic analysis and initial molecular characterization of five new K81 mutations are described here. Hemizygous conditions and heteroallelic combinations of the alleles were associated with male sterility caused by defects in embryogenesis. No other mutant phenotypes were observed. Thus, the K81 gene acted as a strict paternal effect gene. Moreover, the biphasic pattern of developmental arrest was common to all the alleles. These findings strongly suggested that the unusual embryonic phenotype caused by all five new alleles was due to loss of function of the K81+ gene. The K81 gene is therefore the first clear example of a strict paternal effect gene in Drosophila. Based on the embryonic lethal phenotypes, we suggest that the K81+ gene encodes a sperm-specific product that is essential for the male pronucleus to participate in the first few embryonic nuclear divisions.

Heterochromatin and gene expression in Drosophila.

Heterochromatin is both necessary for the expression of heterochromatic genes and inhibitory for the expression of euchromatic genes. These two properties of heterochromatin have been elucidated from the study of chromosome rearrangements that induce position effect variegation (PEV) in Drosophila melanogaster. Novel euchromatin-heterochromatin junctions can affect the expression of euchromatic and heterochromatic genes located several megabases away, distinguishing higher order chromatin structure from most other regulatory mechanisms. Studies of PEV promise insights into the basis for heterochromatin formation and the role of higher order chromatin and chromosome structure in gene regulation. We evaluate the models and experimental data that address the mechanisms of PEV in different cell types, the potential functions of modifiers of PEV, and the relationship of PEV to other phenomena associated with variegated gene expression in Drosophila.

The effect of modifiers of position-effect variegation on the variegation of heterochromatic genes of Drosophila melanogaster.

Dominant modifiers of position-effect variegation of Drosophila melanogaster were tested for their effects on the variegation of genes normally located in heterochromatin. These modifiers were previously isolated as strong suppressors of the variegation of euchromatic genes and have been postulated to encode structural components of heterochromatin or other products that influence chromosome condensation. While eight of the modifiers had weak or no detectable effects, six acted as enhancers of light (lt) variegation. The two modifiers with the strongest effects on lt were shown to also enhance the variegation of neighboring heterochromatic genes. These results suggest that the wild-type gene products of some modifiers of position-effect variegation are required for proper expression of genes normally located within or near the heterochromatin of chromosome 2. We conclude that these heterochromatic genes have fundamentally different regulatory requirements compared to those typical of euchromatic genes.

The organization and expression of the light gene, a heterochromatic gene of Drosophila melanogaster.

The light (lt) gene is located in the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster. This gene is necessary for normal levels of pigmentation in a number of adult and larval tissues and is required for viability. Hybrid dysgenic and X-ray induced mutations have been used to identify the gene and compare its organization to that of euchromatic genes. Molecular mapping of lt mutations and its major transcripts has shown that the lt gene is at least 17 kb. By injecting cosmid clones that include this region into lt mutant embryos, we have defined a 30-kb region that can transiently rescue the pigmentation defect in the Malpighian tubules. The major transcription unit of this gene is comprised of exons that are single copy. It is unusual in its organization in having a heterogeneous array of middle repetitive DNA sequences within its intronic and flanking regions.

The effects of chromosome rearrangements on the expression of heterochromatic genes in chromosome 2L of Drosophila melanogaster.

The light (lt) gene of Drosophila melanogaster is located at the base of the left arm of chromosome 2, within or very near centromeric heterochromatin (2Lh). Chromosome rearrangements that move the lt+ gene from its normal proximal position and place the gene in distal euchromatin result in mosaic or variegated expression of the gene. The cytogenetic and genetic properties of 17 lt-variegated rearrangements are described in this report. We show that five of the heterochromatic genes adjacent to lt are subject to inactivation by these rearrangements and that the euchromatic loci in proximal 2L are not detectably affected. The properties of the rearrangements suggest that proximity to heterochromatin is an important regulatory requirement for at least six 2Lh genes. We discuss how the properties of the position effects on heterochromatic genes relate to other proximity-dependent phenomena such as transvection.

Characterization and functional expression in mammalian cells of genomic and cDNA clones encoding a Drosophila muscarinic acetylcholine receptor.

