The ZW10 and Rough Deal checkpoint proteins function together in a large, evolutionarily conserved complex targeted to the kinetochore.

The zeste-white 10 (zw10) and rough deal (rod) genes of Drosophila both encode kinetochore components, and mutations in either gene greatly increase the missegregation of sister chromatids during mitosis. Here, we present genetic, cytological and biochemical evidence for a close, evolutionarily conserved relationship between the ROD and ZW10 proteins. We show that the phenotypes caused by disruption of either gene's function are similar in Drosophila and in C. elegans. No additive effects are observed in zw10; rod double null mutants. In flies, the two proteins always colocalize and, moreover, require each other for their recruitment to the mitotic apparatus. The human ROD and ZW10 homologs also colocalize on HeLa cell kinetochores or kinetochore microtubules throughout most but not all of mitosis. Finally, we show that in both Drosophila and human cells, ROD and ZW10 are in fact physically associated, and in Drosophila these proteins are together constituents of a large (700-900 kDa), soluble macromolecular complex.

Rough deal and Zw10 are required for the metaphase checkpoint in Drosophila.

The metaphase-anaphase transition during mitosis is carefully regulated in order to assure high-fidelity transmission of genetic information to the daughter cells. A surveillance mechanism known as the metaphase checkpoint (or spindle-assembly checkpoint) monitors the attachment of kinetochores to the spindle microtubules, and inhibits anaphase onset until all chromosomes have achieved a proper bipolar orientation on the spindle. Defects in this checkpoint lead to premature anaphase onset, and consequently to greatly increased rates of aneuploidy. Here we show that the Drosophila kinetochore components Rough deal (Rod) and Zeste-White 10 (Zw10) are required for the proper functioning of the metaphase checkpoint in flies. Drosophila cells lacking either ROD or Zw10 exhibit a phenotype that is similar to that of bub1 mutants - they do not arrest in metaphase in response to spindle damage, but instead separate sister chromatids, degrade cyclin B and exit mitosis. These are the first checkpoint components to be identified that do not have obvious homologues in budding yeast.

Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila.

We describe a new mitotic locus of Drosophila melanogaster required for the progression through mitosis in the syncytial embryo and in late larval development. The locus aar (abnormal anaphase resolution) maps to the cytological interval 85E7-F16 and was identified by two alleles. The aar1 allele causes pupal lethality. Larval neuroblasts show an elevated mitotic index with high chromosome condensation and stretched and lagging chromatids during anaphase. aar2 produces fully viable but sterile females. aar1/aar2 females lay eggs that develop mitotic figures with similar abnormalities to those observed in neuroblasts. Indirect immunofluorescence of these embryos indicates that the centrosome cycle appears normal, although some abnormal spindle microtubules can be seen during mitosis.

Structurally distinct genes for the surface protease of Leishmania mexicana are developmentally regulated.

gp63 is a highly abundant glycosylphosphatidylinositol (GPI)-anchored membrane protein expressed in both the promastigote and the amastigote forms of Leishmania species. In Leishmania mexicana, gp63 exists as a heterogeneous family of proteins that are differentially processed and localized during the 2 developmental stages. In this study we determined the molecular organization of the L. mexicana gp63 gene family, demonstrating that the gp63 genes fall into 3 linked families of tandemly repeated, but structurally distinct, entities designated as C1, C2 and C3. The C1 and C2 gene clusters contain 4-5 copies each, while the C3 gene may be single copy. Whilst promastigotes contain transcripts from all 3 gene classes, the intracellular amastigote only expresses detectable transcript from the C1 gene class. Moreover, the sequence of the C1 genes predicts a unique carboxy terminus substantially different from the GPI anchor addition signal sequence found in other Leishmania spp. and which has characteristics incompatible with substitution with a GPI anchor. These findings have significance for both the diversity of gp63 and for the regulation of tightly clustered, tandem gene arrays.

polo encodes a protein kinase homolog required for mitosis in Drosophila.

We show that mutation in polo leads to a variety of abnormal mitoses in Drosophila larval neuroblasts. These include otherwise normal looking mitotic spindles upon which chromosomes appear overcondensed; normal bipolar spindles with polyploid complements of chromosomes; bipolar spindles in which one pole can be unusually broad; and monopolar spindles. We have cloned the polo gene from a mutant allele carrying a P-element transposon and sequenced cDNAs corresponding to transcripts of the wild-type locus. The sequence shows that polo encodes a 577-amino-acid protein with an amino-terminal domain homologous to a serine-threonine protein kinase. polo transcripts are abundant in tissues and developmental stages in which there is extensive mitotic activity. The transcripts show no obvious spatial pattern of distribution in relation to the mitotic domains of cellularized embryos but are specifically concentrated in dividing cells in larval discs and brains. In the cell cycles of both syncytial and cellularized embryos, the polo kinase undergoes cell cycle-dependent changes in its distribution: It is predominantly cytoplasmic during interphase; it becomes associated with condensed chromosomes toward the end of prophase; and it remains associated with chromosomes until telophase, whereupon it becomes cytoplasmic.

