BSCI410-Liu/SP09 Midterm Exam #2/Apr. 14 Your Name: 1. (28 points total) Scientists are trying to unlock the molecular basis of a mysterious hereditary disease named DEAN SYMDROM (DS). ds mutant mice show a lack of memory and ds/ds mutant mice, when crossed with wild type (+/+) mice, produces progeny that also show memory loss. (a) (4 points) Is the ds mutation recessive or dominant? dominant (b) (4 points) Using molecular markers such as SNPs, scientists have mapped the putative DS gene to a small chromosomal region. This region contains three genes: X, Y, and Z. Design an experiment to test the expression of X, Y, and Z genes. Which tissue do you expect the candidate DS gene to be expressed in? Northern blot, RT-PCR, in situ hybridization to see which gene is expressed in brain tissue (c) (8 points) Through the above experiments, scientists identified gene Y that is expressed in the expected tissue. List two additional experiments that will definitely prove that gene Y is the DS gene. 1) sequence mutant mice DNA and compare with WT. If one detects a mutation in Y in the mutant mice, it would prove it. 2) Transgenic study - Introduce the mutant gene into the WT mouse to see if it causes memory loss (cannot introduce WT into mutant mouse to rescue because the mutation is dominant) I also took other reasonable answers. NOTE: Testing RNA expression will not definitely prove that gene Y is the DS gene. (d) (8 points) After identifying the mouse DS gene, you now have the DNA sequence of the mouse DS gene. How will you find out if there is any human gene that is homologous to the mouse DS gene? What database will you check to see if the same gene in human underlies a human disease? 1)BLAST 2)OMIM (e) (4 points) If the work described in (d) revealed that the mouse DS gene is homologous to a human gene which is known to encode an inhibitor of memory synapse formation and subsequently you discovered that the ds mice produced too much of the inhibitor. How will you devise a strategy to cure (or lessen the effect of) this disease in the mutant mice? Use RNAi or homologous recombination to knock down or knock out, respectively, the mutant DS gene in mutant mice 1 BSCI410-Liu/SP09 Midterm Exam #2/Apr. 14 Your Name: 2. (10 points) Following is a diagram illustrating that the maize (corn) gene 1 and maize gene 2 are derived from a gene duplication event of a precursor gene X'. Similarly, rice gene 1 and rice gene 2 are derived from duplicating a precursor gene X". The maize X' and rice X" are likely derived from gene X which existed in a common ancestor. Maize (Corn) Maize gene 1 (X') Maize gene 2 X Rice gene 1 (X'') Rice Rice gene 2 Use the terms (orthologous and paralogous) to describe the relationship between (A) Maize gene X' and Rice gene X" ortholog (B) Maize gene 1 and Maize gene 2 paralog (C) Maize gene 1 and Rice gene 2 ortholog (D) Maize gene 2 and Rice gene 2 ortholog (E) Rice gene 1 and Rice gene 2 paralog 3. (32 points) Circle correct answers for each question. Sometimes, you may need to circle more than one correct answer in each question. (A) Which of the following method can be used to determine the level and size of RNA transcripts for a specific gene? (a)qRT-PCR (b) FISH; (c) Southern blot; (d) in situ hybridization; (e) Northern NOTE: qRT-PCR does not test size of RNA because it only tests the size of the fragment in between the primers, not the entire RNA. (B) In the human genome, the repetitive DNA is largely consisted of (a) ribosomal RNA genes; (b) Retrotransposons; (c) Olfactory receptor genes (C) One way to predict if a specific segment of human DNA sequence encodes a gene or not is by determining: (a) If this sequence is conserved in mouse or not (b) If an EST corresponding to this piece of DNA can be identified or not (c) If open reading frames can be found in this DNA sequence or not (d) a-c (D) High throughput approach to scientific research is accomplished by (a) Hiring more experienced workers (b) Hiring a larger number of workers (c) Processing a large number of samples at the same time. (d) Using robotics 2 BSCI410-Liu/SP09 Midterm Exam #2/Apr. 14 Your Name: (E) Synteny is a term used to describe (a) Repetitive DNA sequences in different but related genomes (b) Synthesis of DNA using a light-directed chemistry (C) Evolutionary conserved organization and arrangement of single copy genes (F) The DNA finger printing technique used in forensic/paternity applications mostly relies on the molecular marker(s) of (a) SSR (microsatellite); (b) Minisatellite; c) Insertion/Deletion; (d) a-c (G) What is the size of human genome? NOT GRADED (a) 100 million base pair; (b) 3 billion base pair; (C) 3 billion kb (H) Example(s) of discovery-based science as opposed to hypothesis-based science is/are (a) Test if the putative testis determining factor Y can indeed induce gender change in females; (b) Sequencing human genome; (c) Collecting data on daily temperature change and its impact on environment. 