Where is dna replicated in meiosis

Prophase is the first phase of mitosis. During this phase, the chromosomes inside the cell's nucleus condense and form tight structures. In fact, the chromosomes become so dense that they appear as curvy, dark lines when viewed under a microscope Figure 1. Because each chromosome was duplicated during S phase, it now consists of two identical copies called sister chromatids that are attached at a common center point called the centromere.

Figure 2: The mitotic spindle white begins to form outside the cell's nucleus. Important changes also take place outside of the nucleus during prophase. In particular, two structures called centrosomes move to opposite sides of the cell during this phase and begin building the mitotic spindle. The mitotic spindle plays a critical role during the later phases of mitosis as it orchestrates the movement of sister chromatids to opposite poles of the cell Figure 2.

After prophase is complete, the cell enters prometaphase. During prometaphase, the nuclear membrane disintegrates and the mitotic spindle gains access to the chromosomes. During this phase, a protein structure called the kinetochore is associated with the centromere on each sister chromatid.

Stringlike structures called microtubules grow out from the spindle and connect to the sister chromatids at their kinetochores; one microtubule from one side of the spindle attaches to one sister chromatid in each chromosome, and one microtubule from the other side of the spindle attaches to the other sister chromatid Figure 3a. Figure 3: a Metaphase and b Anaphase. In metaphase a , the microtubules of the spindle white have attached and the chromosomes have lined up on the metaphase plate.

During anaphase b , the sister chromatids are pulled apart and move toward opposite poles of the cell. Figure Detail. After metaphase is complete, the cell enters anaphase. During anaphase, the microtubules attached to the kinetochores contract, which pulls the sister chromatids apart and toward opposite poles of the cell Figure 3c.

At this point, each chromatid is considered a separate chromosome. Figure 4: During telophase, two nuclear membranes form around the chromosomes, and the cytoplasm divides. Finally, once anaphase is complete, the cell enters the last stage of the division process — telophase.

During telophase, the newly separated chromosomes reach the mitotic spindle and a nuclear membrane forms around each set of chromosomes, thus creating two separate nuclei inside the same cell.

As Figure 4 illustrates, the cytoplasm then divides to produce two identical cells. Why is mitosis important? As previously mentioned, most eukaryotic cells that are not involved in the production of gametes undergo mitosis. These cells, known as somatic cells , are important to the survival of eukaryotic organisms, and it is essential that somatic parent and daughter cells do not vary from one another. With few exceptions, the mitotic process ensures that this is the case.

The histograms were analysed using the Cylchred Software from Cardiff University developed by Terry Hoy, which is a cell cycle analysis software based on previously developed algorithms [45] , and allows removing the cell debris marker from the histograms. To study the progression of replication during early meiosis in wheat, one anther per floret was carefully checked to determine the meiotic stage using a light microscopy.

The identification, selection and isolation of anthers was carried out until a total of anthers were accumulated in each meiosis stage of prophase I leptotene, zygotene, pachytene, diplotene and diakinesis and metaphase I, either in the presence or in the absence of the Ph1 locus.

Each sample was then separated in three aliquots of 50 anthers each with the aim of having three independent replicates of each stage of meiosis, either in the presence or in the absence of the Ph1 locus for three independent experiments. In addition, three different flow cytometric measurements were taken from each sample in each experiment to account for equipment deviations. The small peak 4C corresponded to those cells that had already finished replication and cells going under active replication were detected between the 2C and 4C peaks Figures 1 and 2.

The replication value obtained for the somatic tissue 3. Flow cytometric analysis was carried out in wheat anthers to establish the temporal sequence of replication during early meiosis in wheat. To correctly stage the meiocytes during early meiosis, in situ hybridisation was carried out to allow the visualisation of chromosome dynamics by labelling centromeres and telomeres Figure 1a.

Interestingly enough, the number of cells under active replication in wheat anthers in premeiosis was much higher and significantly different than in the somatic control In fact, the level of replication in wheat anthers in premeiosis was almost ten times higher than in the somatic tissue which reveals that replication is occurring during premeiosis in wheat. In addition, replicating cells S value were also detected in leptotene, zygotene and pachytene Figure 1 , Table 2.

