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Interstrand crosslinks (ICLs) present a major threat to genome integrity, preventing both the correct transcription of active chromatin and complete replication of the genome. This is exploited in genotoxic chemotherapy where ICL induction is used to kill highly proliferative cancer cells. Repair of ICLs involves a complex interplay of numerous proteins, including those in the Fanconi anemia (FA) pathway, though alternative and parallel pathways have been postulated. Here, we investigate the role of the 3’-5’ exonuclease, EXD2, and the highly related WRN exonuclease (implicated in premature ageing human Werner syndrome), in repair of interstrand crosslinks in the fruit fly, Drosophila melanogaster. We find that flies mutant for EXD2 (DmEXD2) have elevated rates of genomic instability resulting from chromosome breakage and loss of the resulting acentric fragments, in contrast to WRN exonuclease (DmWRNexo) mutants where excess homologous recombination is the principal mechanism of genomic instability. Most notably, we demonstrate that proliferating larval neuroblasts mutant for either DmWRNexo or DmEXD2 are deficient in repair of DNA interstrand crosslinks caused by diepoxybutane or mitomycin C, strongly suggesting that each nuclease individually plays a role in repair of ICLs in flies. These findings have significant implications not only for understanding the complex process of ICL repair in humans, but also for enhancing cancer therapies that rely on ICL induction, with caveats for cancer therapy in Werner syndrome and Fanconi anemia patients.

‘Big Data’ and a surge in quantitative methodologies increasingly urge bioresearchers to train and improve their mathematical and computational skills. In response to this challenge the Biotechnology and Biological Sciences Research Council in the United Kingdom offered funds to develop web-based training in mathematics and computation for life scientists in all fields. The SysMIC consortium won the bid and implemented a new type of online course, which so far has been delivered to more than 1400 scientists, mostly in the UK. Researchers train self-paced or in cohorts in a media rich environment with materials based around intuitive biological examples. We describe the course, its trainee spectrum, and the modes of trainee engagement. We propose that tailored interactive online training is a scalable model for continuing professional development in the interdisciplinary life sciences in the 21st century.

Werner syndrome (WS) is a rare late-onset premature ageing disease showing many of the phenotypes associated with normal ageing, and provides one of the best models for investigating cellular pathways that lead to normal ageing. WS is caused by mutation of WRN, which encodes a multifunctional DNA replication and repair helicase/exonuclease. To investigate the role of WRN protein’s unique exonuclease domain, we have recently identified DmWRNexo, the fly orthologue of the exonuclease domain of human WRN. Here, we fully characterise DmWRNexo exonuclease activity in vitro, confirming 3′–5′ polarity, demonstrating a requirement for Mg2+, inhibition by ATP, and an ability to degrade both single-stranded DNA and duplex DNA substrates with 3′ or 5′ overhangs, or bubble structures, but with no activity on blunt ended DNA duplexes. We report a novel active site mutation that ablates enzyme activity. Lesional substrates containing uracil are partially cleaved by DmWRNexo, but the enzyme pauses on such substrates and is inhibited by abasic sites. These strong biochemical similarities to human WRN suggest that Drosophila can provide a valuable experimental system for analysing the importance of WRN exonuclease in cell and organismal ageing. Electronic supplementary material The online version of this article (doi:10.1007/s11357-012-9411-0) contains supplementary material, which is available to authorized users.

Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ-type helicase with additional exonuclease activity. While biochemical studies in vitro have proven invaluable in determining substrate specificities of the WRN exonuclease and helicase, it has been difficult to dissociate the two key enzyme activities in vivo. We are developing Drosophila as a model system for analysis of WRN function; the suitability of Drosophila for extensive and sophisticated genetic manipulation permits us to investigate regulatory pathways and the impact of WRN loss at organismal, cellular, and molecular levels. BLASTP screening of the Drosophila genome with human WRN sequence allowed us to identify three RecQ helicases with strong homology to human WRN, a presumed helicase component of the spliceosome, and two DEAH-box putative RNA helicases with weaker WRN homology. None of these helicases contain a WRN-like exonuclease domain, but two potential WRN-like exonucleases in flies encoded by the loci CG7670 and CG6744 were also identified in the BLAST search. CG6744 and CG7670 are more closely related to human WRN than to each other. We have obtained a fly strain with a piggyBac insertional mutation within the CG6744 locus, which decreases expression of the encoded mRNA. Such flies show elevated levels of somatic recombination. We suggest that WRN-like exonuclease activity is critical in maintaining genomic integrity in flies.

