Dominant-negative mutant dynein allows spontaneous centrosome assembly, uncouples chromosome and centrosome cycles.

Cleavage cycles commence and chromosome and centrosome cycles proceed in harmony following fertilization of Drosophila eggs and completion of the meiotic divisions. The sperm-introduced centrioles replicate, separate, and while recruit pericentriolar material centrosomes (CS) form. The CS nucleate asters of microtubules (MT). Spindles form following interaction of some astral MT with kinetochores. In unfertilized eggs, chromosomes do not replicate, and CS and MT asters never form, although their components are present in the egg cytoplasm; unknown mechanisms prevent chromosome replication and CS and MT assembly. In unfertilized Laborc(D) eggs, rudimentary CS assemble spontaneously and instantaneously and nucleate small MT asters. In fertilized Laborc(D) eggs, normal CS form and organize normal asters. However, the CS replicate prior to accomplishment of the first mitosis, and spindles with multiple CS develop. In fertilized Laborc(D) eggs, while the chromosome cycles cease, CS cycles proceed as in wild type. Knowing that Laborc(D) is a dominant-negative mutation and encodes the formation of mutant cytoplasmic dynein heavy chain molecules, we show here that cytoplasmic dynein is involved in prevention of CS assembly in unfertilized eggs and establishing harmony between the chromosome and the CS cycles.

The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation.

The Ketel(D) dominant female-sterile mutations and their ketel(r) revertant alleles identify the Ketel gene, which encodes the importin-beta (karyopherin-beta) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel(D) eggs deposited by the Ketel(D)/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel(D) eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel(+) transgenes slightly reduce effects of the Ketel(D) mutations. The paternally derived Ketel(D) alleles act as recessive zygotic lethal mutations: the Ketel(D)/- hemizygotes, like the ketel(r)/ketel(r) and the ketel(r)/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel(D)-related defects originate most likely following association of the Ketel(D)-encoded mutant molecules with a maternally provided partner. As in the Ketel(D) eggs, embryogenesis does not commence in eggs of germline chimeras with ketel(r)/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel(D) mutations. The ketel(r) homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.

Recombination and disjunction in female germ cells of Drosophila depend on the germline activity of the gene sex-lethal.

Gametogenesis in males and females differs in many ways. An important difference in Drosophila is that recombination between homologous chromosomes occurs only in female meiosis. Here, we report that this process relies on the correct functioning of Sex-lethal (Sxl) which is primarily known as the master gene in somatic sex determination. Certain alleles of this gene (Sxl(fs)) disrupt the germline, but not the somatic function of Sxl and cause an arrest of germ cell development during cystocyte proliferation. Using dominant suppressor mutations that relieve this early block in Sxl(fs) mutant females, we discovered additional requirements of Sxl for normal meiotic differentiation of the oocyte. Females mutant for Sxl(fs) and carrying a suppressor become fertile, but pairing of homologous chromosomes and formation of chiasmata is severely perturbed, resulting in an almost complete lack of recombinants and a high incidence of non-disjunction events. Similar results were obtained when germline expression of wild-type Sxl was compromised by mutations in virilizer (vir), a positive regulator of Sxl. Ectopic expression of a Sxl transgene in premeiotic stages of male germline development, on the other hand, is not sufficient to allow recombination to take place, which suggests that Sxl does not have a discriminatory role in this female-specific process. We propose that Sxl performs at least two tasks in oogenesis: an 'early' function in formation of the egg chamber, and a 'late' function in progression of the meiotic cell cycle, suggesting that both events are coordinated by a common mechanism.

The Tomaj mutant alleles of alpha Tubulin67C reveal a requirement for the encoded maternal specific tubulin isoform in the sperm aster, the cleavage spindle apparatus and neurogenesis during embryonic development in Drosophila.

The three dominant TomajD and their eleven revertant (TomajR) alleles have been localized to the alpha Tubulin67C gene of Drosophila melanogaster. Although the meiotic divisions are normally completed in eggs laid by TomajD/+, TomajD/-, TomajR/- females, embryogenesis arrests prior to the gonomeric division. The arrest is caused by: (1) the failure of prominent sperm aster formation; and (2) a consequent lack of female pronuclear migration towards the male pronucleus. Concomitant with the sperm aster defect, the four female meiotic products fuse (tetra-fusion), similar to what is seen in eggs of wild-type virgin females. In eggs of females heterozygous for weaker TomajR alleles, embryogenesis comes to a cessation before or shortly after cortical migration of cleavage nuclei. The apparent source of embryonic defect is the cleavage spindle apparatus. One of the three TomajD alleles is cold-sensitive and its cold-sensitive period coincides with the completion of female meiosis and pronuclear migration. Disorganized central and peripheral nervous systems are also characteristic of embryos derived from the temperature-sensitive TomajD/+ females. The Tomaj mutant phenotypes indicate an involvement of the normal alpha Tubulin67C gene product in: (1) the formation of the sperm aster; (2) cleavage spindle apparatus formation/function; and (3) the differentiation of the embryonic nervous system. The TomajD alleles encode a normal-sized alpha Tubulin67C isotype. Sequence analyses of the TomajD alleles revealed the replacement in different positions of a single negatively charged or neutral amino acid with a positively charged one. These residues presumably identify important functional sites.

