Exam 8: Analyzing Cells, Molecules, and Systems

Indicate whether each of the following descriptions better applies to the determination of atomic structure of proteins by x-ray diffraction (X) or by nuclear magnetic resonance (NMR) spectroscopy (N). Your answer would be a five-letter string composed of letters X and N only, e.g. NNXNN. ( ) It is more suitable for complexes larger than about 100 kD. ( ) It requires samples to crystallize. ( ) It can better reveal dynamics of molecular motions. ( ) It involves the calculation of electron-density maps. ( ) It is used to determine the solution structure of proteins.

You have isolated five mutations (1 to 5) in the yeast Saccharomyces cerevisiae that make the haploid cells unable to grow in the absence of histidine. Each haploid mutant can be mated with any of the other ones, forming diploid cells that either can (+) or cannot (-) grow in the absence of histidine, as indicated in the following complementation table. How many complementation groups do these mutations represent? Each complementation group typically corresponds to a separate gene.

Consider a promoter sequence with n binding sites for an activator protein. Which Hill coefficient (h) describes noncooperative binding, i.e. independent, noninteracting binding sites?

Consider a transcription regulatory protein (A) that can bind to a promoter (p). After reaching an initial steady state, the concentration of A is suddenly increased tenfold. The following graph shows the formation of A:p over time after this change for two systems, one of which is described by higher k?ff and k?n values compared to the other. Which curve (1 or 2) corresponds to this system? Write down 1 or 2 as your answer.

In sequence alignments such as those generated by a BLAST search, the significance of the alignment can be presented as an E-value, which specifies how likely it is for a match this good to be found in a database of a given size by chance. You have scanned a large sequence database with a sequence query, hoping to find a significant match. Which one would you look at:

Indicate whether ordinary agarose-gel electrophoresis (A), polyacrylamide-gel electrophoresis (P), or pulsed-field gel electrophoresis (F) is used to better separate each of the following DNA molecules. Your answer would be a three-letter string composed of letters A, P, and F only, e.g. APF. ( ) Small DNA oligonucleotides such as hybridization probes and PCR primers ( ) Plasmid DNA ( ) Bacterial chromosomes

You have used fluorescence-activated cell sorting to separate blood T lymphocytes from a healthy person and a patient infected with a virus. To sort the cells, you used antibodies against two cell-surface receptors, CD4 and CD8, each conjugated to a different fluorescent dye. Based on the results below, which cell type do you think is depleted by the viral infection: CD4+ helper T cells (H) or CD8+ cytotoxic T cells (C)? In these dot plots, each cell is represented by a dot whose coordinates are the intensities of CD4 and CD8 signals. Write down H or C as your answer.

If two genomic polymorphic sites were randomly and independently associated with each other, one would expect the frequency of observing a combination of their allelic forms in an individual to be equal to the multiplication product of the probability of the alleles occurring separately. In many cases, however, this is not true. The degree of deviation from random association can be quantified and summarized in diagrams such as the one below. In this diagram, eight SNPs in a genomic region are shown and their pairwise deviation from random association is color-coded by different shades of red, such that the darkest red shade indicates the furthest deviation from random association. In contrast, those pairs that associate randomly are colored white. For example, SNPs A and F seem to associate randomly as indicated by the corresponding box marked with black borders. From this diagram, two haplotype blocks can be readily detected. What subset of the shown SNPs is not part of any of the two blocks? Write down the letter(s) as your answer, in alphabetical order, e.g. CEH.

You have discovered a mutant protein that forms promiscuous disulfide bonds with many other cellular proteins in the endoplasmic reticulum. Using an antibody against the protein, you immunoprecipitate proteins that interact with it. You then separate these proteins using a special type of two-dimensional polyacrylamide-gel electrophoresis (PAGE): in this method, separation in the first dimension is carried out by SDS-PAGE in the absence of β-mercaptoethanol, while separation in the second dimension is carried out by SDS-PAGE in the presence of β-mercaptoethanol. The result is shown in the following schematic drawing. Apparent molecular weights are indicated on the left. What do you think is the molecular weight of the mutant protein? What is the molecular weight represented by the question mark?

Consider the transcriptional circuits depicted in the following schematic drawings. These circuits share identical kinetic parameters but have a different number of stages. Which circuit (A or B) creates more robust oscillations? Write down A or B as your answer.

The quantitative output of a gene depends in part on the relative affinities of transcription regulators. In the following graphs, the red color indicates high gene expression and blue color indicates low gene expression. The expression level is plotted as a function of the concentrations of two transcription regulatory proteins, an activator (A) and a repressor (R). Every other parameter being identical, which graph corresponds to a system where the affinity of protein A for the cis?-regulatory sequences is higher than that of protein R? Which graph corresponds to a system that shows a "leakier" expression?

Consider a transcription regulatory protein (A) that can bind to the promoter (p) of a target gene. After reaching an initial steady state, the concentration of A is suddenly increased tenfold. The following graph shows the formation of A:p over time after this change. What is the slope of the curve at time zero?

You mated male and female heterozygous Oct4 knockout mice to study the function of this important gene. Among the progeny, one-third were homozygous wild type and the rest were heterozygous. You then repeated the cross, but this time you genotyped several embryos at different stages of early development and grouped them as either homozygous wild type (+/+), heterozygous (+/-), or homozygous null (-/-). You have summarized your results in the following table, in which the number of embryos in different groups and at different developmental stages are presented. According to these results, complete loss of Oct4 is …

Indicate whether each of the following better applies to a coherent (C) or incoherent (I) feed-forward motif. Your answer would be a three-letter string composed of letters C and I only, e.g. CCC. ( ) It can provide cellular responses with robustness despite perturbations in the input signal. ( ) A cellular pathway with such a motif generally requires a sustained (long) input signal to generate a full response. ( ) A cellular pathway with such a motif can generate a brief pulse of response in the presence of a sustained input signal.

Which of the following chromatography methods resembles immunoprecipitation in the purification of proteins from mixtures?

Imagine a bistable system composed of two repressor proteins X and Y, each of which binds to the cis-regulatory sequences of the gene encoding the other protein, i.e. X represses the expression of Y, and Y represses the expression of X. A researcher engineers the genes such that they encode protein X fused to green fluorescent protein (GFP) and protein Y fused to red fluorescent protein (RFP). Cells containing the fusion constructs are then grown and sorted by fluorescence-activated cell sorting (FACS) based on their GFP and RFP signals. The results are presented in the following dot plot. Each cell is represented by a dot whose coordinates correspond to GFP and RFP fluorescence in the cell. According to these results, which protein (X or Y) do you think binds to its target DNA sequence with a higher degree of cooperativity? Write down X or Y as your answer.

Bacterial artificial chromosomes (BACs) …

The following schematic graph shows temperature change over time in a cycle of a PCR assay. Which steps (A to C) in the cycle correspond to hybridization (annealing), strand separation (denaturation), and DNA synthesis (extension), respectively? Your answer would be a three-letter string composed of letters A to C, e.g. ACB.

Which of the following methods provides the most sensitive and accurate way of measuring and comparing mRNA levels of a set of genes in two different tissues. The genes are expressed at relatively low levels in these tissues, and do not necessarily code for protein.

In the schematic graphs below, the red color indicates high gene expression and blue color indicates low gene expression. The expression level is plotted as a function of the concentrations of two transcription regulatory proteins, A1 and A2. Which graph (1 or 2) do you think corresponds to an "AND NOT" combinatorial logic? Write down 1 or 2 as your answer.