Mitotic protein kinases in Drosophila embryos.
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Animals↗
Biomedical subjects
D M Glover
Publications and source records attributed to D M Glover.
P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F.
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 approximately 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 alphagamma 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.
Alleles↗
P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development.
To identify novel genes and to isolate tagged mutations in known genes that are required for the development of the peripheral nervous system (PNS), we have screened a novel collection of 2460 strains carrying lethal or semilethal P element insertions on the third chromosome. Monoclonal antibody 22C10 was used as a marker to visualize the embryonic PNS. We identified 109 mutant strains that exhibited reproducible phenotypes in the PNS. Cytological and genetic analyses of these strains indicated that 87 mutations affect previously identified genes: tramtrack (n = 18 alleles), string (n = 15), cyclin A (n = 13), single-minded (n = 13), Delta (n = 9), neuralized (n = 4), pointed (n = 4), extra macrochaetae (n = 4), prospero (n = 3), tartan (n = 2), and pebble (n = 2). In addition, 13 mutations affect genes that we identified recently in a chemical mutagenesis screen designed to isolate similar mutants: hearty (n = 3), dorsotonals (n = 2), pavarotti (n = 2), sanpodo (n = 2), dalmatian (n = 1), missensed (n = 1), senseless (n = 1), and sticky ch1 (n = 1). The remaining nine mutations define seven novel complementation groups. The data presented here demonstrate that this collection of P elements will be useful for the identification and cloning of novel genes on the third chromosome, since >70% of mutations identified in the screen are caused by the insertion of a P element. A comparison between this screen and a chemical mutagenesis screen undertaken earlier highlights the complementarity of the two types of genetic screens.
Alleles↗
Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis.
Mutations in a gene (Klp38B) encoding a novel kinesin-like protein in Drosophila melanogaster lead to the formation of polyploid cells in the larval central nervous system and in the follicle cells of adult egg chambers. Some homozygous mutants survive to adulthood and also exhibit morphological defects indicative of abnormal cell cycle progression, including rough eyes, missing bristles, and abnormal abdominal cuticles. In larval brains, there is no accumulation of mitotic cells and the frequency of anaphase figures is comparable to wild type, suggesting that nuclear division is not affected. Such brains contain polyploid cells with metaphase and anaphase chromosomes associated with bipolar spindles. Such spindles have a number of unseparated centrosomes at their poles reflecting the degree of polyploidy of the cell. Follicle cells frequently contain two nuclei of roughly equal size. Taken together, we conclude that these Klp38B mutations lead to a failure of cytokinesis resulting in polyploidy, and discuss whether or not this is a direct effect of the mutation.
Amino Acid Sequence↗
Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II.
We have isolated a Drosophila gene, barren (barr), required for sister-chromatid segregation in mitosis. barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human. Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons. Centromeres move apart at the metaphase-anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging. This phenotype is similar to that described in TOP2 mutants in yeast. Barren protein localizes to chromatin throughout mitosis. Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II. We propose that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase.
Anaphase↗
The conserved mitotic kinase polo is regulated by phosphorylation and has preferred microtubule-associated substrates in Drosophila embryo extracts.
The Drosophila gene polo encodes a protein kinase required for progression through mitosis. Wild-type polo protein migrates as a tight doublet of 67 kDa which is converted to a single band by phosphatase treatment, which also inactivates the kinase. We have determined putative polo substrates in a cell-free system derived from mutant embryos. Exogenous polo protein kinase phosphorylates proteins of sizes 220 kDa, 85 kDa and 54 kDa, to a greater extent when added to extracts of polo(1)-derived embryos compared with extracts of wild-type embryos, which in both cases have been subject to mild heat treatment to inactivate endogenous kinases. Proteins of the same size are predominantly phosphorylated by the endogenous kinases present in wild-type extracts, and are either not phosphorylated or are poorly phosphorylated in extracts of polo(1)-derived embryos. We show that a specific monoclonal antibody to beta-tubulin precipitates the phosphorylated 54 kDa protein together with an associated 85 kDa protein also phosphorylated by polo protein kinase. Moreover polo binds to an 85 kDa protein which is enriched in microtubule preparations. We discuss the extent to which these in vitro phosphorylation results reflect the effects of mutations in polo on microtubule behaviour during the mitotic cycle.
Animals↗
Polo kinase: the choreographer of the mitotic stage?
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Animals↗
A maternal requirement for glutamine synthetase I for the mitotic cycles of syncytial Drosophila embryos.
We describe the maternal effect phenotype of a hypomorphic mutation in the Drosophila gene for glutamine synthetase I (GSI). The extent of development of embryos derived from homozygous mutant females is variable, although most mutant embryos fail to survive past germband elongation and none develop into larvae. These embryos are characterised by an increase in the number of yolk-like nuclei following nuclear migration to the cortex. These nuclei appear to fall into the interior of the embryo from the cortex at blastoderm. As they do so, the majority continue to show association with PCNA in synchrony with nuclei at the cortex, suggesting some continuity of the synchrony of DNA replication. However, the occurrence of nuclei that have lost cell cycle synchrony with their neighbours is not uncommon. Immunostaining of mutant embryos revealed a range of mitotic defects, ultimately resulting in nuclear fusion events, division failure or other mitotic abnormalities. A high proportion of these mitotic figures show chromatin bridging at anaphase and telophase consistent with progression through mitosis in the presence of incompletely replicated DNA. GSI is responsible for the ATP-dependent amination of glutamate to produce glutamine, which is required in the formation of amino acids, purines and pyrimidines. We discuss how the loss of glutamine could depress both protein and DNA synthesis and lead to a variety of mitotic defects in this embryonic system that lacks certain checkpoint controls.