4. (10 points) Using yeast two hybrid, one discovers that GA1 protein interacts with GA79 and GA5 proteins. GA1, GA5, and GA79 all interact with a forth protein MY6. Could you draw a diagram summarizing the interaction network among these four proteins? Please indicate (ie. give examples of) nodes and edges in this interaction network. GA1 Edges GA5 GA79 Nodes MY6 5. (16 points) True or false (A) When comparing similarities between two sequences, the lower the E-value, the more significant the similarity is between the two. True (B) The C-value paradox describes the phenomenon that the percentage of repetitive DNA in an organism's genome increases as the genome size increases. True (C) One surprising finding from sequencing different genomes is that Arabidopsis genome has significantly more genes than human. False (D) RNAi is a process a cell uses to eliminate unwanted and aberrant RNA. True 3 BSCI410-Liu/SP09 Midterm Exam #2/Apr. 14 Your Name: 6. (10 points) Calculate the percentage positive and percentage identity between these two protein sequences. Query 1 LKPVYEPGMGAQRLTQYMYRQQHRPEDNNIEFWRKFVAEYFAPNAKKRWCVSMYGS 56 LK ++ G+ A+RL QY+Y Q+ R +N I +WRKFV+EY++P AKKRWC+S+Y Sbjct 1 LKRPFDGGICARRLMQYLYHQRQRLAENTIAYWRKFVSEYYSPRAKKRWCLSLYNV 56 % identity = 29/56 51.8% % positive = (29+13)/56 75% 7. (20 points) Compare and contrast (explain what each term/method is about and then highlight the differences) (A) Northern blot vs. in situ hybridization Both used to observe mRNA expression Northern – Run RNA on a gel, which separates by size. Use probe to hybridize to desired RNA to detect size and level In situ – Use probe to hybridize to RNA in fixed tissue. Detects spatial information like in which cells or which location the mRNA is expressed. (B) Forward genetics and reverse genetics Forward: Identify mutants (mutation phenotype) first, then hunt for the gene that underlies that mutant Reverse: Know the gene at hand first then identify its function (phenotype) through mutation (knockout) (C) Transcriptome and proteome Transcriptome: the entire collection of RNA in a cell or tissue Proteome: the entire collection of proteins in a cell or tissue (D) siRNA and dsRNA siRNA: small interfering RNA, about 21 bp long, serves as a guide to target mRNA for degradation dsRNA: double stranded RNA, triggers RNA silencing (RNAi), it gets cleaved by DICER to produce siRNA 4 BSCI410-Liu/SP09 Midterm Exam #2/Apr. 14 Your Name: 8. (10 points) Dr. Goodey studies TIEDIE gene from Arabidopsis and would like to know if TIEDIE protein is secreted from the root into nearby surroundings. He has made two types of reporter genes: pTIEDIE::GUS (Tiedie promoter drives GUS) and pTIEDIE::TIEDIE-GUS (Tiedie promoter drives a fusion protein between Tiedie and GUS). Which reporter gene construct should he use? Explain why. Use pTIEDIE::TIEDIE-GUS. It is a protein fusion, so GUS will be located where TIEDIE will be located. The other construct will only show where TIEDIE is expressed, not where it is secreted to. 9a. (27 points) Apply following methods to accomplish the goals in (a) to (e). You can apply more than once the method listed below: Microarray hybridization; ChIP-chip; FISH; MASS-Spec; ASO (Allele-specific Oligonucleotide) hybridization; RNAi; Yeast two hybrid; Blast; Southern blots to analyze minisatellite markers; (a) Measure and/or compare mRNA levels between cancer and normal cells microarray (b) Detection of a few SNPs between two individuals ASO hybridization (C) Determine if Dolly the sheep is really a clone of her mother sheep Southern blots to analyze minisatellite markers (d) Determine where a gene is located on a chromosome FISH (e) Computational approach to identify genes with sequence similarity to the HD (Huntington Disease) gene (encoding a transcription factor) Blast (f) Identify HD target binding sites in the human genome ChIP-chip (g) Determine pair wise protein-protein interactions among all proteins in yeast genome Yeast two hybrid (h) Determine the identity of a protein detected in a 2-dimentional (2D) protein gel Mass-spec (i) Knockout (or down-regulate) all genes in the C. elegans genome RNAi 9b (5 points) Which of the above (a-i) should utilize a "High throughput" approach? a f g i NOTE: I tried to grade this question based on the answers you gave, if different from the correct answers.