BSCI410-LIU/Spring 2007 Homework #2 Due March 27 (Tu), 07 KEY 1. What are each of the following molecular markers? (Indicate (a) what they stand for; (b) the nature of the molecular polymorphism and (c) Methods of detection (such as gel electrophoresis, PCR, restriction digest etc.); and (d) their primary applications). RFLP SNP a) Restriction Fragment Length Polymorphism b) Single base change leading to loss/gain of restriction site c) Restriction digestion, gel electrophoresis and southern blot d) Mapping, linkage analyses, genotyping a) Single Nucleotide Polymorphism b) single base pair substitution c) PCR & allele-specific oligonucleotide (ASO) hybridization or PCR followed by Mass Spec or using RFLP if the change create or destroy an enzyme site d) diagnosis, gene mapping/linkage mapping Micro-satellite marker (SSR) a) Simple Sequence Repeat b) repeated units about 2-5 bp in length due to replication error c) PCR & gel electrophoresis d) linkage mapping Mini-satellite b)repeating units of 20-100 bp long that give a total length of 1-20kb c)restriction digest, southern blot d) DNA fingerprinting, mapping 1
2. In corn, the allele A- (AA or Aa) allows for the deposition of anthocyanin (blue) pigment in the kernels (seeds), while aa plants have yellow kernels. At a second gene, W- (ie. WW or Ww) produces smooth kernels, while ww kernels are wrinkled. A plant with blue smooth kernels was crossed to a plant with yellow wrinkled kernels. The progeny consists of 1447 blue smooth, 169 blue wrinkled, 186 yellow smooth, and 1510 yellow wrinkled. a. Are the a and w loci linked? If so, how far apart are they? (169+186)/3312 = 10.7% - yes they are linked b. What was the genotype of the blue smooth parent? AaWw 3. b- is an Arabidopsis mutation that causes plants to be short. To determine the map distance between b- and the microsatellite marker L (both b- and L are located on the same chromosome), Dr. Liu made following cross: She let the F1 plants self-cross and then isolated DNA from 19 F2 short plants (b-/b-). The PCR primers were used to PCRamplify the L locus from these 19 b- /b- mutant plants. The PCR reactions were run on an agarose gel, an image of which is shown below (L/L and l/l lanes are controls; 1-19 are the b-/b- plants) (a) Which of these 19 plants (use number) show show L/L Pattern and which showed heterozygous l/l pattern? Parents F1 F2 L/L: 15 L/l: 4,7 (b) Calculate the distance (in % recombination) between L and b- [(2x1)+(1x2)]/(19x2) = 0.105 = 10.5% 2
4. Dr. Liu's lab works on two different genes named LEUNIG and SEUSS. (a) Use Pubmed search to find out how many journal articles describe research on the LEUNIG gene First, when entered LEUNIG in search window, how many articles are there? 232 Second, click Preview/Index tab on top, then scroll down to see "Add terms or query". Select Author from the pulldown menu, enter leunig to the right, then click "Not" In the search box, it will show "leunig NOT leunig [author], hit Go. 23 (b) How many journal articles describe both the LEUNIG and SEUSS genes? 