The premature human ageing Werner's syndrome is caused by loss or mutation of the WRN helicase/exonuclease. We have recently identified the orthologue of the WRN exonuclease in flies, DmWRNexo, encoded by the CG7670 locus, and showed very high levels of mitotic recombination in a hypomorphic PiggyBac insertional mutant. Here, we report a novel allele of CG7670, with a point mutation resulting in the change of the conserved aspartate (229) to valine. Flies bearing this mutation show levels of mitotic recombination 20-fold higher than wild type. Molecular modelling suggests that D229 lies towards the outside of the molecule distant from the nuclease active site. We have produced recombinant protein of the D229V mutant, assayed its nuclease activity in vitro, and compared activity with that of wild type DmWRNexo and a D162A E164A double active site mutant we have created. We show for the first time that DmWRNexo has 3'-5' exonuclease activity and that mutation within the presumptive active site disrupts exonuclease activity. Furthermore, we show that the D229V mutant has very limited exonuclease activity in vitro. Using Drosophila, we can therefore analyse WRN exonuclease from enzyme activity in vitro through to fly phenotype, and show that loss of exonuclease activity contributes to genome instability.

The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3?�5? exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5? UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hypomorphic; although adults show no gross morphological abnormalities, females are sterile. Like human WS cells, we show that the DmWRNexo mutant flies are hypersensitive to the topoisomerase I inhibitor camptothecin. Furthermore, these mutant flies show highly elevated rates of mitotic DNA recombination resulting from excessive reciprocal exchange. This study identifies a novel WRN ortholog in flies and demonstrates an important role for WRN exonuclease in maintaining genome stability.

From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D- structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extra-corporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed. (C) 2004 Elsevier Ltd. All rights reserved

Aerobic cells produce reactive oxygen species as a consequence of normal cellular metabolism, and an array of antioxidant systems are in place to maintain the redox balance. When the redox equilibrium of the cell is upset by pro-oxidant environmental stimuli, adaptive responses to the redox stress take place, which can result in up-regulation of antioxidant proteins and detoxification enzymes. Over the past few years, it has become apparent that members of the CNC (cap 'n' collar)-basic leucine zipper family of transcription factors are principal mediators of defensive responses to redox stress. In mammals, the CNC family members nuclear factor-erythroid 2 p45-related factors 1 and 2 (Nrf1 and Nrf2) have been shown to be involved in the transcriptional up-regulation of cytoprotective genes including those encoding glutamate cysteine ligase, NAD(P)H:quinone oxidoreductase, glutathione S-transferases and aldo-keto reductases. An evolutionarily conserved system exists in Caenorhabditis elegans, and it is possible that Drosophila melanogaster may also utilize CNC transcription factors to induce antioxidant genes in response to pro-oxidant chemicals. The advent of microarray and proteomic technologies has advanced our understanding of the gene batteries regulated by oxidative insult, but has highlighted the complexity of gene regulation by environmental factors. This review focuses on the antioxidant response to environmental stress, and the impact that microarrays and proteomics have made in this field.

