Metaphase During metaphase , spindle fibers fully attach to the centromere of each pair of sister chromatids. The blue lines are spindles, and the orange rectangles at the cell poles are centrioles. Some spindles from the opposing centrioles attach with each other, and some spindles attach to the kinetochores of the sister chromosomes from their respective sides. Each chromosome is attached to two spindles. Anaphase During anaphase , sister chromatids separate and the centromeres divide.
The newly separated sister chromatids are called chromosomes now. Telophase The chromosomes reach the opposite poles and begin to decondense unravel , relaxing once again into a stretched-out chromatin configuration. Cytokinesis Cytokinesis is the final stage of cell division in eukaryotes as well as prokaryotes. It occurs differently in animal left and plant right cells. You can see a microfilament ring forming at the center of the elongated animal cell. This creates a depression called cleavage furrow.
This invagination ultimately separates the cell cytoplasm into two cells. A cell plate forms at the center of the elongated plant cell. Then a new plasma membrane and cell wall form along each side of the cell plate. Review Describe the different forms that DNA takes before and during cell division in a eukaryotic cell.
Identify the four phases of mitosis in an animal cell, and summarize what happens during each phase. Explain what happens during cytokinesis in an animal cell.
In order to be studied by TEM, chromosomes must go through a sectioning procedure, which disrupts the internal structure, especially the winding of the DNA structure, which is of the most interest. Scanning electron microscopy SEM can be used to look at chromosomes in their entirety; however, being a surface-sensitive technique, very little information about the internal structure can be observed.
There have been many previous attempts to image chromosomes with SEM, which have mainly been concerned with their gross morphology [ 4 ] and structural changes through mitosis [ 5 ]. This has also been observed in atomic force microscopy AFM studies [ 6 ].
Sample preparation has a critical effect on the presence and the shape of these globular substructures. Studies show evidence that its shape and size change with sample preparation conditions [ 7 , 8 ]. The effect of different buffers used in sample preparation on the globular structure has been studied with SEM by Sone et al. They observed that surface structure was different between chromosomes prepared in a polyamine buffer and those prepared in a citric acid buffer.
However, chromosomes with the ribosomes removed showed no peaks at this level of structure [ 10 ]. Another key step in sample preparation for SEM is chemical, freeze-drying or critical point drying, because chromosomes are often viewed in vacuum and therefore cannot be imaged in a wet state. It has been found to date, in our experience and by others [ 4 , 5 ], that the globular structure cannot be detected in chromosomes that have been air-dried; therefore, this suggests that drying in this way causes a loss of the surface structural features.
Careful drying, such as critical point drying, can be used to slowly dry the chromosome and preserve the surface structures.
Insulating samples build up charge in the SEM, causing artefacts in the image; these can be reduced by making the sample surface conductive. This is usually achieved by coating the sample in a layer of carbon or metal a few nanometres thick.
Osmium impregnation has been used in many studies both to improve conductance and to enhance contrast [ 4 , 9 ]. However, it was found that the application of osmium impregnation produces swelling in the chromosome, disrupting the fine surface structure [ 11 ].
A sample preparation developed by Wanner and Formanek preserves the surface structure of the chromosome and stains with a contrast-enhancing dye, avoiding toxic osmium impregnation [ 12 ]. The stain used in this protocol is a polymer of platinum II bis acetamide complex [ 13 ], which binds to the minor groove of DNA. This type of stain has an advantage over osmium-based stains, because it binds only to the DNA and not to the proteins [ 14 ]. During the condensation of the chromosomes, the chromomere loops attach to the matrix of parallel fibres, which contract upon condensation.
This protocol has not been successfully applied to mammalian chromosomes because of a mesh-like layer over the chromosomes, which is not present in plants, making barley an ideal subject to study chromosome structure with SEM. In studies by Schroeder-Reiter, this nucleoplasmic layer was digested away with certain proteases; however, it is likely that the surface structure of the chromosome is damaged or modified by this treatment [ 16 ].
This layer was also reported by Gautier et al. SEM remains a useful tool to look at the effects of sample preparation on chromosomes owing to its high-resolution imaging of changes in surface structure and the ability to look at the uptake of metal-based stains. This is especially important for X-ray imaging, where the application of heavy metals will increase the scattering power of the object and therefore increase the obtainable resolution.
While three-dimensional information was present, this resolution is not sufficient to see the inner structural detail of interest.
The chromosomes studied here were unstained and comparison with fluorescence microscopy images was used to analyse structure seen in the X-ray images. In one set of preparations, Cytoclear, a commercially available mixture of proteases, lipases and detergents, was added to the cell culture step to try and remove the mesh-like layer observed in human chromosomes.
The surface structure of the Cytoclear-treated chromosomes was compared to preparations that were obtained from standard cell cultures.
Secondary electron SE images are analysed to look at the effects of sample preparation. The suitability of this sample preparation protocol for X-ray microscopy methods is discussed. Calculations of estimated phase shift are made from the images obtained in order to provide an estimation of the suitability of ptychography as an imaging method for chromosomes.
