Luke Alphey

twine, a cdc25 homolog that functions in the male and female germline of drosophila

twine is the second homolog of the fission yeast gene cdc25 to be found in Drosophila. Both string and twine cDNAs can rescue a temperature-sensitive cdc25 mutation in fission yeast, but not a deletion. We detect the expression of string but not twine transcripts in the proliferating cells of newly cellularized embryos, in third instar larval brains, and in imaginal discs. Both genes are abundantly expressed in nurse cells during oogenesis, the maternal transcripts persisting throughout the syncytial stage of embryonic development. In the testis, twine transcripts are seen in the growing stage of premeiotic cysts. Analysis of a twine mutant suggests a requirement for the gene during oogenesis, during syncytial embryonic development, and for male meiosis. Meiosis does not occur in homozygous twine males, which produce cysts containing 16 rather than 64 spermatids.

Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string.

We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first ‘adapt’ pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1‐LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25‐22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase I

cDNA coding for protein phosphatase 2A (PP2A) has been isolated from Drosophila head and eye imaginal disc libraries. Drosophila PP2A mRNA is expressed throughout development, but is most abundant in the early embryo. The cDNA hybridises to a single site on the left area of the second chromosomes at position 28D2‐4. The deduced amino acid sequence (309 residues) of Drosophila PP2A shows 94% identity with either rabbit PP2Aα or PP2Aβ, indicating that PP2A maybe the most conserved of all known enzymes.

A Drosophila homologue of oxysterol binding protein OSBP - implications for the role of OSBP

The identification of a Drosophila homologue (OSBP-Dm) of mammalian oxysterol binding protein (OSBP) is reported. OSBP-Dm was identified by its ability to overcome the cell cycle arrest induced by over-expression of Wee1p in fission yeast. OSBP-Dm has an overall sequence identity of 52% with mammalian OSBP, and shows a number of highly conserved regions of functional significance. Insects are unable to biosynthesize the steroid core, relying instead on dietary sterols to satisfy their requirements. It is therefore unlikely that OSBP-Dm is involved in feedback inhibition of the mevalonate pathway, as has previously been suggested for its mammalian homologues.

Complete reversions of a gypsy retrotransposon-induced cut locus mutation in Drosopbila melanogaster involving jockey transposon insertions and flanking gypsy sequence deletions

SummaryWe have analysed the structures of three phenotypic revertant alleles of a gypsy retrotransposon-induced mutation at the cut locus of Drosophila melanogaster. All three revertants are associated with the insertion of jockey transposons into a common region of gypsy. Two of these alleles are complete reversions to wild type. One complete revertant (ct+D) is derived from a third allele, a partial revertant (ctMRPD) by a deletion of part of the gypsy sequence flanking the jockey transposon. Sequence differences between the jockey elements in ctMRpD and ct+D suggest that this deletion may have been created by the insertion of a second jockey near to the first, followed by recombinational excision of a composite jockey and the region between the two genetic elements. The other complete revertant also carries a deletion of gypsy DNA flanking the jockey insertion. The deleted regions of both complete revertants and the target region for all the jockey insertions contain a repeated sequence that resembles a transcriptional enhancer. The strength of the cut phenotype in these mutants correlates with the proportion of this region remaining near the gypsy transcriptional start site, suggesting that the jockey insertions relieve the gypsy-induced mutation at cut by interfering with a region which is required for the transcriptional competence of gypsy.

Instability in the ctMR2 strain of Drosophila melanogaster: Role of P element functions and structure of revertants

SummarySimultaneous multiple transpositions and longterm genetic instability have been described in the ctMR2 strain of Drosophila melanogaster and its derivatives. This strain originated from a cross that was dysgenic in the P-M system. While spontaneous instability declined over 2 years, instability has been reactivated by backcross to the progenitor P element bearing strain MRh12/Cy. We show here using germline transformation that active P factor alone cannot mimic the effect of this cross, suggesting that MRh12/Cy contains some other activator. In addition, we have observed that ct+ exceptional progeny arise in the F1 s well as the F2 generations. Molecular analysis of X chromosomes from some ct+ progeny indicates that phenotypic reversion of the ct mutation can arise through two unrelated mechanisms.

Cell cycle genes of Drosophila

Publisher Summary This chapter discusses the cell cycle genes of Drosophila. The chapter provides new insights into various aspects of the process of cell division and describes what is known about genes whose function is required for the cell cycle to a greater or lesser extent and the choice of Drosophila as a model system. The chapter discusses various aspects of Drosophila development that are more relevant for genetic analysis of cell division in this organism. The genetic analysis of cell division in Drosophila is the subject of several recent reviews. A most striking case of cell cycle control during development is provided by the tip cell that controls mitosis during development of the Malpighian tubule in D. melanogaster. The tip cell does not incorporate BUdR or divide during the stage at which other cells in the tubule are proliferating. Ablation experiments have shown that tubules without a tip cell do not undertake cell proliferation. Some gene products contain highly conserved motifs that are known to perform various biochemical activities and prompt a review and reinterpretation of the understanding of the phenotypes resulting from mutations in such genes.

The Meiotic Role of twine, A Drosophila Homologue of cdc25

cdc25 was first identified in fission yeast as a positive regulator of cdc2 (Russell and Nurse, 1986). Homologues of cdc25 have since been found in higher eukaryotes, including two from Drosophila. These are named string and twine (Edgar and O’Farrell, 1989, Jimenez et al., 1990). Bacterially expressed proteins from both of these genes have been shown to have tyrosine phosphatase activity and can activate the p34cdc2 kinase in vitro (Kumagai and Dunphy, 1991; Gautier et al., 1991; Alphey and Clarke, unpublished). Furthermore, both homologues are functional in fission yeast in that they will rescue a temperature-sensitive mutant, cde 25–22. This inter-specific complementation was the basis of the original isolation of twine (Jimenez et al., 1990), although the sequence similarity between cdc25 homologues from different species allowed Courtot et al. (1992) to obtain twine by PCR.