The Drosophila kinesin-like protein KLP3A is required for proper behavior of male and female pronuclei at fertilization.

Drosophila melanogaster females homozygous for mutations in the gene encoding the kinesin-like protein KLP3A are sterile (Williams et al., 1995). We have investigated the basis of this sterility. The eggs produced by KLP3A mutant mothers are fertilized by sperm, and female meiosis appears to occur normally. However, the large majority of these embryos arrest their development soon thereafter with a characteristic phenotype. The four nuclei produced by female meiosis associate together in a polar body-like structure, while a bipolar spindle is established around the metaphase-arrested male pronucleus. Thus, the major defect caused by depletion of the KLP3A protein is either in specification of the female pronucleus, or in migration of the male and female pronuclei toward each other. We have also found that the KLP3A protein is located throughout the metaphase spindle during meiosis and the early embryonic mitotic divisions, but later accumulates specifically at the midzone of these same spindles during telophase. The protein is also present on two other microtubule structures: the sperm aster; and the radial, monastral array of microtubules established between the two meiosis II spindles. We discuss these results in light of possible functions of the KLP3A protein in pronuclear specification and migration.

Horka, a dominant mutation of Drosophila, induces nondisjunction and, through paternal effect, chromosome loss and genetic mosaics.

Fs(3)Horka (Horka) was described as a dominant female-sterile mutation of Drosophila melanogaster. Genetic and cytological data show that Horka induces mostly equational nondisjunction during spermatogenesis but not chromosome loss and possesses a dominant paternal effect: the X, second, third and the fourth chromosomes, but not the Y, are rendered unstable while undergoing spermatogenesis and may be lost in the descending zygotes. The frequency of Horka-induced chromosome loss is usually 2-4% but varies with the genetic background and can be over 20%. The X chromosome loss occurs during the gonomeric and the initial cleavage divisions. Loss of the X and fourth chromosomes shows no correlation. We propose, based on similarities in the mutant phenotypes with the chromosome destabilizing mutations nonclaret disjunctional and paternal loss, that the normal Horka+ product is required for function of the centromeres and/or nearby regions. Horka is a convenient tool for the generation of gynandromorphs, autosome mosaics and for the study of gene expression in mosaics.

Differential mechanisms governing segregation of a univalent in oocytes and spermatocytes of Drosophila melanogaster.

In tricomplex heterozygotes in Drosophila melanogaster three metacentric autosomes (the TRI chromosomes) appear as a trivalent in meiosis while one autosome consisting of two homologous arms attached to the same centromere (a compound) behaves as an obligatory univalent. Cytological analysis of meiosis of tri-complex heterozygotes indicates that in oocytes the univalent compound behaves non-independently in relation to segregation of the trivalent. The compound is distributed preferentially to the same pole as one TRI chromosome. In spermatocytes the compound is distributed at random. In some oocytes the directed segregation is shown to be due to a disjunctional interaction between the compound and one partner of the trivalent at the same time as the other two chromosomes of the trivalent are separating from each other. The basic difference between the segregational mechanisms in the two sexes is discussed with a review of evidence indicating that in males segregation is determined by physical linkage that produces a stable orientation of the homologues at metaphase I. On the other hand, both genetic and cytological evidence indicate that in females a physical linkage (a chiasma) is non-essential for maintenance of co-orientation and stability after the onset of prometaphase. Genetic and cytological evidence support the hypothesis that disjunction is predetermined by non-random arrangement of the centromeric regions of chromosomes in the chromocentre - a suprachromosomal organization characteristic of maturing oocytes.

Mechanisms contributing to non-recovery of translocations induced in meiotic cells.

A cytological analysis of 22 and 7 autosomal (2;3) translocations induced, respectively, in mature and immature oocytes of Drosophila melanogaster appeared to be random in the distribution of the breaks. This finding is contrary to that expected if, as suggested by the mechanism of directed disjunction, the chromosomes involved in interchange at the centric region tended to be distributed to opposite poles at division I of meiosis. In each arm, the breaks were distributed more or less at random between the centromere and the telomere. However, in the translocations from the immature oocytes, the break-telomere distances of the segments interchanged showed a positive correlation, indicating that translocations induced in meiotic prophase and having a highly disproportionate length of segments interchanged are prone to be eliminated at division II by non-random disjunction of heteromorphic dyads.

Terminal chromosome attachments.

Descriptions are presented of four cases of attachment of chromosome material at the ends of normal chromosomes in Drosophila. Since no material appears to be missing from the polytene chromosomes and there are no ill effects to the organism in morphology, viability, or fertility when the chromosome is made homozygous, it is argued that the attachment occurred without the loss of any essential genetic material and that, in all probability, the break at the end of the chromosome occurred within the telomere of the chromosome. These cases may serve as a parallel to cases of apparent terminal breakage and reunion in certain rearrangements in man.