Chromosomes were prepared from b-lymphocyte cells from a Yoruba cell line GM which were at passage 4 following a protocol described in [ 19 ]. After hypotonic treatment using 0. One set of chromosomes was prepared with Cytoclear from Genial Genetics product no. This product is designed to remove cytoplasm during cell culture. Here, it was applied during the methanol—acetic acid fixation stage to remove the mesh-like layer. The spreads were fixed in 2. Finally the chromosomes were chemically dried using hexamethyldisilazane HMDS from Sigma Aldrich, which has been shown to have a similar effect as critical point drying [ 20 ].
The slides were cut and mounted on SE images were taken using the gentle beam mode settings, with 3. The globule size was measured from the images by the following analysis. The images were first treated with a Gaussian filter to improve the sharpness over the edges of the globules. A threshold was applied to obtain just the contribution of the globules by removing the background. Measurements of the diameter of the globules were made from these thresholded images with ImageJ.
This analysis was performed over different regions of interest in the image. Data from these regions were then combined to produce a histogram of globule size. In a coherent diffraction imaging experiment, a diffraction pattern is measured from a coherently illuminated sample. In measuring the intensity of the diffraction pattern, the phase information is lost but can be retrieved using computed algorithms and an image can be inverted from the diffraction data.
In CDI, the spatial extent of the beam provides the necessary constraint on the phase retrieval algorithms; therefore it cannot be used for imaging extended samples. Ptychography is an extension of CDI that uses overlapping diffraction patterns to provide a constraint and therefore allows for extended objects to be imaged.
From these algorithms, a quantitative measure of amplitude and phase shift through the object is obtained. Phase contrast imaging is the most promising aspect of CDI methods because the phase information obtained by measuring the relative phase shift through an object provides quantitative information about its composition. We can therefore use the information from the SEM about the sample preparation to make estimates of this phase shift. Since we do not know the sample thickness, we can obtain it indirectly from the known mass of the chromosomes and their area, which can be measured from the SEM images.
This means different tissues and organs have different ages Box 1. Source: Cell Biology by the Numbers. At any one time, most cells in the body are not in an active state of division but in interphase — a stable state between phases of cell division. This is the time when cells are growing, maturing and carrying out their normal physiological functions.
During interphase, the nucleus of a cell has a granular appearance due to the presence of chromatin see part 1 for more details. At this time deoxyribonucleic acid DNA is quite loosely arranged, with no visible chromosomes in the nuclear envelope. Just before cell division, DNA replication takes place — this ensures an identical copy of the genetic blueprint genome can be passed on to the future daughter cells.
The first article in this series explored the base pairing of nucleotides and described the complementary nature of the purine and pyrimidine bases Knight and Andrade, The DNA complementary base pairing rule is:. The process of DNA replication is incredibly fast and random errors often occur. Cell division occurs either through mitosis or meiosis.
Most nucleated human cells have 46 chromosomes visible during cell division — this is called the diploid number see part 1. During mitosis, the diploid number is rigorously maintained and, provided there are no DNA replication errors, all daughter cells receive a complement of DNA identical to that of their parent cells.
In prophase, the normal transcription and translation of DNA required for protein synthesis see part 3 stops and the loosely arranged DNA in the nucleus, characteristic of the interphase, becomes tightly wound up by enzymes including DNA polymerase topoisomerases. This results in the DNA condensing into chromosomes see part 1. The appearance of chromosomes in the nucleus during prophase indicates imminent cell division.
At this stage, since DNA has already been replicated, each chromosome consists of two identical sister chromatids exact copies of the replicated chromosome joined at a central region, the centromere. The nuclear membrane gradually breaks down, leaving the chromosomes floating free in the cytoplasm. Cytoplasmic organelles called centrioles produce thin contractile spindle tubules that are attached to each chromosome at its centromere, forming a scaffold.
The centrioles and spindle tubules manoeuvre each chromosome into the central region equator of the cell. The spindle tubules contract, thereby pulling each chromatid apart from its identical sister and towards opposite poles of the cell. The separated chromatids are now isolated at the two opposite poles of the cell, where they form two sets of 46 chromosomes each.
New nuclear membranes begin to form around each diploid set of chromosomes. The cytoplasm between the two new nuclei begins to cleave through a process called cytokinesis, which eventually results in complete separation into two new cells. Cytokinesis ensures that each daughter cell receives a portion of cytoplasm including its essential organelles, such as mitochondria and endoplasmic reticulum. This ensures that each new cell has the intracellular components to build its own molecules and undertake cellular metabolism, allowing it to grow, mature and survive independently.
Gradually, the chromosomes in each nucleus become less distinct as they de-condense, resulting is less densely arranged DNA. The characteristic banding of chromosomes you will see in pictures here is obtained by staining with various dyes. The banding of chromosomes by using dyes was discovered in the late 's and before that cytogeneticists depended on chromosome length and position of a constriction to identify the individual chromosomes. The band width and the order of bands is characteristic of a particular chromosome - a trained cytogeneticist can identify each chromosome 1,2, What is a chromosome?
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