Exam 8: Analyzing Cells, Molecules, and Systems

In isoelectric focusing, is the low-pH end close to the anode (A; positive electrode) or cathode (C)? Would you use octylglucoside (O; a nonionic detergent) or SDS (S) for separation of proteins by this technique? Would a highly basic protein be near the high-pH (H) or low-pH (L) end of the strip at the end of the experiment? Write down your answer as a three-letter string using the letters in the parentheses above, e.g. ASH.

The specificity of nucleic acid hybridization is remarkable. Even a single mismatch between a probe and a target sequence can be detected depending on hybridization conditions. This sensitivity can be employed in applications such as SNP haplotyping. Consider a probe that is complementary to a 20-nucleotide-long genomic region including a single polymorphic nucleotide. The probe is perfectly complementary to the more common allele, but has one mismatch in the case of the minor allele. The temperature-dependent hybridization of the probe to the single-strand genomic fragment is schematically presented in the following graph. To detect hybridization, a fluorescent dye is added to the reaction. The dye fluoresces strongly only when intercalated between the bases of double-stranded DNA, and is therefore used to quantify double-strand formation or dissociation. Which "melting curve" (1 or 2) in the graph do you think corresponds to the more common SNP allele? Which curve shows a higher melting temperature?

In which of the following organisms is systemic RNAi possible through feeding the animal with the RNA?

Indicate whether each of the following descriptions better applies to cDNA libraries (C) or genomic DNA libraries (G). Your answer would be a five-letter string composed of letters C and G only, e.g. CGCGC. ( ) Their production requires use of reverse transcriptase. ( ) They are enriched in protein-coding genes. ( ) Different sequences are represented in them based on their transcription level. ( ) They contain intronic sequences. ( ) They are cell-type specific.

Migration of proteins during separation by SDS polyacrylamide-gel electrophoresis (SDS-PAGE) is generally related to the logarithm of their molecular weight, as depicted in the qualitative graph below. If a protein mixture containing proteins of evenly distributed molecular weights (e.g. 10, 20, 30, 40, etc.) is separated by SDS-PAGE, which of the following staining patterns would you expect to observe?

You have used a reporter gene system to investigate the contribution to gene expression of three cis-regulatory DNA sequences (A to C) from a key developmental gene. Reporter gene expression from constructs containing all three or only a subset of the regulatory sequences is summarized in the diagram below. According to these findings, indicate whether each of the following conclusions is (Y) or is not (N) acceptable. Your answer would be a four-letter string composed of letters Y and N only, e.g. NNNN. ( ) Region B is likely to normally bind an activator and turn the gene on. ( ) Region C is sufficient for activation of gene expression. ( ) Region A is necessary for activation of gene expression. ( ) Region C is necessary for activation of gene expression.

Each cycle of PCR doubles the amount of DNA synthesized in the previous cycle. A pair of primers is used to direct the specific synthesis of a desired DNA segment. The following diagram shows the products of PCR amplification after three cycles. The DNA strands of the desired segment are indicated in yellow boxes in the diagram. As shown, at the end of the third cycle, 8 out of 16 strands have the desired length and are boxed. After four more cycles (i.e. at the end of the seventh cycle), how many strands of DNA are expected to be synthesized? How many of them have the desired length? Write down the two numbers and separate them with a comma, e.g. 16,8.

Due to friction with molecules of the surrounding solution, sedimenting particles in a mixture reach a terminal velocity when subjected to ultracentrifugation. The sedimentation coefficient (S) for a particle is defined as the ratio of terminal velocity (vt) to the centrifugal acceleration applied to the particle (ac). S = vt / ac If each Svedberg unit (S value) is defined as 10-¹³ seconds, approximately how long would it take for a free 50S ribosomal subunit to travel the length of a 9 cm ultracentrifuge tube subjected to a centrifugal force of 50,000 times gravity (g)? Recall that g is approximately 10 m/s².

Consider a genetic network consisting of gene A whose product activates the transcription of gene R. Imagine that the concentration of protein A is suddenly changed by a significant amount. The following graphs show how the concentration of protein R would then change over time to reach steady state again. The two curves (1 and 2) in each graph correspond to systems that have different transcription rates for A. For one of the two graphs (A or B) a negative feedback loop is present, in which protein R represses the transcription of gene A. Which conclusion is correct according to these results?

Which equation better describes the steady-state concentration of a protein (X) that is regulated by the simple binding of a transcription repressor (R) to its cis?-regulatory DNA sequences? β is the transcription rate, m is the translation rate, K is the association constant in the binding of R to the regulatory sequences, and τX is the mean lifetime of the protein X.

You have studied mutations in a transcription regulatory protein from Bacillus subtilis by an in vivo reporter gene assay carried out in Escherichia coli. The cis-regulatory sequence that the protein normally recognizes was inserted upstream of a gene encoding an enzyme that can produce a visible blue product.

What are the advantages of monoclonal antibodies over antisera?

Three subunits of a protein complex have been resolved in a two-dimensional polyacrylamide gel, as shown in the schematic drawing below. Indicate whether each of the following better describes the a, b, or c subunit. Your answer would be a three-letter string composed of letters a, b, and c only, e.g. cca. ( ) It has the smallest apparent molecular weight. ( ) It has the highest negative charge. ( ) It has the lowest isoelectric point.

The EcoRI restriction enzyme recognizes and cuts at the sequence GAATTC. Such an enzyme is expected to cut a typical double-stranded DNA at 1 in every … sites.

In a forensic lab, DNA samples from a mother (M) and her child (C) have been analyzed at three short tandem repeat (STR) loci using PCR and gel electrophoresis. The same analysis has been performed for five men (1 to 5). According to the results below, which men can be eliminated as possible candidates for the biological father of the child? Your answer would be a number composed of digits 1 to 5 only, e.g. 145.

Which of the following is NOT correct regarding both Cas9 and EcoRI?

A researcher has labeled the 5? end of a DNA primer with ³²P radioisotopes. The primer can hybridize to a complementary sequence near the 3? end of a long, spliced mRNA molecule composed of three exons. The mRNA molecule can interact with two RNA-binding proteins: one of them (protein 1) binds to the first exon and the other (protein 2) binds to the second exon. The researcher uses reverse transcriptase to extend the primer in the presence of the mRNA and either of these proteins, then separates the cDNA products by gel electrophoresis, and finally visualizes the labeled DNA bands by autoradiography. The results are presented schematically in the following drawing. Assuming that the reverse transcriptase falls off the template when confronted by bound proteins, which lane (A or B) in the gel corresponds to the reaction in the presence of protein 1? Write down A or B as your answer.

How is tandem affinity purification distinct from other purification schemes such as simple affinity or ion-exchange chromatography?

How are antibody-producing hybridoma cell lines immortalized to provide an unlimited source of monoclonal antibodies?

In Sanger sequencing, does each labeled DNA molecule have a fluorescently labeled dideoxyribonucleotide at its 5′ end or its 3′ end? Do the labeled nucleotides lack the 2′ or 3′ hydroxyl group?