Ouabain, a sodium pump (Na+/ K+-ATPase) inhibitor, has been shown to act as a hormone and is possibly involved in the pathogenesis of hypertension. The mechanism by which ouabain may act was investigated using primary cultures of human umbilical artery endothelial cells (HUAECs), which are known to express and release the vasoconstrictive hormone endothelin (ET-1). Five minutes after application, low concentrations of ouabain induced Ca2+ oscillations and stimulated ET-1 release from endothelial cells into the medium. To investigate whether the observed effects were due to inhibition of the sodium pump, the effects of ouabain on the uptake of 86Rb+ by HUAECs were examined. Unexpectedly, ouabain concentrations below 10 nm stimulated 86Rb+ uptake by 15–20%, and in some experiments by 50%, results that are consistent with a stimulation of the pump. Within the concentration range 1–10 nm, ouabain induced a 2.5-fold stimulation (phosphorylation) of mitogen-activated protein kinase (MAP kinase). After incubation of HUAECs with ouabain for 12 h, the glycoside stimulated cell growth by 49 ± 4%, as measured by cell number, with a maximum response at 5 nm. At similar concentrations, ouabain also increased ET-1 mRNA abundance by 19.5 ± 3.1%. The results indicate that, by influencing ET-1 expression and release, ouabain may contribute to the regulation of vascular tone. The data also confirm that it is not a global inhibition of the sodium pump that is involved in the mechanism of action of this cardiac glycoside.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.

The Rev protein of human immunodeficiency virus is a nuclear shuttling protein that promotes nuclear export of mRNAs that encode the viral structural proteins Gag, Pol, and Env. Rev binds to a highly structured RNA motif, the Rev-responsive element (RRE), that is present in all Rev-responsive viral transcripts and facilitates their entry into a nuclear export pathway by recruiting cellular export factors. In mammalian and yeast cells, the principal export receptor engaged by Rev has been identified as the importin/transportin family member CRM1/exportin 1. CRM1 binds directly to a leucine-rich nuclear export sequence (NES) present in Rev, and similar motifs have been identified in a variety of cellular nuclear shuttling proteins. We and our colleagues previously demonstrated that, in transfected Drosophila cells, HIV-1 Rev is fully functional and promotes expression of the viral envelope glycoprotein. We now demonstrate that the fundamental mechanism of Rev action in insect cells is identical to that observed in the mammalian systems. In particular, we show that Drosophila cells express a leptomycin B-sensitive homologue of human CRM1 that supports Rev-dependent gene expression and is required for nuclear export of NES-containing proteins in insect cells.

We have established a collection of 2460 lethal or semi-lethal mutant lines using a procedure thought to insert single P elements into vital genes on the third chromosome of Drosophila melanogaster. More than 1200 randomly selected lines were examined by in situ hybridization and 90% found to contain single insertions at sites that mark 89% of all lettered subdivisions of the Bridges' map. A set of chromosomal deficiencies that collectively uncover ~25% of the euchromatin of chromosome 3 reveal lethal mutations in 468 lines corresponding to 145 complementation groups. We undertook a detailed analysis of the cytogenetic interval 86E-87F and identified 87 P-element-induced mutations falling into 38 complementation groups, 16 of which correspond to previously known genes. Twenty-one of these 38 complementation groups have at least one allele that has a P-element insertion at a position consistent with the cytogenetics of the locus. We have rescued P elements and flanking chromosomal sequences from the 86E-87F region in 35 lines with either lethal or genetically silent P insertions, and used these as probes to identify cosmids and P1 clones from the Drosophila genome projects. This has tied together the physical and genetic maps and has linked 44 previously identified cosmid contigs into seven “super-contigs” that span the interval. STS data for sequences flanking one side of the P-element insertions in 49 lines has identified insertions in the αγ element at 87C, two known transposable elements, and the open reading frames of seven putative single copy genes. These correspond to five known genes in this interval, and two genes identified by the homology of their predicted products to known proteins from other organisms.

abnormal spindle , a gene required for normal spindle structure and function in Drosophila melanogaster , lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp 1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of ∼124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH 2 -terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34 cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.

A physical map of the Drosophila melanogaster genome is being assembled, consisting of ordered overlapping cosmid clones. The map is constructed in steps, separately for each chromosomal division. Gaps in this map are to be bridged with yeast artificial chromosome clones. Hybridization to previously cloned genes and extensive use of in situ hybridization to polytene chromosomes ensure that the cosmid map is firmly anchored to the wealth of available genetic and cytogenetic information. The intention is to make the physical map widely available as part of an overall, integrated genetic resource for the Drosophila research community.

