BSCI410-Liu/SP07 Exam #2 Apr. 5, 2007

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1 Your Name: KEY UID# 1. (20 points) Dr. Liu has isolated a recessive Arabidopsis mutation; mutants homozygous for this mutation produce small seeds. She named this mutant tiny. To map and clone the corresponding wild type TINY gene, Dr. Liu discovered that TINY is linked to molecular marker J on chromosome I. Specifically, a tiny- mutant was crossed into a wild-type plant of a different genetic background (and thus contains a DNA polymorphism j ). The F1 progeny of this cross is wild type in phenotype. She let the F1 plants self-cross and then isolated DNA from 18 / mutant plants in the F2 generation. This process is diagrammed below: J J TINY + TINY + j j F1 TINY + j J Self cross F2 Test 18 plants To determine if these 18 F2 tiny plants are J/J, J/j, or j/j, a primer pair was used to PCR-amplify the J locus from these 18 individual F2 tiny-/tiny- mutants. The PCR fragments were digested with BamHI and then run on a 1% agarose gel, an image of which is shown below: J j (a) (10 points) Calculate the distance (in % recombination) between TINY and J. [(3x1) + (1x2)] / (18x2) = 5/36 = = 13.9% (b) (5 points) If 1% recombination equals ~200 kb in physical distance, what is the physical distance between TINY and J? (5/36) x 200 kb = 2780 kb (c) (5 points) What type of molecular marker is J (RAPD, RFLP, CAPS, SSR)? 1

2 2. (28 points) Through above mapping experiments, Dr. Liu mapped TINY to a small chromosomal region. This region contains three genes: X, Y, and Z. Dr. Liu needs to further determine which of the three genes corresponds to TINY. (a) (10 points) Design an experiment to test the expression of these three candidate genes X, Y, and Z. Which tissue of the plants do you expect the candidate TINY gene to be expressed in? 1) Northern blot testing RNA from different tissues using X, Y, Z as probes, respectively. (or RT-PCR or in situ hybridization examining X, Y, Z expression). 2) TINY is expected to be expressed in seeds (b) (10 points) Through experiment in (a), Dr. Liu identified gene Y, which is expressed in the expected tissue. List two additional experiments she can perform to further prove that gene C is indeed the TINY gene. 1) Sequence DNA of Y gene from WT and from tiny-; tiny- should show mutation in gene Y (or changes in nucleotide sequence from WT in gene Y) 2) Transform WT gene Y into tiny- mutant to rescue phenotype (complementation test) (C) (5 points) Assuming that the mutation is a recessive and loss-of-function mutation that causes small seeds, is the wild type protein of TINY involved in stimulating or inhibiting seed size growth? TINY WT gene promotes seed size growth. (D) (3 points) When Dr. Liu finally proves that the gene C is indeed TINY, she would like to engineer crop plants to produce bigger seeds using the TINY gene. How will she accomplish this goal using the TINY gene? Increase Y gene expression (by transforming 35S::Y into plants) 2

3 3. (16 points) Following is a diagram illustrating that the human gene 1 and human gene 2 are derived from a gene duplication event of a precursor gene X'. Similarly, ape gene 1 and ape gene 2 are derived from duplicating a precursor gene X". The human X' and ape X" are likely derived from gene X which existed in a common ancestor. X Human (X') Human gene 1 Human gene 2 Ape gene 1 Ape (X'') Ape gene 2 Use the terms (orthologous and paralogous) to describe the relationship between (A) Human gene X' and Ape gene X" ortholog (B) Human gene 1 and Ape gene 1 ortholog (C) Human gene 1 and Ape gene 2 ortholog (E) Ape gene 1 and Ape gene 2 paralog 4. (36 points) Circle correct answers for each question. Sometimes, you may need to circle more than one correct answer in each question. (A) Which of following method can be used to determine the level of RNA transcripts for a specific gene? (a)qrt-pcr (b) FISH; (c) Southern blot; (d) in situ hybridization (B) The C-value paradox describes the phenomenon that the percentage of repetitive DNA in an organism's genome is (a) fixed; (b) increases as the genome size increases; (c) decreases as the genome size increases. (C) In human genome, the repetitive DNA is largely consisted of genes (a) Genes encoding ribosomal RNAs; (b) Retrotransposons; (c) Olfactory receptor (D) 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 3

