Cytogenetics. Chromosome is composed of 2 identical structures. And there is a constriction which called the centromere.

Transcription

1 Cytogenetics This sheet contains just the extra notes of 1-56 slides Slide 3 Cytogenetics is the study of the cell genetics that involve number of the chromosomes, their structure, their function, and how they behave in inheritance and how the pass the genetic material from one generation to the other. Slide 4 Chromosome is composed of 2 identical structures. And there is a constriction which called the centromere. Chromosomes are microscopic not macroscopic structures. Slide 5 As we know DNA (primarystructure) is double-stranded and coiled around histones to formnucleosomes (secondary structure). Nucleosomes +DNA = will form a Chromatin Fiber (tertiary structure). The fibers will be coiled around each other to produce a loop. The loop is a one segment of the chromosome. Nucleosomes will pose the nucleoproteins which they're important for the analysis. This makes the DNA compact in a very small structure to fit inside the nucleus, otherwise something wrong may happen. Slide 6 To see chromosomes, we have to culture the cells and keep the cell at one stage of mitosis. Mitosis consists of: prophase, prometaphase, metaphase, anaphase, telophase. Slide 7 So, to study the chromosome we would stop the mitosis of the cell either in metaphase (because the chromosomes will be separated from each other) or sometimes in prometaphase (because at this state the chromosomes are very thin & very long). Slide 8 Genetic errors in combination of each other at the end they will cause a certain abnormality and certain disease. Slide 9 At 1959, scientists found that there is a relation between number of chromosomes and certain clinical picture like Down syndrome. Banding technique is used for identification of individual chromosomes and addressing the abnormalities.

2 Analysis is more detailed, where we use some dyes, fluorescence insitu hybridization (FISH) where we can examine simpler& smaller segments of the chromosome where we can study by these type of characteristics. Slide 10 Telomeres are important for keeping the integrity and the age of the chromosome, otherwise the chromosome will be shortened and abnormality might happen there. Slide 11 Chromatids in the same chromosome are called sister chromatids. Chromatids in different chromosomes are called non-sister chromatids. Slide 13 Telomeres length decrease with age. Slide 14 How do scientists study the chromosomes, group & classify them? We use 3 different characteristics: Size, Banding pattern & Centromere position. Slide 15 According to the 3 characteristics in the previous slide we can classify the chromosomes into 3 classes: 1- Metacentric: the centromere in the middle, p arm (short arm) equals q arm (long arm) in length. 2- Submetacentric : the centromere is closer to the p arm, p arm is shorter than q arm 3- Acrocentric : no p arm Slide 16 The banding can be generated by certain conditions. These certain conditions depend on certain characteristics. Solid staining: If we just culture the cells and look for chromosomes and stains them, we will see dark chromosomes. When we do banding which means after culturing the cell we treat the chromosomes with proteolyticenzymes like trypsin which will destroy the nucleoproteins. The nucleoproteins will be released and we will start to see some dark areas and light areas in the chromosome. We said that we can stop the cell mitosis at the metaphase or at the prometaphase. At the prometaphase, in comparison to the metaphase, the chromosome is longer and we see more bands and more thin thread lines in these chromosomes. In banding we can use many different bands, such as G, R, C, Q-Banding & Ag-NOR, each one has different characteristics. Slide 17 There are many differences between the dark and the light areas.