7 (What are the Genbank accession numbers for the Arabidopsis LEUNIG protein? (List at least three accession numbers). Hint: select "Protein from All Databases", then perform the search. (make sure from Arabidopsis thaliana) AAG32022, Q9FUY2, NP_567896, BAD67819, BAD67818, XP_550319, XP_550318, XP_468366, BAD53056, BAE98785, NP_565594 (d) What is the amino acid sequence of the LEUNG protein of the accession Q9FUY2? Please print it out in the FASTA format and attach it to this homework. >gi 30580400 sp Q9FUY2 LEUNG_ARATH Transcriptional corepressor LEUNIG MSQTNWEADKMLDVYIHDYLVKRDLKATAQAFQAEGKVSSDPVAIDAPGGFLF EWWSVFWDIFIARTNEKHSEVAASYIETQMIKAREQQLQQSQHPQVSQQQQQQQ QQQIQMQQLLLQRAQQQQQQQQQQHHHHQQQQQQQQQQQQQQQQQQQQHQ NQPPSQQQQQQSTPQHQQQPTPQQQPQRRDGSHLANGSANGLVGNNSEPVMRQ NPGSGSSLASKAYEERVKMPTQRESLDEAAMKRFGDNVGQLLDPSHASILKSAA ASGQPAGQVLHSTSGGMSPQVQTRNQQLPGSAVDIKSEINPVLTPRTAVPEGSLI GIPGSNQGSNNLTLKGWPLTGFDQLRSGLLQQQKPFMQSQSFHQLNMLTPQHQQ QLMLAQQNLNSQSVSEENRRLKMLLNNRSMTLGKDGLGSSVGDVLPNVGSSLQ PGGSLLPRGDTDMLLKLKMALLQQQQQNQQQGGGNPPQPQPQPQPLNQLALTN PQPQSSNHSIHQQEKLGGGGSITMDGSISNSFRGNEQVLKNQSGRKRKQPVSSSG PANSSGTANTAGPSPSSAPSTPSTHTPGDVISMPNLPHSGGSSKSMMMFGTEGTG TLTSPSNQLADMDRFVEDGSLDDNVESFLSQEDGDQRDAVTRCMDVSKGFTFTE VNSVRASTTKVTCCHFSSDGKMLASAGHDKKAVLWYTDTMKPKTTLEEHTAMI TDIRFSPSQLRLATSSFDKTVRVWDADNKGYSLRTFMGHSSMVTSLDFHPIKDDL ICSCDNDNEIRYWSINNGSCTRVYKGGSTQIRFQPRVGKYLAASSANLVNVLDVE TQAIRHSLQGHANPINSVCWDPSGDFLASVSEDMVKVWTLGTGSEGECVHELSC NGNKFQSCVFHPAYPSLLVIGCYQSLELWNMSENKTMTLPAHEGLITSLAVSTAT GLVASASHDKLVKLWK (e) Use the LEUNIG protein (accession: Q9FUY2) as a query to perform a Blastp search. How many types of protein domains does the LEUNIG protein have? What are the names of these domains? 3: SSDP & WD40 & LisH 3
(f) The STYLOSA protein from a different plant called Antirrhinum majus is highly similar to LEUNIG from Arabidopsis thaliana. What are the score and the e-value for the alignment between STYLOSA and LEUNIG? Score: 1079, e-value: 0.0 What is the percent identity between these two proteins? 554/759 (72%) What is the percentage positive between these two proteins? 637/759 (83%) 5. Use OMIM to search Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)" (a) What is the name of disease that mutant CFTR causes? Cystic Fibrosis (b) What are the disease symptoms? (List at least 2) Liver disease Pancreatic insufficiency Pulmonary disease Infertility Carcinoma Etc. (c) Briefly describe the function of this protein. ATP binding cassette transporter, functions as a chloride channel & controls regulation of other transport pathways (d) Indicate the exact chromosomal location of this gene in human. Chromosome 7q31.2 (e) What are names of its neighboring genes on the human chromosome map? (Provide one protein at each side) Full zoom: FRA7G on one side and ACTBP5 on the other side (f) [2 pts] Is the mutation causing the disease dominant or recessive? Recessive 4
6. Search the "Structure" database with "Drosophila AND Homeodomain" as a query. (a) How many different homeodomain structure entries did you obtain? 33 (b) (2 points) Look further into the structure of 1JGG and subsequently look into the 3D structure using the Cn3D program. The Cn3D program shows two homeodomains that bind to a short stretch of double stranded DNA. How many beta-sheets or alpha-helices are in each homeodomain? 3 alpha helices 0 beta-sheets (c) What is the DNA sequence bound by the two homeodomains shown in 1jGG? 5 naattgaatt3 3 attaacttan5 (d) Indicate whether the following amino acids (NYVS) are located in the beta-sheets or alpha-helices, or in the interconnecting regions. Interconnecting regions 5