Question

3 of 5 Part II (25 points) Problem 3 (25 pts) Components from the cytosol can be separated on a sucrose gradient by equilibrium equilibrium (Remember: a similar method was used to demonstrate that DNA replication is semi- conservative). After centrifugation, heavy (high density) protein complexes will sediment further away from the top of the gradient than light (low density) complexes. When the gradient is collected into fractions of increasing density, protein complexes that migrated at different level of the gradient can be separated. Note: the cytosol corresponds to the cytoplasmic fraction minus all the organelles All the figures listed in this question are included in the Appendix at the end of your home assignment 1. Figure 1 (Panel A) shows the measure of the absorbance at 260 nm of the fractions collected after centrifugation of the cytosol obtained from frog previtellogenic oocytes. Four peaks of absorbance corresponding to RNA-protein complexes of different densities (75, 425, 80S, and 100S) were detected. a) Give a reason supporting the hypothesis that proteins found in the absorbance peak are associated with nucleic acids. (2 pts) b) Give a reason supporting the hypothesis that the associated nucleic acids are RNA molecules and not DNA. (2 pts) 2. RNA is extracted from the 7S, 42S, and 80S fractions and resolved by gel electrophoresis. The RNA molecules are visualized by staining the gel with silver nitrate. The different fractions give different patterns of bands (Figure 2, Panel A), these bands are labeled a, b,c,d and e. Bands, a, c, d, and e are very sharp suggesting a single type of RNA whereas band b is relatively broad suggesting a moderate heterogeneity in the RNA content. A pulse-chase experiment is performed to identify the RNA found in band b. A mixture of radioactive UTP is injected inside the oocytes. After the chase, the cytosol is submitted to centrifugation, the RNA contained in the 7S, 42S, 80S fraction is purified, and resolved by gel electrophoresis. An X-ray film is placed in contact with the gel and then developed. The result of the film development is shown in the panel B of figure 2 a) Based on this result, identify the type of RNA contained in band b. Briefly explain. (3 pts) b) Why is the band b broad? (2 pts) c) Would the appearance of the band detected on the film change if the pulse is performed with a single radiolabeled tRNA instead of a radiolabeled UTP? Explain. (2pts).

Answer 1

3 of 5 Part II (25 points) Problem 3 (25 pts) Components from the cytosol can be separated on a sucrose gradient by equilibrium density-gradient equilibrium (Remember: a similar method was used to demonstrate that DNA replication is semi- conservative). After centrifugation, heavy (high density) protein complexes will sediment further away from the top of the gradient than light (low density) complexes. When the gradient is collected into fractions of increasing density, protein complexes that migrated at different level of the gradient can be separated. Note: the cytosol corresponds to the cytoplasmic fraction minus all the organelles All the figures listed in this question are included in the Appendix at the end of your home assignment Question-1 1. Figure 1 (Panel A) shows the measure of the absorbance at 260 nm of the fractions collected after centrifugation of the cytosol obtained from frog previtellogenic oocytes. Four peaks of absorbance corresponding to RNA-protein complexes of different densities (75, 425, 80S, and 100S) were detected. a) Give a reason supporting the hypothesis that proteins found in the absorbance peak are associated with nucleic acids. (2 pts) Yolk protein formation is under progress during the development of vitellogen in oocytes to participate in female reproductive system. Gene expression performed via transcription to generate mRNA transcript followed by translation of the mRNA for protein synthesis on ribosomal subunits. Mainly 7S (svedberg) RNA sedimentation observed at the top of the centrifugation associated with proteins compared to 42S, 100S and 80S. Absorbance is higher at 260nm due to more proteins in complexation with rRNA and mRNA that are present in the protein synthesis of previtellogenic oocytes; therefore higher concentration with higher density observed (7S) Fractionation of oocyte cytosol by equilibrium 5 mM KC 805 425 1005 Top Bottom b) Give a reason supporting the hypothesis that the associated nucleic acids are RNA molecules and not DNA. (2 pts) Nucleic acids such s rRNA, mRNA and tRNA have identified during centrifugation in combination with protein sbut not DNA. It is mainly due to translation process took place during protein synthesis. It has found that transcription process almost finished The density of the subcellular fractions are varies and density of DNA differs compared to the RNA along with proteins

Answer 2

a)   Based on these data (gel and absorbance peaks) what is the identity of the RNA-protein complex in the 80S, 60S and 40S peaks? Briefly explain (2pts)
 
b)   What are the identities of the bands a, c, d and e? Briefly explain (4pts)
 
c)   If the same fractionation were performed using a bacterial extract, which RNA band would not have any equivalent in bacteria? Explain. Note: An equivalent would be a RNA molecule that plays an identical role in bacteria and eukaryote but could have a slightly different size. (1pts)
 
d)   Give two distinct reasons suggesting that the 42S RNA-protein complex found in oocytes is distinct from the 40S RNA-protein complex identified in HeLa cells. (2pts)
 
e)   Pulse chase experiments using radioactive UTP (32P-UTP) are performed on HeLa cell and oocytes (O). After chases for variable length of time (1, 6, and 12 hours) the cytosols are incubated with oligo (dT) conjugated to sepharose beads. The content of the radioactive fractions bound to sepharose-beads isshowedinFigure3(PanelB).Brieflydescribethreeexperimentalevidencesthatallowyoutoidentify the class of nucleic acid detected (DNA or RNA) and the type of molecule in this class (chromosome, plasmid, cDNA, phage DNA, tRNA, rRNA, mRNA snRNA). (3pts.)