The replication values decreased from premeiosis Then, replication remained constant from pachytene to diakinesis, but higher These results suggested that residual synthesis of DNA occurred in wheat after pachytene, when chromosomes are already associated in pairs. At the onset of meiosis the number of replicated cells increased from premeiosis The number of replicated cells from pachytene to metaphase I remained constant and almost double compared with the somatic control These results confirm that replication occurs during early meiosis in wheat, as an increment in the number of replicated cells was clearly detected from premeiosis to pachytene.

Hence, flow cytometry is an efficient tool to successfully detect and quantify replication during early meiosis in wheat, and shows that replication occurs actively from premeiosis until pachytene, when chromosomes are paired and telomeres are clustered at the bouquet.

Replication was also studied by flow cytometry in early meiosis in wheat in the absence of the Ph1 locus, which affects replication and controls chromosome pairing during meiosis. Chromosome dynamics was tracked using in situ hybridisation during early meiosis by labelling centromeres and telomeres to correctly stage meiosis Figure 2a. Cells going under active replication S phase were also clearly detected in early meiosis in wheat in the absence of the Ph1 locus using flow cytometry Figure 2.

In fact, the number of cells detected in replication was Replication was also detected in leptotene and zygotene 9 and 7. Thus, replication decreased sharply from premeiosis until reaching zygotene Table 3. The level of replication remained constant after zygotene but slightly higher than the somatic cell control Table 3 , suggesting that residual synthesis of DNA also occurred after zygotene in wheat anther in the absence of the Ph1 locus.

Therefore, active replication was detected by flow cytometry in wheat in the absence of the Ph1 locus, with particularly high levels of replication in premeiosis and in early meiosis leptotene and pachytene. Moreover, the number of replicated cells did also increase during leptotene up to zygotene, where the level of replicated cells detected was almost double the number of replicated cells in premeiosis Finally, the number of replicated cells remained constant from zygotene to metaphase I, being double the number of replicated cells detected either in premeiosis or in the somatic control Table 3.

Thus, these flow cytometric results clearly confirm that replication occurs during early meiosis in wheat in the absence of the Ph1 locus, particularly in leptotene and zygotene, and can be monitored and quantified at each stage of meiosis.

The effect of the Ph1 locus on replication during early meiosis was analysed by flow cytometry. The amount of DNA was measured and compared for each meiotic stage in the presence and in the absence of the Ph1 locus Table 4.

The only significance differences were found in metaphase I between unreplicated cells of wheat lines in the presence and in the absence of the Ph1 locus These differences may be due to the differences in mature anther size in relation to the presence and absence of the Ph1 locus, given that anthers in the ph1 mutant are slightly smaller than anthers in the presence of the Ph1 locus. Each value represents the mean of 9 measurements at each meiotic stage.

Differences were found in pachytene in the presence and in the absence of the Ph1 locus. Differences were found in zygotene in the presence and in the absence of the Ph1 locus. No differences were found for the slope of the line either in the presence or in the absence of the Ph1 locus. The slope of the line for replication was higher in absolute value in the absence of the Ph1 locus which means that the rate of replication is higher in its absence.

The slope of the line was higher in the absence of the Ph1 locus which implies that new replicated cells appear faster than in the presence of the Ph1 locus. In contrast, differences in replication during early meiosis in wheat have been revealed by flow cytometric analysis in the presence and in the absence of the Ph1 locus. Our results showed that at the onset of meiosis replication occurred similarly in both wheat lines with no statistical differences in the amount of DNA either in the presence or in the absence of the Ph1 locus Figure 3b , Table 4.

However, differences in the level of replication were observed in pachytene in wheat in the presence and absence of the Ph1 locus.

Thus, in the presence of the Ph1 locus replication is still occurring in pachytene Therefore, active replication seemed to terminate earlier zygotene in wheat in the absence of the Ph1 locus. After pachytene replication remained similar in both wheat lines Figure 3b ; Table 4.

