Mammalian (cytosine-5) methyltransferases cause genomic DNA methylation and lethality in Drosophila.

CpG methylation is essential for mouse development as well as gene regulation and genome stability. Many features of mammalian DNA methylation are consistent with the action of a de novo methyltransferase that establishes methylation patterns during early development and the post-replicative maintenance of these patterns by a maintenance methyltransferase. The mouse methyltransferase Dnmt1 (encoded by Dnmt) shows a preference for hemimethylated substrates in vitro, making the enzyme a candidate for a maintenance methyltransferase. Dnmt1 also has de novo methylation activity in vitro, but the significance of this finding is unclear, because mouse embryonic stem (ES) cells contain a de novo methylating activity unrelated to Dnmt1 (ref. 10). Recently, the Dnmt3 family of methyltransferases has been identified and shown in vitro to catalyse de novo methylation. To analyse the function of these enzymes, we expressed Dnmt and Dnmt3a in transgenic Drosophila melanogaster. The absence of endogenous methylation in Drosophila facilitates detection of experimentally induced methylation changes. In this system, Dnmt3a functioned as a de novo methyltransferase, whereas Dnmt1 had no detectable de novo methylation activity. When co-expressed, Dnmt1 and Dnmt3a cooperated to establish and maintain methylation patterns. Genomic DNA methylation impaired the viability of transgenic flies, suggesting that cytosine methylation has functional consequences for Drosophila development.

DNA replication control through interaction of E2F-RB and the origin recognition complex.

The E2F transcription factor and retinoblastoma protein control cell-cycle progression and DNA replication during S phase. Mutations in the Drosophila dE2F1 and dDP genes affect the origin recognition complex (DmORC) and initiation of replication at the chorion gene replication origin. Here we show that mutants of Rbf (an retinoblastoma protein homologue) fail to limit DNA replication. We also show that the dDP, dE2F1 and Rbf proteins are located in a complex with DmORC, and that dE2F1 and DmORC are bound to the chorion origin of replication in vivo. Our results indicate that dE2F1 and Rbf function together at replication origins to limit DNA replication through interactions with DmORC.

Separation anxiety at the centromere.

During mitosis, replicated sister-chromatids must maintain cohesion as they attach to the mitotic spindle. At anaphase, cohesion is lost simultaneously along the entire chromosome, releasing sisters from one another and allowing them to segregate to opposite poles. During meiosis, sisters separate in a two-step process. At anaphase of meiosis I, cohesion is lost along the chromosome arms but is maintained at centromeric regions. Not until meiosis II are sister chromatids able to break the connection at the centromere and separate away from one another. Recent studies suggest that the centromere exhibits dynamics that are very different compared with those of the chromatid arms during both mitosis and meiosis. This review discusses the nature of the specialized chromatid cohesion seen at the centromere.

The cohesion protein MEI-S332 localizes to condensed meiotic and mitotic centromeres until sister chromatids separate.

The Drosophila MEI-S332 protein has been shown to be required for the maintenance of sister-chromatid cohesion in male and female meiosis. The protein localizes to the centromeres during male meiosis when the sister chromatids are attached, and it is no longer detectable after they separate. Drosophila melanogaster male meiosis is atypical in several respects, making it important to define MEI-S332 behavior during female meiosis, which better typifies meiosis in eukaryotes. We find that MEI-S332 localizes to the centromeres of prometaphase I chromosomes in oocytes, remaining there until it is delocalized at anaphase II. By using oocytes we were able to obtain sufficient material to investigate the fate of MEI-S332 after the metaphase II-anaphase II transition. The levels of MEI-S332 protein are unchanged after the completion of meiosis, even when translation is blocked, suggesting that the protein dissociates from the centromeres but is not degraded at the onset of anaphase II. Unexpectedly, MEI-S332 is present during embryogenesis, localizes onto the centromeres of mitotic chromosomes, and is delocalized from anaphase chromosomes. Thus, MEI-S332 associates with the centromeres of both meiotic and mitotic chromosomes and dissociates from them at anaphase.

Stopping and starting the meiotic cell cycle.

The meiotic cell cycle arrests in response to both perturbations and developmental signals. Recent research suggests that meiosis has checkpoints to monitor the completion of meiotic recombination and the attachment of chromosomes to the spindle. New insights have been gained into how meiosis resumes after normal developmental arrests, and new genes have been identified that are required for proper meiotic progression.

Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere.

Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis [1,2]. Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin. The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region [3]. By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives [4], we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote. The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins. Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA. We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation. These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast [5], the requirements for cohesion are separable from those for kinetochore assembly.

The Drosophila RAD21 cohesin persists at the centromere region in mitosis.

'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.

Developmental modification of the Drosophila cell cycle.

The cell cycle is subject to extrinsic regulation that coordinates cell division with developmental events. This important area at the interface of developmental biology and research into the cell cycle has yielded recent exciting advances. Developmental modification of the cell cycle is exemplified during Drosophila embryogenesis. Early in embryogenesis, regulation of the cell cycle becomes progressively more complex as a simple S-M cycle is altered to include gap phases. Later in embryogenesis, the cycle alters again, to include only an S and a gap phase. These developmental alterations are partly effected by regulating the expression of genes that control key transitions in the cell cycle. Certain controls that are unique to the variant cell cycles have also been defined. The field is now poised for the identification of the intervening steps between the developmental signals and their ultimate effects on known cell cycle regulators.

Multiple, tandem plasmid integration in Saccharomyces cerevisiae.

Nonreplicating plasmids transform Saccharomyces cerevisiae by recombining with a homologous site in the genome. Frequently, multiple copies of the plasmid integrate in a tandem array. We show that, after transformation with restriction enzyme-cut plasmids, most, if not all, multimers arise by sequential integration of plasmid molecules into the same genomic location.

Drosophila chorion gene amplification requires an upstream region regulating s18 transcription.

A cluster of Drosophila melanogaster chorion genes at locus 66D on the third chromosome amplifies 60-fold in the ovarian follicle cells prior to the onset of gene expression. A 3.8-kilobase (kb) region of the gene cluster can induce tissue-specific amplification in transformants. Previous models postulated that amplification is activated in follicle cells by transcription of one of the two chorion genes (s15 and s18) located within the 3.8-kb essential region. In this study, we showed that neither s15 nor s18 chorion gene transcription was required for amplification. However, a 510-bp region upstream from s18 contained sequences essential for both amplification and s18 transcription. No other region within the 3.8-kb fragment was required for amplification. We propose that upstream transcription control elements rather than transcription per se are involved in controlling amplification during development.

Gene conversion adjacent to regions of double-strand break repair.

The repair of double-strand breaks and gaps can be studied in vegetative yeast cells by transforming the DNA with restriction enzyme-cut plasmids. Postulated models for this repair process require the formation of heteroduplex DNA on either side of the region of break or gap repair. We describe the use of restriction site mutations in the his3 gene to detect conversion events flanking but outside of a region of a double-strand break repair. The frequency with which a mutation was converted declined with increasing distance between the mutation and the edge of the gap repair region. The data are consistent with heteroduplex DNA tracts of at least several hundred base pairs adjacent to regions of double-strand break repair.

The Drosophila ACE3 chorion element autonomously induces amplification.

The Drosophila chorion genes amplify in the follicle cells by repeated rounds of reinitiation of DNA replication. ACE3 (amplification control element from the third chromosome) has been identified by a series of deletion experiments as an important control element for amplification of the third-chromosome chorion cluster. Several elements that quantitatively enhance amplification also have been defined. We show that a single 440-bp ACE3 sequence is sufficient to regulate amplification with proper developmental specificity autonomously from other chorion DNA sequences and regulatory elements. Although ACE3 is sufficient for amplification, the levels of amplification are low even when ACE3 is present in multiple copies. When controlled solely by ACE3, amplification initiates either at ACE3 or within closely linked sequences. Amplification of an ACE3 transposon insertion produces a gradient of amplified DNA that extends into flanking sequences approximately the same distance as does the amplification gradient at the endogenous chorion locus. The profile and extent of the amplified gradient imply that the low levels of amplification observed are the result of limited rounds of initiation of DNA replication. Transposon inserts containing multiple copies of ACE3 in a tandem, head-to-tail array are maintained stably in the chromosome. However, mobilization of the P-element transposons containing ACE3 multimers results in deletions within the array at a high frequency.

Drosophila PLUTONIUM protein is a specialized cell cycle regulator required at the onset of embryogenesis.