Genomic and cDNA clones encoding a muscarinic acetylcholine receptor from Drosophila melanogaster have been isolated. Sequence analysis demonstrates that this gene encodes a receptor with a high degree of amino acid identity to the mammalian muscarinic acetylcholine receptors and has three introns in the portion of the gene encoding the third putative cytoplasmic loop. A full-length cDNA clone has been placed under the control of the mouse metallothionein promotor and transfected into mouse Y1 adrenal cells. The receptor expressed in these cells exhibits the high-affinity binding for the antagonists quinuclidinyl benzilate and atropine expected of a muscarinic receptor. The Drosophila muscarinic receptor, when expressed in Y1 cells, is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism.

Amplification of the X-linked Drosophila chorion gene cluster requires a region upstream from the s38 chorion gene.

Genomic sequences controlling follicle cell-specific amplification of the X-linked Drosophila chorion gene cluster were mapped by P element-mediated transformation. Several DNA fragments containing the s38 gene and flanking sequences induced tissue-specific amplification, although replication levels were subject to position effects. Deletion analysis identified a 467-bp region upstream from the s38 transcription start site that contained sequences essential in cis for amplification. The essential region shared 32 bp of imperfect sequence homology with a previously identified region necessary for third chromosome chorion gene cluster amplification. This homologous segment contained a repetitive motif consisting of perfect and imperfect AATAC repeats; it was localized near the boundary of the essential domain since most, but not all, the repeats could be deleted without eliminating transposon-induced amplification. The repetitive region was not required for developmentally regulated s38 transcription, therefore our results identified at least one element required for amplification but not for chorion gene transcription. The homologous repetitive sequences within the amplification-essential regions may constitute part of the replication origins used to differentially replicate the two chorion domains during oogenesis.

Replication and expression of an X-linked cluster of Drosophila chorion genes.

Two 80- to 100-kb chromosomal replicons containing clustered chorion genes amplify in the ovarian follicle cells during the final 22 hr of Drosophila oogenesis. We have studied the relationship between amplification and transcription within one of these domains, located at 7E10-7F3,4 on the X chromosome. A tandem cluster of six genes, encoding chorion structural proteins s36-1, s38-1, and four putative minor chorion protein mRNAs, was mapped in the central 18 kb of the amplified domain, a region showing the highest levels of amplification. The regions both proximal and distal to this gene cluster, where lower levels of amplification occur, were also transcribed in ovary, but mRNAs produced specifically during choriogenesis were not detected. Thus, differences in amplification do not appear to modulate differential RNA accumulation. Instead, the gradient of amplification observed in egg chamber DNA may simply reflect the mechanism of amplification. In the female sterile mutation, In(1)ocelliless, a chromosomal rearrangement separates the central gene cluster into two parts, only one of which retains the capacity to amplify. Genes located within the unamplified portion of the ocelliless chromosome were expressed at the appropriate time during oogenesis, but at a 5- to 10-fold reduced level of RNA per gene. Thus neither cluster integrity nor amplification are required for the normal developmental program of gene expression within the cluster.

Developmentally regulated expression of Drosophila chorion genes introduced at diverse chromosomal positions.

Drosophila chorion genes are organized into two clusters that are selectively amplified in the ovarian follicle cells. During oogenesis the transcription of individual genes is temporally regulated, resulting in distinct, stage-specific profiles of chorion mRNA accumulation. P element-mediated gene transfer was used to study the regulation of genes encoding the major chorion proteins s15-1 and s38-1. Transformed chorion genes integrated at diverse chromosomal locations exhibited proper tissue-specific and stage-specific expression, despite separation from the gene clusters. Qualitatively normal expression was not dependent on the ability of the inserted DNA to undergo amplification. However, chromosome position quantitatively influenced the RNA produced by the transformed genes. The level of RNA per gene copy produced by individual transformed genes varied approximately tenfold, after correction for differences in gene dosage due to the amplification of some inserted sequences. Transformation experiments with an s38-1-lacZ fusion gene demonstrated that cis-regulatory sequences sufficient for the stage-specific program of s38-1 expression were confined to a 1.3 X 10(3) base-pair segment between -748 and +573 relative to the s38-1 initiation site. Finally, egg chamber-specific amplification was induced at the site of two s38-1 insertions, suggesting that an amplification control element resides near this gene.