Premeiotic S-phase Research Articles

Meiosis in Coprinus. IX. The influence of premeiotic S-phase arrest and cold temperature on the meiotic cell cycle.

ABSTRACT Arrest of premeiotic 5 phase by high temperature and light causes the meiotic cell cycle time to become shortened by 4 h after release of the arrest. Genetical and cytological evidence shows that diplotene is drastically reduced from 4·5 h to less than 1 h. Cycloheximide (50 μg/ml) treatment of early and late diplotene cells in normal cultures prevents the meiotic divisions even though the chromosomes are condensed. The same treatment given within 1 h of metaphase I allows both divisions to be completed but fails to allow sporulation. This is interpreted to mean that the cellular programme for meiotic division (division programme) is made during diplotene. In the arrest-release cultures, however, the division programme is made during pachytene. This finding is interpreted to mean that the cellular programme in meiosis is prepared one step at a time. When the nuclear cycle is arrested, the cytoplasmic programmes continue. As a result, the division programme is precociously induced at pachytene. Since the diplotene programme is complete, the cells proceed directly from pachytene into metaphase I with the diplotene stage drastically reduced. Cold treatment at 5 °C at pachytene temporarily suspends the progress of meiosis and hence prolongs the pachytene stage. This prolongation causes an increase in recombination frequency. This finding supports the argument that recombination frequency is a function of time.

High frequency recombination in centromeric and histone regions of Drosophila genomes.

DNA SYNTHESIS in eukaryotes is asynchronous, with heterochromatin replicating later than euchromatin1. In Drosophila, autoradiographic studies of metaphase chromosomes from embryonic2 and cultured3 cells have shown that the centromeric heterochromatin of chromosome 2 is late replicating. The histone genes have been localised to subsections 39D and 39E in the left arm chromosome 2, close to the centromere4, and may be part of this late-replicating region for the following reasons. First, the five tandemly arranged histone coding sequences are reiterated approximately 50 times, and late-replicating DNA has been shown to contain significantly more repeated (but non-satellite) sequences than the genome as a whole5. Second, the histone genes are transcriptionally active in all but the final hours of the S phase, suggesting that their replication may be delayed6. Third, they are in close proximity to the centromere. Asynchrony has been detected in the recombination process as well. Heat treatment of Drosophila oocytes at sequential intervals during premeiotic-S phase elicits increases in recombination that follow a well-ordered pattern7. To investigate the possibility that the pattern of heat response parallels that of replication, the centromeric heterochromatin of chromosome 2 and the contiguous histone region have been examined for the time of their recombinational response to heat during the S phase. We present here evidence for late response in both regions. In addition, a level of response roughly two orders of magnitude greater than the average encountered in the genome has been found in these regions.

The Relation of Chromosome Compaction to DNA Synthesis During Meiosis in <I>Lilium</I>

Three intervals of DNA synthesis in Lilium microsporocytes-premeiotic S-phase, delayed zygotene replication and pachytene repair synthe-sis-were analyzed with respect to chromosome compaction. Chromatin was prepared from isolated nuclei and fractionated into a condensed and a diffuse component. Glutaraldehyde was used in a parallel procedure to crosslink chro-matin that might be compacted but unstable in the fractionation procedure. Nuclei in premeiotic S-phase had a much larger fraction of condensed chro-matin rescued by glutaraldehyde than those in any other stage. Virtually no DNA replication occurred in this fraction until the last interval of S-phase. Zygotene replication occurred mainly in the diffuse chromatin fraction and the distribution remained so after completion of replication. Pachytene repair syn-thesis occurred mainly in condensed chromatin. Generally, sequences with high C0t value fractionated preferentially with diffuse chromatin but the opposite was the case for pachytene nuclei. Digestion with staphylococcal nuclease revealed no significant differences in nucleosome organization of condensed and diffuse chromatin.

Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.