3 Slide 18 G-banding: we treat the chromosome with trypsin then we stain it with Giemsa stain. We call it G-banding because there are 7 groups of chromosomes (A, B, C, D, E, F, G) and we stain chromosomes from group G. In Q-banding we use specific important stains such as quinicrine&hoecght. If we want to know it is male or female, we can use these stains to see the Y chromosome because Y chromosome will be stained very clearly with these stains. R-banding is reverse to the G-banding; which means the dark areas in G-banding will appear as light areas in R-banding& light areas in G-banding will appear as dark areas in R-banding. We also use Differential staining of sister chromatids (SCE). Sometimes we have some diseases (unstable chromosome syndromes like fanconianemia) that will be exchanged between 2 sister chromatids and we use these stains to see if there is a disease or not. DAPI banding is used for chromosomes 15,1,9& 16. Slide 19 After classifying the chromosomes according to the length, banding & centromere position these groups are what we get. Slide 21 Group A is composed of chromosomes 1, 2, 3 Group B is composed of chromosomes 4, 5 Group C (largest group) is composed of chromosomes 6-12 Group D (Acrocentric chromosomes) is composed of chromosomes 13, 14, 15 And so on. Slide 23 Each eukaryotic cell contains all the genetic material. So, theoretically we can use any cell to study the chromosomes. But, there is a difference in their growth ability; some cells grow slowly (such as nerve cell & muscle cell) & others grow quickly (such as lymphocyte). So, when we study the chromosomes we use lymphocytes because we can stimulate them by certain molecules. To study the chromosomes we can use G-banding (the simplest one) or FISH or Molecular techniques. Slide 24 We don't use sperm or ovum for studying the chromosomes. We use somatic cell for that. Slide 25 During the division all cell will duplicate. At the end of mitosis we will have sister chromatids. Whereas, at the end of meiosis we will have half number of what we have in the original cell.

4 Mitosis differ from meiosis in the number of the cells produced, but both have the same number of chromosome in the original cell. Slide 26 How we do the test? Firstly, we draw blood from any person. Then, we put the blood in depolarized tube. The heparin should contain no toxic material, so we use lithium heparin. Then, we put it in a culture media and add phytohemagglutinin which stimulate the lymphocytes. When the lymphocytes are stimulated we culture them for 2-3 days. After that we stop their division by adding a cytotoxic material such as mitomycinc which stop the division at the metaphase. Then, we centrifuge the tubes and take a palate that contains all the cells (including RBCs). Then, we add the cells to a hypotonic solution and the fluid will go inside the cell and destroy the RBCs, but WBCs will be swallowed and the chromosomes will be separated from each other. Then, we will wash the cells and add a fixative (such as alcohol &glacial acetic acid) to fix the cell at this stage. Then, we put it in a slide, stain it with Giemsa stain and put it under the microscope. Slide 27 As we said, in order to classify the chromosomes we look at the centromere location and the length. Then we take the chromosomes which look exactly like each other and then we group them. If we do this manual, it'll take one week for a single specimen. Slide 28 Now, there's an automated machine (composed of microscope, special camera & computer) where the machine do all the previous steps and group the chromosomes for you. If you're not satisfied with the machine's grouping, you can edit it and group them according to what you think is right. This is called Karyotyping. And at the end of it we will get a karyogram of chromosomes of a person (he is a male in this case). Slide 29 Q-banding: It stains the area that are more florescence than the other R-banding: It is important to study the centromere pairing.sometimes, there's a dicentric chromosomes ( 2 chromosomes that aren't separated from each other, so the centromeres are stick to each other). If we study them in one condition, it is difficult to see them. But if we do the reverse we can see the differences and we can study them. Slide 31

5 The number of bands that we can get from the 46 chromosomes is bands in normal conditions. That's enough to study the large abnormalities, but to study more detail in minor abnormalities that's not enough and it would be difficult to see. That's why we do a High resolution band where we stop the cell division at the pometaphase. We see here that q21 (one band) is now 3 bands. So, we can study that area better. Even we can do more resolution and we can study one of these three bands (q21.1 \ q21.2 \ q21.3). So here we get 5 bands (q21.1&q21.21&q21.22&q21.23&q21.3). So, we started with 500 bands and now we have about 800 bands. If we get more bands, we can study the chromosomes in more details. For example if we suspect minor deletion we do high resolution. Each band contains Kb. Each gene contains around 3000 base. So, each band contains a high number of genes. Slide 32 The acid and the alkali will destroy all regions except the centromere Slide 34 In FISH we will do the reaction with the DNA of the chromosome. We use fluorescent to see the segment which we are going to study under the microscope. The fluorescent will do nothing to the reaction except making it visible. We can use it for centromere, specific chromosome, whole chromosome, reverse painting. Slide 35 Why we use this technique? For translocation and sequencing (we will talk about later) Slide 36 How this is done? We'll prepare a segment of chromosome or DNA labeled with fluorescein and that segment is complementary to the segment that we interested in. Slide 37 How we do the test? We take the chromosome from the patient and we take the probe that we prepared (which is double stranded). Then, we denature both by heating them to 100. The DNA Strands separate from each other. Then we cool them and each of these strands will react with its complementary. Because the labeled probe is complementary to the natural strand, the natural strand can react with it natural complementary or with the probe. Then we examine this under the fluorescent microscope. Slide 39