At the transition from meiosis to cleavage mitoses, Drosophilarequires the cell cycle regulators encoded by the genes, giant nuclei(gnu), plutonium (plu) and pan gu(png). Embryos lacking Gnu protein undergo DNA replication and centrosome proliferation without chromosome condensation or mitotic segregation. We have identified the gnu gene encoding a novel phosphoprotein dephosphorylated by Protein phosphatase 1 at egg activation. Gnu is normally expressed in the nurse cells and oocyte of the ovary and is degraded during the embryonic cleavage mitoses. Ovarian death and sterility result from gnu gain of function. gnu function requires the activity of pan gu and plu.

Glutamate-cysteine ligase (GCL) plays an important role in regulating glutathione homeostasis. In mammals, it comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence of a modifier subunit in invertebrates has not been described to date. We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM). A putative DmGCLM was obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC (DmGCLC) and the candidate DmGCLM were expressed separately in Escherichia coli, purified, mixed, and then subjected to gel filtration, where they eluted as an approximately 140-kDa complex. DmGCLC co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting that they also associate in vivo. Enzyme kinetic analyses showed that DmGCLC has a K(m) for glutamate of 2.88 mm, but when complexed with DmGCLM, the K(m) for glutamate is 0.45 mm. Inhibition of DmGCLC activity by glutathione was found to be competitive with respect to glutamate (K(i) = 0.03 mm), whereas inhibition of the GCL complex was mixed (K(i) = 0.67 mm), suggesting allosteric effects. In accordance with this, DmGCLC and DmGCLM have the ability to form reversible intermolecular disulfide bridges. A further mechanism for control of D. melanogaster GCL was found to be induction of DmGCLC by tert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.

We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome ofDrosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites ofP-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through “shotgun” sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.[All of the sequences analyzed in this paper have been deposited in the EMBL-Bank database under the following accession nos.: AL009146,AL009147, AL009171, AL009188–AL009196, AL021067, AL021086,AL021106–AL021108, AL021726, AL021728, AL022017, AL022018, AL022139,AL023873, AL023874, AL023893, AL024453, AL024455–AL024457, AL024485,AL030993, AL030994, AL031024–AL031028, AL031128, AL031173, AL031366,AL031367, AL031581–AL031583, AL031640, AL031765, AL031883, AL031884,AL034388, AL034544, AL035104, AL035105, AL035207, AL035245, AL035331,AL035632, AL049535, AL050231, AL050232, AL109630, AL121804, AL121806,AL132651, AL132792, AL132797, AL133503–AL133506, AL138678, AL138971,AL138972, and Z98269. A single file (FASTA format) of the 2.6-Mb contig is available fromftp://ftp.ebi.ac.uk/pub/databases/edgp/contigs/contig_1.fa.]

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101lcd. To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIβ. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the ∼120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes ∼13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Mutations in the rough deal (rod) gene of Drosophila greatly increase the missegregation of sister chromatids during mitosis, suggesting a role for this gene product in spindle or kinetochore function. The activity provided by rod also appears to be necessary for the recruitment of two known kinetochore components, Zw10 and cytoplasmic dynein. In this paper we describe the cloning of rough deal and an initial cytological characterization of its product. The Rod protein shares no identifiable structural motif with other known proteins, although apparent homologs exist in the genomes of nematode and man. By immunocytochemistry we show that Rod displays a dynamic intracellular staining pattern, localizing first to kinetochores in prometaphase, but moving to kinetochore microtubules at metaphase. Early in anaphase the protein is once again restricted to the kinetochores, where it persists until the end of telophase. This behavior is in all respects similar to that described for Zw10, and suggests that the proteins function together.