Unfertilized eggs and fertilized embryos from Drosophila mothers mutant for the plutonium (plu) gene contain giant polyploid nuclei resulting from unregulated S-phase. The PLU protein, a 19-kDa ankyrin repeat protein, is present in oocytes and early embryos but is not detectable after the completion of the initial rapid S-M cycles of the embryo. The persistence of the protein during the early embryonic divisions is consistent with a direct role in linking S-phase and M-phase. When ectopically expressed in the eye disc, PLU did not perturb the cell cycle, suggesting that PLU regulates S-phase only in early embryonic development. The pan gu (png) and giant nuclei (gnu) genes also affect the S-phase in the unfertilized egg and early embryo. We show that functional png is needed for the presence of PLU protein. By analyzing png mutations of differing severity, we find that the extent of the png mutant phenotype inversely reflects the level of PLU protein. Our data suggest that PLU protein is required at the time of egg activation and the completion of meiosis.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Sister-chromatid misbehavior in Drosophila ord mutants.

In Drosophila males and females mutant for the ord gene, sister chromatids prematurely disjoin in meiosis. We have isolated five new alleles of ord and analyzed them both as homozygotes and in trans to deficiencies for the locus, and we show that ord function is necessary early in meiosis of both sexes. Strong ord alleles result in chromosome nondisjunction in meiosis I that appears to be the consequence of precocious separation of the sister chromatids followed by their random segregation. Cytological analysis in males confirmed that precocious disjunction of the sister chromatids occurs in prometaphase I. This is in contrast to Drosophila mei-S332 mutants, in which precocious sister-chromatid separation also occurs, but not until late in anaphase I. All three of the new female fertile ord alleles reduce recombination, suggesting they affect homolog association as well as sister-chromatid cohesion. In addition to the effect of ord mutations on meiosis, we find that in ord2 mutants chromosome segregation is aberrant in the mitotic divisions that produce the spermatocytes. The strongest ord alleles, ord2 and ord5, appear to cause defects in germline divisions in the female. These alleles are female sterile and produce egg chambers with altered nurse cell number, size, and nuclear morphology. In contrast to the effects of ord mutations on germline mitosis, all of the alleles are fully viable even when in trans to a deficiency, and thus exhibit no essential role in somatic mitosis. The ord gene product may prevent premature sister-chromatid separation by promoting cohesion of the sister chromatids in a structural or regulatory manner.

Identification of genomic regions required for DNA replication during Drosophila embryogenesis.

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G1 phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.

Mutational analysis of the Drosophila sister-chromatid cohesion protein ORD and its role in the maintenance of centromeric cohesion.

The ord gene is required for proper segregation of all chromosomes in both male and female Drosophila meiosis. Here we describe the isolation of a null ord allele and examine the consequences of ablating ord function. Cytologically, meiotic sister-chromatid cohesion is severely disrupted in flies lacking ORD protein. Moreover, the frequency of missegregation in genetic tests is consistent with random segregation of chromosomes through both meiotic divisions, suggesting that sister cohesion may be completely abolished. However, only a slight decrease in viability is observed for ord null flies, indicating that ORD function is not essential for cohesion during somatic mitosis. In addition, we do not observe perturbation of germ-line mitotic divisions in flies lacking ORD activity. Our analysis of weaker ord alleles suggests that ORD is required for proper centromeric cohesion after arm cohesion is released at the metaphase I/anaphase I transition. Finally, although meiotic cohesion is abolished in the ord null fly, chromosome loss is not appreciable. Therefore, ORD activity appears to promote centromeric cohesion during meiosis II but is not essential for kinetochore function during anaphase.

Double or nothing: a Drosophila mutation affecting meiotic chromosome segregation in both females and males.

We describe a Drosophila mutation, Double or nothing (Dub), that causes meiotic nondisjunction in a conditional, dominant manner. Previously isolated mutations in Drosophila specifically affect meiosis either in females or males, with the exception of the mei-S332 and ord genes which are required for proper sister-chromatid cohesion. Dub is unusual in that it causes aberrant chromosome segregation almost exclusively in meiosis I in both sexes. In Dub mutant females both nonexchange and exchange chromosomes undergo nondisjunction, but the effect of Dub on nonexchange chromosomes is more pronounced. Dub reduces recombination levels slightly. Multiple nondisjoined chromosomes frequently cosegregate to the same pole. Dub results in nondisjunction of all chromosomes in meiosis I of males, although the levels are lower than in females. When homozygous, Dub is a conditional lethal allele and exhibits phenotypes consistent with cell death.