DNA scission and repair during pachytene in Lilium

Sedimentation characteristics of native and denatured DNA were determined for sequential stages of meiosis in Lilium. Degradation of DNA during its preparation for analysis was minimized by extracting it from meiocytes that had been converted to protoplasts. Native DNA sedimented with a major peak in the 250s region of a glycerol gradient. The profiles for all meiotic stages were essentially the same. Denatured DNA showed a bimodal profile at pachytene (104s and 62s) and a more or less unimodal profile at other stages (104s). The difference was consistently observed regardless of the particular techniques used for preparation and measurement. The 62s component was not observed in achiasmatic cells or in cells which had been arrested by cycloheximide at prepachytene stages. Isotopic studies of pachytene DNA synthesis showed that DNA label accumulated in the 100s region and that it was present in both the old and new strands derived from premeiotic S-phase. The significance of the endogenous nicking of DNA is related to the timing and mechanics of crossing-over.

DNA replication and RNA transcription of euchromatic and heterochromatic chromosome regions during grasshopper meiosis.

The sequence of chromosome replication and the extent of RNA transcription were determined for pre-meiotic S-phase and first meiotic prophase in males of Myrmeleotettix maculatus and Chorthippus parallelus. Some heterochromatic chromosome segments are replicated early in S-phase while others are replicated late. In general, heavily condensed heterochromatic regions of the chromosomes show little or no RNA transcription as defined by 3H-uridine incorporation. Extra (i.e. in excess of 2) M4 autosomes in Ch. parallelus are slightly more condensed (heterochromatic) than their two homologues and this change in coiling behaviour is paralleled by a reduction in the rate of transcription and a change in the time of its replication, more of the DNA being synthesised at the end of S-phase. These findings are discussed in relation to the definition of heterochromatin.

Repeated DNA synthesized during pachytene in Lilium henryi.

CROSSING over probably occurs at pachytene and is subsequently seen as chiasmata1. In microsporocytes of Lilium, a repair synthesis of about 0.1% of the nuclear DNA occurs at this stage, separate from the pre-meiotic S-phase synthesis2. As pachytene synthesis and crossing over seem to occur at the same time, the synthesis may reflect limited repair at the breakpoints of DNA molecules involved in crossing over3.

An ultrastructural and radioautographic study of early oogenesis in the toad Xenopus laevis.

ABSTRACT Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still more- or-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ‘halo’ of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.

Replication of DNA in the chromosomes of eukaryotes.

The evidence that each chromatid of a eukaryotic organism contains only one DNA double helix comes from a variety of observations. It begins with the autoradiographic demonstration by J. H. Taylor that tritiated thymidine, incorporated into chromosomes during one round of DNA synthesis, is present in both chromatids at the first division after labelling, but in only one chromatid after a further round of DNA synthesis accomplished in the absence of label. Further evidence comes from those experiments which demonstrate that when two sister chromatids break and fuse one with the other, each chromatid behaves as though it contained two chains of opposite polarity, fusion between chains being restricted to those of like polarity. J. G. Gall’s study of the kinetics of digestion of lampbrush chromosomes by pancreatic DNase also supports the view that each chromatid contains only two polynucleotide chains which are cleaved by this enzyme independently of one another; while O. L. Miller’s observations on the dimensions of the fibres remaining after lampbrush chromosomes have been digested by trypsin only allow for there being two polynucleotide chains per chromatid. By means of the technique of DNA fibre autoradiography devised by J. A. Huberman and A. D. Riggs, the units involved in replicating the chromosomal DNA of somatic cells ofXenopushave been compared with those ofTriturus. Both these organisms have initiation points for DNA replication that are arranged in tandem, and from each initiation point replication proceeds in opposite directions at divergent forks. The intervals between initiation points inXenopusrange from about 20 to 125µm apart, whereas those ofTriturusare much more widely separated. At 25 °C replication of DNA inXenopussomatic cells proceeds at 9µm per hour one-way at each fork, whereas the corresponding rate inTriturusis 20µm per hour.Triturussomatic cells take about 4 times longer than comparable cells ofXenopusto replicate their DNA. TheTriturusgenome contains about 10 times as much DNA as theXenopusgenome, and comparison of the replication process in these two organisms indirectly adds weight to the view that theTriturusgenome is 10 timeslongerthan that ofXenopus, rather than that it contains 10 times as many DNA double helices per chromatid. DNA fibre autoradiography has also been used to study replication inTriturusspermato-cytes. The round of DNA synthesis just before meiosis inTriturusis an exceptionally long-drawn-out process, taking 9 to 10 days for completion at 16 °C. This lengthy S-phase is not occasioned by abnormally slow replication, the rate being 12µm per hour one-way at 18 °C, nor is it the result of an exceptional staggering of replication starts. Instead it appears to be correlated with a gross reduction in the number of initiation points for replication. i.e. with an increase in the lengths of the replicating units. A rough calculation suggests that each meiotic chromomere may correspond to a unit of replication during the pre-meiotic S-phase.