Complementation analysis had suggested that the Drosophila melanogaster genome contains approximately 5000 genes, but it is now generally accepted that the actual number is several times as high. We report here an analysis of 1788 anonymous sequence tagged sites (STSs) from the European Drosophila Genome Project (EDGP), totalling 463 kb. The data reveal a substantial number of previously undescribed potential genes, amounting to 6.1% of the number of Drosophila genes already in the sequence databases.

Polytene chromosomes were first described, in Chironomus, by Balbiani (1881). However it was not until the 1930s that their importance in genetic research was realized. Calvin Bridges (1935) created a map of Drosophila melanogaster third instar larval salivary gland polytene chromosomes that is not only still used to this day, but is the model for polytene maps of many other species. Polytene chromosomes are widespread within the class Diptera, and occur in various tissues in different species.

A physical map of the euchromatic X chromosome of Drosophila melanogaster has been constructed by assembling contiguous arrays of cosmids that were selected by screening a library with DNA isolated from microamplified chromosomal divisions. This map, consisting of 893 cosmids, covers approximately 64% of the euchromatic part of the chromosome. In addition, 568 sequence tagged sites (STS), in aggregate representing 120 kb of sequenced DNA, were derived from selected cosmids. Most of these STSs, spaced at an average distance of approximately 35 kb along the euchromatic region of the chromosome, represent DNA tags that can be used as entry points to the fruitfly genome. Furthermore, 42 genes have been placed on the physical map, either through the hybridization of specific probes to the cosmids or through the fact that they were represented among the STSs. These provide a link between the physical and the genetic maps of D. melanogaster. Nine novel genes have been tentatively identified in Drosophila on the basis of matches between STS sequences and sequences from other species.

Complementary DNA encoding a catalytic subunit of protein phosphatase 1, PP1 87B, hybridises at four positions (87B, 9C, 13C and 96A) to Drosophila melanogaster polytene chromosomes, three of which are known to be expressed [Dombradi, V., Axton, J. M., Brewis, N. D., Da Cruz e Silva, E. F., Alphey, L. & Cohen, P. T. W. (1990) Eur. J. Biochem. 194, 739–745]. The fourth gene has been isolated by screening a genomic library of cosmid clones, representing division 13 of the X-chromosome of D. melanogaster, with a PP1 87B probe. This library was constructed as part of the Drosophila genome mapping project [Siden-Kiamos, I., Saunders, R. D. C., Spanos, L., Majerus, T., Trenear, J., Savakis, C., Louis, C., Glover, D. M., Ashburner, M. & Kafatos, F. C. (1990) Nucleic Acids Res. 18, 6261–6270]. The 5′ non-coding region of the isolated gene hybridised to cytological position 13C1–2. By combining reverse transcription and the polymerase chain reaction, the gene was shown to be expressed at a very low level. The PP1 13C gene encodes a protein of 302 amino acids with a predicted molecular mass of 34.5 kDa. It shows 85–94% amino acid identity to the other three protein phosphatase 1 catalytic subunits (PP1 87B, PP1 96A and PP1 9C) described previously, being most closely related to the isoform PP1 87B, which is involved in the control of chromosome separation at cell division and the regulation of chromosome condensation at interphase.

To identify sequences from the centromeric region, we have constructed a Drosophila melanogaster yeast artificial chromosome (YAC) library and screened it with purified DNA from the minichromosome Dp(1;f)1187 derived from the X chromosome. We describe the structure of one clone isolated in this way. This YAC is structurally unstable and contains tandemly repeated G+C-rich 11-mer and 12-mer units, which we call dodeca satellite. Most of this satellite is located near the centromere of an autosome. Cross-hybridizing sequences are found in the genomes of organisms as distant as Arabidopsis thaliana and Homo sapiens.