The regulatory light chain of nonmuscle myosin is encoded by spaghetti-squash, a gene required for cytokinesis in Drosophila.

Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh1, a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis failure and other defects in sqh1. Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.

rough deal: a gene required for proper mitotic segregation in Drosophila.

We describe a genetic locus rough deal (rod) in Drosophila melanogaster, identified by mutations that interfere with the faithful transmission of chromosomes to daughter cells during mitosis. Five mutant alleles were isolated, each associated with a similar set of mitotic abnormalities in the dividing neuroblasts of homozygous mutant larvae: high frequencies of aneuploid cells and abnormal anaphase figures, in which chromatids may lag, form bridges, or completely fail to separate. Surviving homozygous adults are sterile, and show cuticular defects associated with cell death, i.e., roughened eyes, sparse abdominal bristles, and notched wing margins. The morphological process of spermatogenesis is largely unaffected and motile sperm are produced, but meiocyte aneuploidy is common. The nature of the observed abnormalities in mitotic cells suggests that the reduced fidelity of chromosome transmission to the daughter cells is due to a failure in a mechanism involved in assuring the proper release of sister chromatids.

The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage.

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of 'membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface.

Mitosis in Drosophila development.

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.

Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosomal locus and are constitutively transcribed.

The major surface glycoprotein of Leishmania, gp63, is encoded by a small multi-gene family of tandemly linked genes which map to a single chromosome. For Leishmania major, there are five 3.1 kilobasepair (kb) direct repeat units which include a 1.8-kb open reading frame and a 1.3-kb intergenic or spacer region. In addition, there is a single gene copy linked as a direct repeat but separated from the tandem array of gp63 genes by about 8 kb. The restriction enzyme map of the repeat unit is highly conserved among the gene copies. The regions which flank the tandemly repeated genes diverge outside of the 3.1-kb repeat unit. Transcription of the gp63 gene locus is constitutive as the 3-kb transcript was present in promastigotes as well as in amastigotes. A minor 6-kb gp63 gene transcript was also detected in Northern blot analysis which could signify the transcription of the genes as a polycistronic or multigene precursor RNA.

Analysis of P transposable element functions in Drosophila.

We have made a P-element derivative called Pc[ry], which carries the selectable marker gene rosy, but which acts like a nondefective, intact P element. It transposes autonomously into the germline chromosomes of an M-strain Drosophila embryo and it mobilizes in trans the defective P elements of the singed-weak allele. Frameshift mutations introduced into any of the four major open reading frames of the P sequence were each sufficient to eliminate the transposase activity, but none affected signals required in cis for transposition of the element. Complementation tests between pairs of mutant elements suggest that a single polypeptide comprises the transposase. We have examined transcripts of P elements both from natural P strains and from lines containing only nondefective Pc[ry] elements, and have identified two RNA species that appear to be specific for autonomous elements.

A small tandem duplication is responsible for the unstable white-ivory mutation in Drosophila.

The white-ivory (wi) mutation, an unstable allele of the white locus in Drosophila, reverts to wild-type at frequencies of 5 X 10(-5) in homozygous females, and 5 X 10(-6) in males and deletion heterozygous females. We show by molecular cloning and Southern blot analysis of DNA from wi flies that a 2.9 kilobase tandem duplication within the white locus is responsible for the mutation. Phenotypic reversion appears, in most cases, to be due to an exact excision of the extra copy of the sequence. Two derivative alleles of wi, one phenotypically wild-type, the other a partial revertant, carry insertions of moderately repetitive DNA from outside the locus, in addition to suffering deletions of some white locus DNA. Earlier genetic data preclude unequal crossing-over between homologs as an explanation for the precise reversions. Rather, an intrachromosomal meiotic event seems to be responsible. Our results suggest that intrachromosomal recombination may be responsible in other systems for a larger number of rearrangements than has been suspected, and that interallelic recombination frequencies in Drosophila do not always correlate in a simple way with DNA length or extent of homology.

Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus.

Expression of the src gene of Rous sarcoma virus (RSV) in chicken embryo neuroretinal (NR) cells results in morphological transformation and sustained proliferation of a normally resting cell population. We have previously reported the isolation of mutants of RSV which retain full growth-promoting activity while displaying reduced transforming properties. Two such mutants, PA101 and PA104, were used to investigate whether the p60src-associated kinase activity is required for the mitogenic function of src. A comparison of the patterns of phosphorylation of wild-type and mutant p60src revealed that the phosphorylation of tyrosine residues of p60src of PA104 was markedly reduced, whereas the relative amount of phosphotyrosine in p60src of PA101 was comparable to that of the wild-type protein. In vitro kinase activity of p60src immunoprecipitated from NR cells infected with PA101 or PA104 as measured by phosphorylation of the heavy chains of specific immunoglobulin G molecules was 1/10 that of the wild-type molecule. Moreover, when NR cells infected with mutants temperature sensitive for mitogenic capacity were maintained at a temperature either permissive or restrictive for cell growth, quantitation of kinase activity indicated that proliferation of NR cells could not be linked to the absolute level of in vitro kinase activity of p60src. Transformation of NR cells by wild-type RSV resulted in a 10-fold increase in total cellular phosphotyrosine and in the phosphorylation of tyrosine residues of a 34K protein, a possible in vivo substrate for p60src. In contrast, phosphorylation of tyrosine residues of cellular targets was markedly reduced in NR cells infected with PA101 or PA104. These results indicate that the mitogenic capacity of RSV in NR cells does not require elevated levels of p60src kinase activity.