Amino Acid Sequence↗
Mutations in new cell cycle genes that fail to complement a multiply mutant third chromosome of Drosophila.
We have simultaneously screened for new alleles and second site mutations that fail to complement five cell cycle mutations of Drosphila carried on a single third chromosome (gnu, polo, mgr, asp, stg). Females that are either transheterozygous for scott of the antartic (scant) and polo, or homozygous for scant produce embryos that show mitotic defects. A maternal effect upon embryonic mitoses is also seen in embryos derived from females transheterozygous with helter skelter (hsk) and either mgr or asp. cleopatra (cleo), fails to complement asp but is not uncovered by a deficiency for asp. The mitotic phenotype of larvae heterozygous for cleo and the multiple mutant chromosome is similar to weak alleles of asp, but there are no defects in male meiosis. Mutations that failed to complement stg fell into two complementation groups corresponding to stg and a new gene noose. Three of the new stg alleles are early zygotic lethals, whereas the fourth is a pharate adult lethal allele that affects both mitosis and meiosis. Mutations in noose fully complement a small deficiency that removes stg, but when placed in trans to certain stg alleles, result in late lethality and mitotic abnormalities in larval brains.
Alleles↗
The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.
We have identified a Schizosaccharomyces pombe gene with homology to the budding yeast gene CDC5, the Drosophila gene polo, and the mammalian family of genes encoding polo-like kinases. Disruption of this gene, plo1+, indicates that it is essential. Loss of plo1+ function leads to a mitotic arrest in which condensed chromosomes are associated with a monopolar spindle or to the failure of septation following the completion of nuclear division. In the latter case, cells show a failure both in the formation of an F-actin ring and in the deposition of septal material, suggesting that plo1+ function is required high in the regulatory cascade that controls septation. The overexpression of plo1+ in wild-type cells also results in the formation of monopolar spindles but also induces the formation of multiple septa without nuclear division. Septation can also be induced in the absence of mitotic commitment and concomitant spindle formation by the overexpression of plo1+ in cdc25-22 or cdc2-33 cells arrested in G2; in G1 cells arrested at Start by the cdc10-V50 mutation, or in cells lacking the cyclin B homolog cdc13 that undergo repeated S phases in the absence of mitosis.
Actins↗
Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles.
We show that female sterile mutations of aurora (aur) are allelic to mutations in the lethal complementation group ck10. This lies in a cytogenetic interval, 87A7-A9, that contains eight transcription units. A 250 bp region upstream of both aur and a divergent transcription unit corresponds to the site of a specific chromatin structure (scs') previously proposed to be a barrier to insulate enhancers of the major hsp70 gene at 87A7. Syncytial embryos derived from aur mothers display closely paired centrosomes at inappropriate mitotic stages and develop interconnected spindles in which the poles are shared. Amorphic alleles result in pupal lethality and in mitotic arrest in which condensed chromosomes are arranged on circular monopolar spindles. The size of the single centrosomal body in these circular figures suggests that loss of function of the serine-threonine protein kinase encoded by aur leads to a failure of the centrosomes to separate and form a bipolar spindle.
Alleles↗
A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen.
BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
The 190 kDa centrosome-associated protein of Drosophila melanogaster contains four zinc finger motifs and binds to specific sites on polytene chromosomes.
Microinjection of a bacterially expressed, TRITC labelled fragment of the centrosome-associated protein CP190 of Drosophila melanogaster, into syncytial Drosophila embryos, shows it to associate with the centrosomes during mitosis, and to relocate to chromatin during interphase. Indirect immunofluorescence staining of salivary gland chromosomes of third instar Drosophila larvae, with antibodies specific to CP190, indicate that the protein is associated with a large number of loci on these interphase polytene chromosomes. The 190 kDa CP190 protein is encoded by a 4.1 kb transcript with a single, long open reading frame specifying a polypeptide of 1,096 amino acids, with a molecular mass of 120 kDa, and an isoelectric point of 4.5. The central region of the predicted amino acid sequence of the CP190 protein contains four CysX2CysX12HisX4His zinc-finger motifs which are similar to those described for several well characterised DNA binding proteins. The data suggest that the function of CP190 involves cell cycle dependent associations with both the centrosome, and with specific chromosomal loci.
Amino Acid Sequence↗
Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase.
We have introduced an N-terminally truncated form of cyclin B into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (< 0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle; they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase.
Anaphase↗
Mutations in the Drosophila Melanogaster gene three rows permit aspects of mitosis to continue in the absence of chromatid segregation.
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Amino Acid Sequence↗
Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2.
The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A.
Alleles↗
A conserved mitotic kinase active at late anaphase-telophase in syncytial Drosophila embryos.
Mutations in the Drosophila gene polo cause abnormal mitotic and meiotic divisions. This gene encodes a 577-amino-acid protein that has an N-terminal putative kinase domain and a 300-residue C-terminal domain. In budding yeast, a homologous kinase is encoded by CDC5 (ref. 3), a gene required for nuclear division late in the mitotic cycle and during meiosis. Murine homologues have also been described. Here we show that the polo gene product immunoprecipitated from extracts of single Drosophila embryos can phosphorylate casein in vitro, and that the kinase activity peaks cyclically at late anaphase/telophase. This contrasts with the cyclical activity of cyclin B-associated p34cdc2 kinase, which is maximal upon entry into mitosis during the rapid cycles of mitosis in the syncytium.
Anaphase↗
The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.
The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.
Alternative Splicing↗