Therefore, our results indicate that replication is occurring during early meiosis in wheat either in the presence or in the absence of the Ph1 locus, although differences in the progression of replication have been detected. Our observations suggested that replication timing is affected by the Ph1 locus as replication finished earlier zygotene in the absence of the Ph1 locus. Moreover, the gradient of the line for the S phase was steeper 1. All these results suggest that replication timing is affected by the presence of the Ph1 locus, in particular the rate of replication during early meiosis in wheat.

The replication rate during meiosis is lower in the presence of the Ph1 locus. Consequently replication during meiosis in wheat lasts longer in the presence of the Ph1 locus. The cell cycle is a much studied process due to its importance in plant growth and development.

The significance of replication during the cell cycle is critical to ensure proper chromosome association, recombination and segregation in meiosis, which is directly related to viability of gametes and therefore to fertility. This paper presents a simple and robust method for the determination of the synthesis of DNA during early meiosis by means of flow cytometric measurements in nuclei released from fixed wheat anthers.

Nevertheless, genome size determination, which can be correlated with the synthesis of DNA, must be carried out with a DNA intercalation dye that allows total DNA staining, such as ethidium bromide, propidium iodide, or DAPI in this work which provides DNA content histograms with high resolution, uses readily available excitation wavelengths and does not require RNAse treatment of samples. Furthermore, flow cytometry is cheaper than other methods for analysing DNA replication and has already allowed a rapid and accurate analysis of large populations of cells [47].

In fact, flow cytometry has already been applied in plants to determine the nuclear replication stages in seeds from Lactuca sativa L. Fixation of the samples is also often convenient in experiments involving multiple and complex samples. However, fixed nuclear preparations often display wider G1 and G2 peaks in flow cytometric histograms [49].

However, and to the best of our knowledge, it is the first time that this approach is used to quantify replication during meiosis in a crop such as wheat. Replication has been studied in early meiosis in wheat-rye hybrids through the incorporation of EdU [25]. Using flow cytometry we established in this work that DNA synthesis is occurring in early stages of meiosis in common wheat, and quantified the rate of replication during meiosis and the stages of meiosis in which replication occurs.

Moreover, using this methodology we have also been able to study the role of the presence of the Ph1 locus, which controls chromosome pairing [27] , on DNA replication during meiosis in wheat. Thus, we observed that chromosome pairing was initiated before the completion of replication, as telomeres started to associate to form a bouquet when replication was still occurring in both wheat lines, either in the presence or in the absence of the Ph1 locus, similarly to the observations of [25].

But differences in timing of replication during meiosis were found in the presence and in the absence of the wheat Ph1 locus.

Thus replication last longer until pachytene during early meiosis in the presence of the Ph1 locus, or in other words, replication finished earlier zygotene when the Ph1 locus was absent.

In fact, our analysis of the slope of the lines at early meiosis indicates that the rate of replication during meiosis is higher in the absence of the Ph1 locus. Due to the fact that the Ph1 locus is similar to Cdk2 [29] and Cdk2 affects replication [50] , our results confirm one the hypotheses proposed previously [25] by studying wheat-rye hybrids that in the absence of the Ph1 locus, in that the activation of origins of replication might be increased and consequently the rate of replication.

Residual replication was also detected in wheat anthers at later meiosis stages, after pachytene and zygotene in wheat in the presence and in the absence of the Ph1 locus, respectively, when chromosomes are associated in pairs. Replication at this stage of meiosis corresponds not only to heterochromatin regions which replicate later in meiosis [25] but also to cell division and therefore residual replication of the somatic surrounding cells.

The structure of chromatin has been shown determinant for the initiation of replication [51]. Moreover, homologous chromosomes usually replicate synchronously although there are some exceptions [52]. Cell Respiration 9. Photosynthesis 3: Genetics 1. Genes 2. Chromosomes 3. Meiosis 4. Inheritance 5. Genetic Modification 4: Ecology 1. Energy Flow 3. Carbon Cycling 4. Climate Change 5: Evolution 1. Evolution Evidence 2. Natural Selection 3. Classification 4. Cladistics 6: Human Physiology 1.

Digestion 2. The Blood System 3.