Viral and cellular src genes contribute to the structure of recovered avian sarcoma virus transforming protein.

Recovered avian sarcoma viruses (rASVs) were obtained from tumors induced by certain transformation-defective (td) mutants of Schmidt-Ruppin strain Rous sarcoma virus of subgroup A (SR-A). The genomes of these td SR-A mutants lack most but not all of the src gene. rASV genomes, however, possess intact src genes, which are largely derived from cellular genetic information, presumably an endogenous cellular gene called c-src, which shares considerable homology with the viral src. To further define the genetic origin of rASV src, we examined by tryptic peptide analysis the product of this gene, pp60src, from rASV and SR-A, as well as the normal cellular homolog pp60c-src. We found peptides unique to each putative "parental" protein present together in maps of rASV p60src, demonstrating that the endogenous cellular c-src gene itself contributes to the structure of rASV pp60src. Certain isolates of rASV encode pp60srcS of altered apparent molecular weight. In these cases, the variation in structure was located in the amino-terminal portion of the protein. That such polymorphism can be tolerated suggests that this region of the protein is less critical to the ability of these agents to transform cells.

Characterization of the transforming gene of Fujinami sarcoma virus.

The src gene present in all avian sarcoma viruses is not present in the genome of Fujinami sarcoma virus, a potent sarcoma-inducing virus in chickens. Fujinami virus is defective and requires helper virus for replication. RNA from a mixture of helper and transforming viruses consists of two components, 35S and 28S. Oligonucleotide fingerprinting of each RNA component revealed that the 35S component was identical to the RNA of the helper virus. Thus, the genome of Fujinami virus must be the 28S RNA, which corresponds approximately to a molecular weight of 1.7 x 10(6) or 5300 nucleotides. Fujinami viral RNA shares several oligonucleotides with helper viral RNA at both 3' and 5' ends but contains a unique sequence of at least 3000 nucleotides in the middle of the genome. Fujinami viral RNA contains no src-specific oligonucleotides of the Rous sarcoma virus genome and did not hybridize with DNA complementary to the src sequences. The 60,000-dalton src protein of Rous sarcoma virus was undetectable in Fujinami virus-transformed cells. Instead, these transformed cells contain a protein of 140,000 daltons precipitable by antisera against virion proteins, which is likely to be the transforming protein of this virus.

Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to the resulting recombinant viruses genetic information for cell transformation.

Tryptic peptide analysis of avian oncovirus gag and pol gene products.

Radiolabeled tryptic peptides of the gag and pol gene products of avian oncoviruses were examined. This analysis included Rous-associated virus 2 structural proteins and the Pr76gag and P180gag-pol proteins in Rous-associated virus 2-infected chicken embryo cells. The methionine- and cysteine-containing tryptic peptides of virion internal structural proteins were present in both Pr76gag and P180gag-pol, suggesting that there was no loss of gag gene-coding sequences during the generation of P180gag-pol. No overlap of gag and pol gene structural information was detected. Analysis of intermediates in the processing of Pr76gag and translation inhibition mapping with pactamycin yielded the following order of structural proteins within the Rous-associated virus 2 Pr76gag precursor: NH2-p19-p12-p27-p15-COOH. The gag and pol sequences missing in the endogenous gsmp120 protein of uninfected gs+ chicken cells were identified by comparison with those of Rous-associated virus 2 P180gag-pol.

Cellular information in the genome of recovered avian sarcoma virus directs the synthesis of transforming protein.

Recovered avian sarcoma viruses, whose sarcomagenic information is largely derived from cellular sequences [Wang, L.-H., Halpern, C.C., Nadel, M. & Hanafusa, H. (1978) Proc. Natl. Acad. Sci. USA 75, 5812-5816], produce the transforming protein p60src in infected cells, in amounts comparable to the amount found in cells transformed by standard strains of avian sarcoma virus. Though displaying some virus-specific differences in electrophoretic mobility, p60srcs from these viruses are similar to those of other avian sarcoma virus strains by the criteria of (i) antigenicity, (ii) partial proteolysis mapping, and (iii) association with protein kinase activity. We also find that p60sarc, a protein present in normal cells at a low level, is associated with a protein kinase activity, and thus it too is similar by the above criteria to p60src of avian sarcoma virus. Possible causes for the pathogenicity of p60src are discussed in light of these similarities.