Lecture 21 SCREENCAST
Lecture 21 SCREENCAST MCB 150
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This 15 page Study Guide was uploaded by Aj Oliver on Saturday March 12, 2016. The Study Guide belongs to MCB 150 at University of Illinois at Urbana-Champaign taught by Bradley G Mehrtens in Summer 2015. Since its upload, it has received 80 views. For similar materials see Molecular and Cellular Biology in Biology at University of Illinois at Urbana-Champaign.
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Date Created: 03/12/16
MCB 150 Lecture 21 SCREENCAST Nuclear Structure and Domains I. Nuclear Structure a. The Nucleus: i. The regions of the nucleus labeled 18: 1. Nucleoplasm – The aqueous compartment inside the nuclear envelope, that is essentially the nuclear counterpart to the cytoplasm 2. Chromatin – Chromatin fibers – most of the interphase nucleus is comprised of molecules that look like beads on a string 3. Nucleolus – the dense feature in the middle of the nucleus 4. Outer membrane of the nuclear envelope 5. Inner membrane of the nuclear envelope 6. Perinuclear space – space between the inner and outer membranes of the nuclear envelope 7. Nuclear pore complexes – how larger molecules are transported in and out of the nucleus 8. Nuclear lamina – provides a point of attachment for heterochromatin and gives the nucleus its shape and structure b. Functional Domains of the Nucleus: i. In 1885, Carl Rabl proposed that each chromosome occupies a distinct territory ii. In the figure, you see a representation of an interphase nucleus of a hypothetical cell with the chromosomes inside colored in different colors 1. This is 2D representation for a spherical 3D figure 2. These chromosomes occupy its own territory and there’s little overlap between each chromosome iii. In 1984, the Rabl model of chromosome organization was confirmed by detailed studies of polytene chromosomes in Drosphila 1. In drosphila, there are some specialized cells in the salivary glands of these animals in which the chromosomes replicate, but don’t get separated – called polytene chromosomes a. Can have hundreds or thousands of identical DNA molecules still linked together b. This allows one to see the interphase chromosomes under light microscopy II. Visualizing Nuclear Domains a. Fluorescence (in situ hybridization) or FISH can visualize and identify chromosomes i. Process is: 1. Isolate some cells, immobilize them on a glass slide, crack open the cells, and get rid of all of the nonDNA parts. After, denature the DNA that’s left a. This means that the DNA will not move from the slide, and also that the strands are next to each other, but they’re not holding on to each other via complementary base pairs b. This denaturing is also important because if we sent in a probe molecule that was complementary to a region on one of those chromosomes, then the probe wouldn’t be hybridized if the molecule was not denatured 2. In a separate part of the experiment, we create the probe molecule a. Will be fluorescent and detectable 3. Will be complementary to the target sequence once we denature the probe 4. Add a solution of our free floating, single stranded probe molecule to the immobilized denatured DNA on the slide and allow the probe to seek out something that it can hybridize to a. If it doesn’t find anything, it will get washed off b. If it does find something that it’s complementary to, it will form hydrogen base pairs to the gene 5. We activate our probe and look at our slides under a fluorescence microscope 6. Can generate a picture to detect the fluorescence ii. In the next figure, 1. Picture of FISH 2. Chromosomes started as mitotic chromosomes 3. General stain dye was red 4. Fluorescent molecule is bright yellow a. 4 bright spots on two chromosomes b. Chromosome Painting: i. Extends process of FISH ii. Every one of the probes is specific for a different region of a particular chromosome iii. If you have enough of these probes, and they’re all specific for the same chromosome, just different regions of it and they overlap somewhat, and they’re all using the same fluorescent dye, then theoretically you could make the entire length of that chromosome light up 1. This is shown in these fluorescent images 2. Each one of the fluorescence represents the maternal and paternal copies of the chromosomes c. Chromosome painting can help us visualize chromosomal domains: i. Use the technique of chromosome painting to paint each unique chromosome a different color d. Using these techniques, i. If Rabl’s hypothesis was incorrect, the chromosomes would overlap each other 1. The colors would melt together to form a graybrown color ii. If Rabl’s hypothesis was correct, then each chromosome occupies its own territory 1. The colors would be unique iii. This figure proves the interphase chromosomes occupy their own territories iv. Also proves that the nucleus is divided into domains that play important roles in how functions are carried out in the nucleus e. Chromosomal domains (or territories): i. Each chromosome occupies a distinct territory within the nucleus, and is arranged in an organized fashion ii. Nuclei are divided into discrete functional domains that play an important role in regulating gene expression and in replication iii. The space that is not part of the chromosomal territory is the inter chromosomal