4 (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 or not (d) a-c (E) 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. (F) 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 (G) 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 (H) One surprising finding from sequencing different genomes is that (a) Human genome has a lot more genes than Arabidopsis (a plant) genome (b) Human and Arabidopsis genomes have similar number of genes (C) Arabidopsis has more genes than human. (I) What is the size of human genome? (a) 100 million base pair; (b) 3 billion base pair; (C) 3 million base pair 5. (10 points) When comparing similarities between two sequences, which is true: (a) The lower the E-value, the more significant the similarity is between the two. TRUE (b) The higher the E-value, the more significant the similarity is between the two. Briefly explain what E-value measures. Likelihood of the two sequences similar to each other by chance. 4

5 6. (10 points) Calculate the percentage positive and percentage identity between these two protein sequences. Query: 707 RLATSSFDKTVRVWDANKGYSLRTFMGHSSMVTSLDFHPIKDDLISCDNDNEIRYWSI 764 LA+ S+D TV++WD + G L+T GH V ++ FH DL S + ++ W + Sbjct: 963 LLASGSYDHTVKIWDVSSGQCLQTLQGHPGSVLAVAFSCDGKTLFSSGYEKLVKQWDV 1020 Positive%: 32/58 = = 55.2% Identity%: 20/58 = = 34.5% 7. (26 points) Explain, compare, and contrast following terms: (A) (10 points) cdna microarray vs Gene chip (Explain each type; list one similarity and three differences) Similarities: Both used for high-throughput gene expression analyses. Both put DNA on small chip/slide. Differences: Gene Chip is expensive, high density, oligo-nucleotide-based/in situ chemical synthesis, can detect SNP, more accurate/higher quality data (any of those three will be fine) (B) (8 points) qrt-pcr vs. in situ hybridization (list two differences such as in method and application) qrt-pcr: quantitative / uses primers and is a PCR-based method in situ: non-quantitative but gives info on where (which tissue or which organ or which cell type) a gene is expressed/ hybridization based (use probes) (C) (8 points) GUS vs. GFP (indicate one similarity and one difference) Similarity: both reporter genes Gus: fix tissue, can t do live imaging, requires substrate, come from bacteria GFP: don t need to fix tissue, can do live imaging, don t require substrate, come from jelly fish 5

6 8. (9 points) Dr. Sam studies BB2 gene expression. He made two types of reporter genes: (1) the reporter gene GUS was fused to the promoter of BB2 (pbb2::gus), and (2) the reporter gene GUS was fused to the BB2 cdna to generate BB2-GUS fusion protein, which was then controlled by the BB2 promoter (pbb2::bb2-gus). He transformed these two different reporter genes into plants. Although both pbb2::gus (type 1) and pbb2::bb2-gus (type 2) transgenic plants show blue staining in flowers, the plants containing reporter type (1) showed blue-staining in the cytoplasm of all floral cells while plants containing reporter type (2) showed blue-staining in the nucleus of all floral cells. What does the result tell you in terms of which tissue/organ BB2 is expressed and which subcellular compartment is BB2 protein localized. BB2 is transcribed (expressed) in flowers. BB2 protein is localized in nucleus. Why did the two types of reporters described above give different results (ie. one is cytoplasm and one in nucleus)? In 1 st fusion type, none of the BB2 protein is present in the reporter, so GUS remains in the cytoplasm In 2 nd fusion type, BB2 protein is fused to GUS since BB2 protein normally targets to nucleus, it brings GUS with it into the nucleus 9. (20 points) Apply following methods to accomplish the goals in (a) to (e). You can apply more than once the method listed below: Gene-chip hybridization; FISH; Blast; Southern blots to analyze minisatellite markers (a) Transcriptome study gene chip (b) Detection of SNP between individuals gene chip (C) Determine if Dolly the sheep is really a clone of her mother sheep Southern Blot (d) Determine where a gene is located on a chromosome FISH (e) Computational approach to identify genes with sequence similariy to the gene of interest BLAST 6

7 10. (15 points) Which of following databases will you search to find following information listed in (a) to (e). Nucleotide; Protein; PubMed; OMIM; Structure (a) Literatures Pubmed (b) 3D structure of a protein Structure (C) Nucleotide sequences of a gene Nucleotide (d) Protein sequence of a gene Protein (e) Information about a human disease OMIM 11. (10 points) List three benefits of human genome sequencing? List two ethical concerns one may have regarding the availability of personal genome sequence data. Advantages: Facilitate diagnostics of genetic disorders/diseases Help to map disease genes that may lead to therapy development Help understand gene expression and responses to drug treatment Etc. Concerns: Insurance Employment Etc. 7

8 Honor Code (rewrite, sign, and date below on dotted line) I, (fill in your name), pledge on my honor that I have neither given nor received any unauthorized assistance on this exam. Signature Date 8

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