6 Here we used C-banding and FISH. Slide 40 We can also do FISH for the telomeres. Slide 41&42 Each chromosome can be stained different from the other because of certain dyes (red,green,blue) that react with certain nucleotides and these dyes when they react with each other in different concentrations, they will give us spectrum. Because there is different nucleotides in different chromosomes. And it is easy for us to study the chromosomes. And it is easy to look for the abnormality in the chromosome. Look at the green chromosome X that has red color from chromosome 14. So, here we have translocation from chromosome 14 to chromosome X. Slide 43 Also, if want to do a quick test for prenatal diagnosis for a pregnant lady suspected to have Down Syndrome and we want to know is that true or not. We can take one of the 2 specimens (amnioticfluid or chorionic villus sample). If we take amniotic fluid sample where we can find exfoliative cells of the fetus. So, we take these cells and look for chromosome 21 (in case of Down syndrome). In the picture above we see that the cells have 3 red spots of 21 chromosomes which means it is a Down syndrome. The most common 5 chromosomes that contains possibility for syndromes are chromosomes 13, 18, 21, X, Y. And this can be done in less than 24 hour Slide 48 Comparative genomic studies (one of the molecular techniques) are used to look if we have the abnormalities or not. And these abnormalities can be classified into 2 conditions: do we have gain or do we have deletion Here we use 2 chromosomes; one is taken from a normal person and the other is taken from a suspected person with a certain abnormality. Procedure: For example we suspected a person to have a tumor, we take cells from him and culture the cells and they'll give us the chromosome. And we take a control sample which is completely normal chromosome. We label one with a color and we label the other with a different color (for example: the normal with red and the abnormal with green). We put the 2 chromosomes with each other and denature them and then we hybridize them. The result can be completely red chromosome or completely green or green & red chromosome. Then we study these chromosome using a spectrum of the color under the microscope (within the microscope there's a spectrum-photo-meter where it can read the green or red or both).

7 Slide 48 This is the image of analysis. We have 1.0 in the center when the red equals the green. If we have more red the spectrum decreases<1 and we have loss of gene in the chromosome 1 and we didn't have enough red genes to react with the green one. If we have more green the spectrum increases >1 and we have gain in the chromosome 1 from the tumor chromosome. This procedure isn't easy as this, we need a high experience &probation. Slide 50 Limited resolution: there's a great amount of the genes gained or lost no information on the nature of the aberrations: we have to do many other tests to tell where is the lost or the gain of the genes. Slide 52 It is a modification of PCR but it is more sensitive & resolution. You just need to know the name of this technique. Slide 54 How we can address a specific location at the chromosome? We start studying the bands from the centromere. So, the centromere is 0 and we start counting from the centromere up and down. The scientists divided each chromosome into certain areas; p arm has 3 regions and q arm has 4 regions. In each region we have bands. For example we can say there is p arm and region 1 and band 1 for addressing a specific location at the chromosome. We said that we can do high resolution to the band and that will give us a sub-band. 1p31.1 means chromosome 1 p arm region 3 band 1 sub-band 1 Slide 55 These are the terms we can use when writing the name. Slide 56 For example: 46, XX,del(5p) 46: chromosomes number \\\ XX: female \\\ Del (5p): deletion at chromosome 5 at the p arm For example: 46, XX,t(2;4)(q21;q21) 46: chromosomes number \\\ XX: female \\\ t: translocation \\\ (2; 4) (q21; q21): between chromosomes 2 at q arm region 2 band 1 and chromosome 4 at q arm region 2 band 1 From here we can address the site of abnormality at the chromosome.