territory (or domain) III. Transcription and Processing a. Nuclear domains provide localized regions of function for the activities that occur in the nucleus: i. DNA replication 1. Is it together and organized or is it randomly dispersed throughout the nucleus? a. To find the answer, we can use fluorescently labeled dNTPs to study where DNA replication is occurring 2. The figure represents a section of chromosome where replication is about to begin a. Because this is a eukaryotic chromosome, we can expect to have multiple oris, and each will be recognized and unwound b. Once each replication bubble unwinds, we allow replication to begin using fluorescently labeled nucleotides shown in red i. This allows us to see where DNA replication is initiating c. Location of the fluorescence tells where DNA replication is occurring at that moment ii. Fluorescence detection of replication foci: 1. The figure shows a white dotted out line that’s supposed to represent the nucleus a. If DNA replication was unorganized, you would see a light red color diffuse throughout the nucleus i. This is because the various origins of replication throughout the human genome are essentially firing randomly, preventing one from seeing a localized region of color b. If DNA replication is organized, we could be able to see clusters of DNA replication i. Data support this hypothesis ii. If you do this experiment, you see spots 1. Each one of the spots is called a focus of replication iii. Each red dot is NOT one origin of replication or one replication bubble iv. Each replication focus is 200300 ori (replicons) 1. Replicon is the unit of DNA replicated from a single origin of replication v. Probably ≥ 100 foci of replication in the human nucleus vi. This means that it is easier to bring the DNA to the machinery rather than scattering the machinery throughout the DNA b. The eukaryotic cell cycle, revisited: i. The cell cycle is separated into interphase and M phase 1. Interphase is separated into G ,1S, and G 2 2. G 1tands for first gap phase a. Phase immediately after M phase b. In this phase, the cell is growing and building up its nutrient base c. Checks for damage in its DNA d. Preparing to launch into the next extremely important thing that’s going to happen to the cell and that’s undertaking DNA replication 3. S phase stands for the synthesis of DNA a. Other processes such as transcription do not shut down during this phase just because DNA is being synthesized b. Lasts a long time i. Because of this, does DNA replication occur immediately at the beginning of S phase, or is DNA replication spread throughout S phase? 4. After S phase ends, the second gap phase initiates, called G 2 a. Makes sure that you’ve fixed problems with the DNA b. Makes sure that you’re ready to go through the next round of mphase ii. DNA replication pattern: 1. Incorporate one fluorescent dye during early Sphase (DNA Synthesis) and a different dye during late Sphase a. First dye is blue and second dye is red b. If first hypothesis is true, we would see all blue fluorescence and no red fluorescence c. If second hypothesis was true, we would see both blue and red fluorescence i. Data support this hypothesis ii. The fluorescence shows the foci of replication iii. Blue dots are scattered throughout the nucleus while the red dots appear around the periphery of the nucleus and in pockets in the nucleus iv. This models the distribution between euchromatin and heterochromatin 1. Gene density—how many genes are there in a given unit length of DNA a. If we have two pieces of double stranded DNA i. Molecule A has 100,000 bp and 15 genes ii. Molecule B has 100,000 bp and 1 gene iii. Molecule A has higher gene density b. Higher the gene density, the more likely it is to be called on the cell to do transcription on one or more of those genes c. The lower the gene density, the more likely the chromatin will be packed as heterochromatin v. Euchromatin more likely to be unwound first compared to heterochromatin because euchromatin is already unpacked IV. Transcription and Processing a. Nuclear domains provide localized regions of function for the activities that occur in the nucleus: i. Transcription 1. Is it organized or randomly distributed? 2. We can test this using fluorescence 3. If we take an interphase nucleus and stain it with a fluorescent dye that is specific for chromatin, then anywhere the dye finds chromatin, it binds and that’s where you’ll see the color 4. In the nucleus, you can see that it has been stained for chromatin and the brighter the white color, the more light it is, the more chromatin there was 5. This shows the difference between the chromosomal territories and the interchromosomal domains a. Areas of brightest white have the most chromatin b. Areas of darker regions have the least chromatin in it c. The two areas that are circles in the middle are the exceptions because they are the nucleoli i. Dye is unable to reach the chromatin in the nucleolus 6. Inside the red rectangle lies an interchromosomal territory a. The dark area in the middle of the red rectangle is interchromosomal territory b. There exists chromatin that is pushed inside the interchromosomal territory i. The cell does this because it needs to work on that region of DNA and in the interchromosomal territories, you find the machinery to do transcription and