biology 4 pages due by 48 hours

1wk and 3-4pages

(PS3)

Answer all questions in RED

Part I

The following diagram represents the organization of a typical eukaryotic gene. (non-template strand shown)

The sequence AATAAA (AAUAAA in pre-mRNA) is the Poly(A) signal sequence recognized by CPSF (cleavage and polyadenylation specificity factor).

1) Nuclear and cytoplasmic fractions were purified from eukaryotic cells and RNA was purified from each fraction. A careless student labeled the RNA samples, #1 and #2, but forgot which one was extracted from the cytoplasmic fraction. To determine the origin of samples #1 and #2, you decide to perform the following experiment:

Each sample is submitted to gel electrophoresis to resolve RNA molecules based on their size. After electrophoresis, the RNA content of the gel is transferred onto a membrane. The membrane is probed with a radiolabeled oligonucleotide: ACAACCACAC and then placed into contact with an X-ray film to determine the position of the RNA molecules recognized by the radioactive probe.

A similar experiment is conducted using a different radioactive probe whose sequence is TTTATT. The developed X-ray films are shown in the following figure.

a) During polyadenylation, what is the function of the pre-mRNA sequence corresponding to “GTGTGGTTGT” on the non-template DNA strand? (4 pts)

b) Identify the sample (#1 or #2) extracted from the cytoplasmic fraction. Give two reasons to explain your answer. (4 pts)

2) mRNA was purified from wild type yeast (WT) and from two mutant strains of yeast (M1 and M2). Note that yeast is a unicellular eukaryote. A series of experiments were performed with these 3 types of yeast cells.

Experiment 1:

B-galactosidase (B-gal) mRNA was purified, submitted to gel electrophoresis and transferred onto membrane as described above. The membrane was probed with either a radioactive oligonucleotide that recognized a sequence unique to B-gal mRNA or radioactive oligo (dT) (Sequence TTTTTTTTTT). Note that the oligo(dT)-target sequence hybrid is stable if the target sequence contains more than 6 adjacent As.

The developed X-ray films are shown in the following figure.

The film shows that the B-gal mRNA produced by wild type yeast is heterogeneous in size while in mutants M1 and M2 -gal mRNA molecules of a single size are produced. The length of the -gal mRNA in M1 is smaller than that in WT. The -gal mRNA is slightly smaller in M2 than in M1. Note: Additional experiments showed that RNA splicing couldn’t account for the observed differences. Experiment 2: The protein -galactosidase is an enzyme that converts X-Gal, a colorless substrate, into a blue product. mRNA extracted from slices of the electrophoresis gel corresponding to the regions of the film labeled a and b (see above) was used for in vitro synthesis of B-galactosidase. The amount of -galactosidase synthesized was measured by adding X-gal to the reaction and by measuring the amount of blue product formed. This experiment shows that identical quantities of mRNA extracted from a and b produced identical quantities of -galactosidase.

a) Explain the size heterogeneity of the B-gal mRNA in the wild type strain. Note: the result from experiment 2 should give you a clue. (4 pts)

b) The genes mutated in strain M1 and M2 are different. In both mutant strains the gene mutation results in the production of an inactive protein. Propose a function for the proteins inactivated in M1 and M2. (4 pts)

3) The gene coding for -galactosidase is under the control of an inducible promoter. Therefore, the gene is transcribed only when a diffusible inducer is added to the yeast culture.

Experiment 3: The inducer was added to the yeast culture for 2 minutes, and then the cells were harvested and resuspended into a fresh culture medium containing a transcriptional repressor of the - galactosidase gene. Samples of yeast cells were taken from the culture at different time intervals before or after the addition of the repressor. The -galactosidase activity in these samples was

immediately measured. Alternatively, mRNA was extracted immediately after the addition of the repressor and stored on ice. At different time intervals, these mRNA samples were translated and the

-galactosidase activity in these in vitro translation reactions was measured.

a) Is experiment 3 a pulse-chase experiment? Briefly explain (3 pts)

b) The following graphs represent the results of experiment 3.

Describe the two biological properties of the polyA tail illustrated by experiment 3. Briefly explain. (6 pts)

Part II

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 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 (7S, 42S, 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 amino acids (RA-AA) 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 radiolabeled cysteine instead of a mixture of radiolabeled amino acids? Explain. (2pts).

3. Figure 1 (Panel B) shows a fractionation using the cytosol of the human cell line HeLa instead of frog oocyte cytosol. A single absorbance peak is observed at 80S in the presence of a low concentration of KCl. However, at high concentration of KCl, two peaks one at 40S and the other one at 60S replace the 80S peak.

The electrophoretic analysis of RNA molecules purified from fractions 40S, 60S and 80S is shown in Figure 3 (Panel A). Bands a, c, and d are the same as those observed in the oocyte cytosol.

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 (2 pts)

b) What are the identities of the bands a, c, d and e? Briefly explain (4 pts)

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. (1 pts)

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. (2 pts)

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 is showed in Figure 3 (Panel B). Briefly describe three experimental evidences that allow you to identify 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). (3 pts.)

f) Additional experiments lead to the following observations:

i. During the early stages of oocyte maturation, in previtellogenic oocytes (those used in the experiments described above) the tRNA, 5S rRNA genes are transcribed at a very high rate. In contrast, the genes coding for the other ribosomal RNA (28S rRNA, 18S rRNA and 5.8S rRNA) and ribosomal

proteins are transcribed at a low rate. Consequently, the latter ribosomal components are the limiting factors during ribosome assembly

ii. Previtellogenic oocytes submitted to a pulse-chase experiment with 32P-UTP and with a 12- hour chase followed by an oligo (dT) affinity purification gives a radioactive band pattern different from that observed with HeLa cells under the same experimental conditions. The pattern is shown on Figure 3, panel B, lane labeled O.

iii. A radioactive band pattern similar to that observed in the lane labeled O is obtained if the oligo(dT) purification is performed on the 100S absorbance peak (see Figure 1, panel A) and not on the unfractionated oocyte cytosol.

From these observations, predict two functions for the 7S, 42S and 100S RNA-protein complex in previtellogenic oocytes. Note that 42S 7S and 100S complexes do not contain any ribosomal proteins. (2 pts)