Analysis of DNA synthesis during meiotic prophase in Lilium

The nature of the DNA synthesized during the zygotene and pachytene stages of meiosis has been analyzed by the techniques of DNA-DNA hybridization, density label substitution, and sensitivity of synthesis to the presence of hydroxyurea. The synthesis of DNA during zygotene is typically semiconservative and represents a delayed replication. No synthesis of “zygotene DNA” is detectable during the premeiotic S-phase which occurs approximately three to five days before zygotene. In all species of Lilium examined, zygotene DNA has a comparatively high G + C content (about 50 to 53%) and is different from the DNA which is homologous with ribosomal RNA. The DNA synthesized during pachytene has the characteristics of a repair replication. It is variable in composition, shows no density shift after bromodeoxyuridine incorporation, and its synthesis is relatively insensitive to inhibition by hydroxyurea. Thus, chromosome pairing is temporally associated with a semiconservative replication of DNA, whereas chiasma formation is temporally associated with a repair type of replication.

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander, Plethodon cinereus cinereus (Green).

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a “diffuse” stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.

A timing study of DNA amplification in Xenopus laevis oocytes.

The time course of meiotic amplification of nucleolar DNA in Xenopus laevis oocytes has been studied autoradiographically. We find that the process is first detectable in zygotene nuclei less than 7 days after the end of premeiotic S-phase. It is completed 3 1/2 weeks later, towards the end of pachytene. Premeiotic S-phase lasts for 1–2 weeks. We are not certain whether it is followed by a short G2 or whether leptotene commences immediately. Leptotene lasts for 5±2 days, zygotene for 7±2 days and pachytene for about 20 days before the oocyte gradually enters the extended diplotene stage. Various molecular mechanisms for amplification are discussed in the light of a 24±3 day amplification time. All are found to be potentially capable of amplifying sufficient nucleolar DNA in the time available.

Analysis of meiotic exchange by tritium autoradiography.

MITOTIC sister chromatid exchanges (SCE) are currently the subject of research and controversial discussion1,2. In contrast, there is uncertainty as to whether this type of exchange ever occurs in meiotic chromosomes, but cytological studies on ring-rod heterozygotes in Zea3 and Antirrhinum4 certainly suggest that sister as well as non-sister crossovers occur during meiosis. A few direct attempts at this problem have been made using the technique of tritium autoradiography5–7, but by and large the results have proved inconclusive. The detection of meiotic exchanges by this method depends on the fact that germ line chromosomes, like those of somatic cells, replicate in a semi-conservative manner. Thus chromosomes labelled with 3H-thymidine during the pre-meiotic S-phase enter meiosis with both chromatids labelled, while chromosomes labelled one cell cycle earlier, that is, during the S-phase of the last gonial mitosis, enter meiosis having one labelled and one unlabelled chromatid. This system clearly provides an opportunity to test for the occurrence of sister chromatid exchanges in meiotic chromosomes using procedures analogous to those widely applied to mitotic chromosomes. On a simple view this would involve the detection of label switches involving sister chromatids, but in practice the situation is far more complex and the detection and estimation of meiotic SCEs are made difficult by a number of complicating factors. The most obvious of these is the occurrence of another class of exchanges in meiotic cells, namely, the crossovers which involve non-sister chromatids of homologous chromosomes. The most irritating feature of these crossovers is that in suitable combinations they can mimic the effect of SCE by producing label switches which are reciprocal between sister chromatids of AI or MII chromosomes (see Fig. la). This fact led Taylor5 to conclude that “… when exchanges are observed they cannot readily be classified as SCEs or as crossovers”. The results presented here permit a more optimistic view of the situation.