Jaakko Puro

Meiotic segregation of chromosomes in Drosophila melanogaster oocytes

A new technique was developed for a light microscopic analysis of meiosis in Drosophila oocytes. — When the nuclear envelope breaks down the bivalents, till then compressed into a karyosome, separate in early prometaphase. The homologues remain associated by chiasmata except for the fourth chromosomes which are no longer associated. Non-homologous chromosomes regularly segregating from each other in genetic experiments are also unconnected after karyosome disintegration but during metaphase I the fourth chromosomes and the heterologous pairs coorient on the same arc of the spindle and move precociously towards opposite poles. Nondisjunction and other irregularities are not infrequent in oocytes having an uneven number of achiasmatic elements. The fourth chromosomes and the Xs or the large autosomes, when lacking chiasmata, may be involved in non-homologous segregation. In c3G homozygotes all chromosomes appear as univalents in prometaphase. Segregation is variable but the observations suggest the polar distribution of equal numbers of chromosomes in variable combinations irrespective of the size. — Coorientation of univalents may be accounted for if the centromeres, whether homologous or non-homologous, are associated in pairs during early meiotic prophase, and that in the karyosome these pairing relationships are preserved until spindle organization at the onset of prometaphase.

The Drosophila Calcipressin Sarah Is Required for Several Aspects of Egg Activation

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila eggs competence to support male pronuclear maturation is acquired during activation.

TEMPORAL DISTRIBUTION OF X-RAY INDUCED RECESSIVE LETHALS AND RECOMBINANTS IN THE POST-STERILE BROODS OF DROSOPHILA MELANOGASTER MALES.

Abstract The distributions of 3rd chromosome recessive lethals and cross-overs were studied in daily post-sterile broods of Drosophila melanogaster males treated as adults with 3000 R X-rays and mated singly and sequentially up to the 24th day or until the male became permanently sterile. Following the period of excessive sterility on the 6th–9th days—with a clear-cut sterile period on the 8th day—a marked increase in fecundity was found on the 10th day. The highest incidence of cross-overs was on the 10th day followed by a progressive decline in the later broods. The decrease in the frequency of lethals was not so evident, possibly because too few flies were tested in the latest broods. Despite the fact that clusters were to be expected, only singles were recovered from the 9th–10th day broods. Clusters were first found on the 11th day, but frequently not before the 12th day. Many clusters continued for several subsequent broods. It was concluded from continuing clusters that predefinitive gonia, through repeated “cambial”-like divisions, preserve the continuous supply of sperm. Thus continuing clusters can be used as a genetic criterion for differentiating between the definitive and predefinitive gonia. With the brood procedure used, treated definitive gonia were sampled on the 9th–11th days, and predefinitive gonia beginning from the 12th day. Hence the temporal pattern after the 12th day reflects the functional activity pattern of the stem cells.

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.

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 tricomplex 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.

Frequency of X-ray-induced crossing-over in structurally different types of chromosome of Drosophila melanogaster females

Abstract In order to test the influence of the proximal heterochromatin upon the induction of recombination in an adjacent euchromatic region, crossing-over experiments were carried out simultaneously with two types of Drosphila melanogaster female irradiated with X-ray doses ranging from 1 to 6 kR. Females of one of the genotypes (designated “N”) were heterozygous for a series of 3rd chromosome marker genes, including st, in, and ri, but otherwise had normal chromosomes. Females of another type (designated “T”) were homozygous for a translocation, T(2:3)spy, and heterozygous for the same three markers. This translocation was used to relocate the markers in respect to the heterochromatin. The frequency of the st-in recombinants in the progeny from eggs laid by the “N” females 5–12 days after irradiation increased proportionally to the dose. The “T” females also showed an increase in the frequency of crossing-over after irradiation, but the effect was maximal after 3 kR and was then progressively decreased after 4 and 6 kR. When the frequency of exchange attributable to the treatment was calculated on the assumption that the crossing-over induction took place independently of meiotic crossing-over, the effect was the same in the two genotypes at 1 and 3 kR. This indicates that the proximity of the heterochromatin had no influence on the frequency at which an exchange was induced by X-rays in normal chromosomes. Evidence was also found for suggesting that the frequency of induced recombination is a mere function of the number of salivary chromosome bands between gene loci, and hence that there is no hypersensitivity of heterochromatin. The difference between the mode of dose dependence of crossing-over in the two genotypes investigated can be explained on the basis of the hypothesis of Bateman and Chandley that synapsis of chromosomes is disrrupted by irradiation which thus interferes with meiotic crossing-over, and that the extent of the disruption, beginning at the distal end, is proportional to dose.

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.