We have cloned the three rows (thr) gene, by a combination of chromosome microdissection and P element tagging. We describe phenotypes of embryos homozygous for mutations at the thr locus. Maternal mRNA and protein appear to be sufficient to allow 14 rounds of mitosis in embryos homozygous for thr mutations. However, a small percentage of cells in syncytial blastoderm stage thr embryos sink into the interior of the embryo as if they have failed to divide properly. Following cellularisation all cells complete mitosis 14 normally. All cells become delayed at mitosis 15 with their chromosomes remaining aligned on the spindle in a metaphase-like configuration, even though both cyclins A and B have both been degraded. As cyclin B degradation occurs at the metaphase-anaphase transition, subsequent to the microtubule integrity checkpoint, the delay induced by mutations at the thr locus defines a later point in mitotic progression. Chromosomes in the cells of thr embryos do not undertake anaphase separation, but remain at the metaphase plate. Subsequently they decondense. A subset of nuclei go on to replicate their DNA but there is no further mitotic division.

Summary Many aspects of the mitotic cycle can take place independently in syncytial Drosophila embryos. Embryos from females homozygous for the mutation gnu undergo rounds of DNA synthesis without nuclear division to produce giant nuclei, and at the same time show many cycles of centrosome replication (Freeman et al. 1986). S phase can be inhibited in wild-type Drosophila embryos by injecting aphidicolin, in which case not only do centrosomes replicate, but chromosomes continue to condense and decondense, the nuclear envelope undergoes cycles of breakdown and reformation, and cycles of budding activity continue at the cortex of the embryo (Raff and Glover, 1988). If aphidicolin is injected when nuclei are in the interior of the embryo, centrosomes dissociate from the nuclei and can migrate to the cortex. Pole cells without nuclei then form around those centrosomes that reach the posterior pole (Raff and Glover, 1989); the centrosomes presumably must interact with polar granules, the maternally-provided determinants for pole cell formation. The pole cells form the germ-line of the developing organism, and as such may have specific requirements for mitotic cell division. This is suggested by our finding that a specific class of cyclin mRNAs, the products of the cyclin B gene, accumulate in pole cells during embryogenesis (Whitfield et al. 1989). Other genes that are essential for mitosis in early embryogenesis and in later development are discussed.

The three yolk proteins (YP1, YP2 and YP3) of Drosophila melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated in the developing oocytes to provide a nutrient source for embryogenesis. We have described the phenotype of a temperature-sensitive female-sterile mutant, fs (1) K313, nad characterised its yolk proteins. This mutation affects the secretion of YP2 and is the first mutation affecting YP2 to be described. Using genetic and molecular tests we argue that the female-sterile phenotype results, at least in part, from the abnormal secretion of YP2 perturbing the follicle cell secretory pathway in general and thus causing defects in chorion protein secretion. The gene coding for YP2 in fs (1) K313 has been cloned and sequenced. Two amino acid substitutions have been found which probably cause the abnormal secretion of YP2 and the resulting female-sterile phenotype.

The female-sterile mutants fs(1) 1163 of Drosophila melanogaster described by Gans et al. (1975) has been characterised as a yolk protein 1 (YP1) secretion mutant (Bownes and Hames 1978b; Bownes and Hodson 1980). We have cloned and sequenced the YP1 gene from this strain, and the strain in which the mutant was induced. One amino acid substitution was found in the predicted polypeptide sequence, an isoleucine to asparagine change at position 92. The sequence of the leader peptide was identical to previously published YP1 sequences. The possible effects of the amino acid change were investigated by computer analysis, which suggests there is no major alteration of secondary structure, but that a hydrophobic region in YP1 is lost in the mutant. This may affect higher order structure.

γ-Glutamylcysteine synthetase (GCS) catalyses a critical, rate-limiting step in glutathione synthesis. In this study we describe the isolation and characterisation of a GCS cDNA (pDmGCS4.3.3) from Drosophila melanogaster by functional complementation of a Saccharomyces cerevisiae gsh1 mutant. Expression of pDmGCS4.3.3 in the yeast mutant partially restored glutathione levels and conferred resistance to methylglyoxal. The pDmGCS4.3.3 cDNA was found to be approx. 4.6 kb in length, containing a 2 kb fragment encoding an open reading frame with a high degree of deduced amino acid sequence identity with previously reported GCS sequences. In situ hybridisation revealed that the Drosophila GCS gene maps to 7D6–9 on the X chromosome.

Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.