processing ii. This proves that it’s easier to put the DNA where the machinery is and illustrates that the additional processes are systematic and not random 7. In the next image, a different colored stain is used for splicing factors a. This includes molecules within snRNPs b. This image shows that the snRNPs are located within clusters in the nucleus b. The interchromosomal domain is highly organized: i. Green is in the chromosomal territories ii. Red is in the interchromosomal domains iii. If there is a region of DNA you need to be working on, you will simply loop it out to an interchromosomal territory because that’s where the machinery is c. A reminder about microscopic techniques: i. Fluorescence microscopy: “brighter” = “more” ii. Electron microscopy: “darker” = “more” d. With “periphery” i. Could refer to the edges of the nucleus near the nuclear lamina ii. Could also refer to the edges between the chromosomal domain and the interchromosomal domain V. Summary of Nuclear Domains a. This image shows some artificial colors superimposed over a fluorescent image staining of chromatin i. The region labeled A, the yellow chromosome, reminds you that every chromosome occupies its own territory 1. Inside the white rectangle, a region of euchromatin is shown that has been unwound, pushed into an interchromosomal territory, in order to transcribe genes that need to be active at that moment 2. The red shaded regions of the yellow loop represent transcriptionally active genes and the loop is a region of euchromatin that has been pushed out into the interchromosomal territory a. The interchromosomal territory is where RNA polymerase II, type II transcription factors, and where the capping enzymes and PolyA polymerase and splicing machinery are located ii. The region labeled B shows that not only does a chromosome occupy its own territory and it doesn’t interfere with another chromosome’s territory, but even the arms of a given chromosome don’t generally overlap 1. This region in green one arm of a chromosome 2. In red, the other arm of that chromosome 3. Centromere region is shaded in gold iii. In the region labeled C, there is heterochromatin vs euchromatin 1. The brighter the yellow, the more likely it is to be euchromatin 2. The darker the red, the more likely it is to be heterochromatin 3. The euchromatin is located on the periphery of the chromosomal territory because it is about to be used 4. The heterochromatin is moved out of the way by moving it to the periphery of the nucleus or buried in the interior of the chromosomal territory iv. The region labeled D shows replication patterns 1. The areas shaded in green show regions of DNA that are going to be replicated early during S phase because they are already unwound 2. The areas shaded in red are going to be replicated late during S phase because they exist as heterochromatin 3. The gold line represents scaffolding proteins that hold chromatin together in a packed state v. The region labeled E is showing the difference between transcriptionally active and transcriptionally silent genes 1. Within the white rectangle, the white circles are transcriptionally active genes 2. Black circles are transcriptionally silent genes 3. Transcriptionally active genes are located in the periphery of the territory because that’s where the transcription and processing machinery is located 4. The gold circles represent nuclear speckles a. These are clusters of transcription and processing machinery, and they are recruited to areas of active transcription vi. The region shaded in green and labeled F is an interchromosomal territory 1. Whenever the chromosome doesn’t occupy space, that’s considered an interchromosomal territory 2. Because the chromatin is long and flexible, the interchromosomal territories look like a web rather than a big block of space 3. Inside the white box, you see larger clusters of processing and transcription machinery and smaller speckles 4. Collect very large aggregations of processing and splicing factors and smaller speckles are sent out vii. The region labeled N is the nucleolus VI. Lecture—Nuclear Lamina a. The nuclear lamina is made up of lamin proteins i. 3 types: A, B, and C ii. Lamin A and Lamin C are alternatively spliced from LMNA gene 1. Supposed to be soluble (not bound to a membrane) iii. In Lamin A (but not C), a lipid entity called a farnesyl group gets attached to the protein 1. Lamin A is initially attached to the nuclear envelope 2. The farnesyl group is then removed, and the resulting protein is free to be an unbound component of the lamina iv. A single point mutation in a 57,000 bplong gene leads to an inability to become defarnesylated 1. This mutation activates a cryptic splice sites 2. This resulting misprocessed protein is called Progerin b. Chromatin organization is critical for cell division i. Progerin stays attached to the envelope, weakening the lamina and the entire nuclear envelope ii. Weakening of the lamina limits the cell’s ability to divide c. A connection with telomeres i. Progerin is found in normal cells in low amounts ii. As cells age, shortened telomere length activates increased Progerin levels, further aging the cells d. Hope for the future? i. Farnesyltransferase inhibitors (FTIs) are in animal testing, and are showing promising leads
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