2_1998 - Spring 1998 Molecular Biology Exam #2 - Real Data...

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Unformatted text preview: Spring 1998 Molecular Biology Exam #2 - Real Data There is no time limit on this test, though I have tried to design one that you should be able to complete within 2.5 hours, except for typing. You are not allowed to use your notes, or any books: nor are you allowed to discuss the test with anyone until noon Monday March 23, 1998. EXAMS ARE DUE AT 8:30 ON MONDAY, March 23. You mu_st use the web as indicated on question #7 and only for #7. The answers to the questions must be typed on a separate sheet of paper unless the question specifically says to write the answer in the space provided. If you do not write your answers on the appropriate pages, I may not find them unless you have indicated where the answers are. -3 PTS IF YOU DO NOT FOLLOW THIS DIRECTION: Please do not write or type your name on any page other than this cover page. Staple all your pages (INCLUDING THE TEST PAGES) together when finished with the exam. .\ Name (please print here): ' \ ,C \ 0 Write out the full pledge and sign: (‘ g \ J How long did this exam take you to complete (excluding typing)? 8 pts. 1) Figures 1 and 2 show the results when a rece with its ligand (IgG). There are 2 naturally occuring alleles of FcRII (B1 and B2), and one engineered e 1 is provided to give you some ptor on the plasma membrane of mammalian cells (FcRII) interacts allele (Tail‘) that has had its cytoplasmic tail deleted completely. Figur background information. Interpret the data from figure 2. 8 pts. 2) A recent discovery was made about individuals who are resistant to the onset of AIDS after they have been infected with HIV. As we all know, there is genetic variation in the population and this included coding as well as non—coding portions of the genome. Some individuals have a chemokine (short protein used in cell to cell communications) that has an altered 3’ UT region. In these individuals, the amount of chemokine is higher than in people who do not have the altered 3’ UT region. A. Formulate an hypothesis to explain what is being observed. B. Devise an experiment to test your hypothesis. 8 pts. 3) As you can see in fi A. What assumption have the experimenters made with this figur B. Let’s assume that the proper controls have been performed, interpret figure 3. gure 3, you can now buy RNA blots already to probe from companies like Clonetech. e? What control would you like to see? 7 pts. 4) Figure 4 was published in Cell . Interpret the results. 7 pts. 5) Interpret figure 5B. Ignore the lines below the graph marked 1, 2, 3. 7 pts. 6) Figure 6 shows a family pedigree and an agarose type, a spot in the middle means carrier genotype, an gel. Interpret these results if an open symbol means wild— d a black shape means diseased status for this recessive disease. 10 pts. 7) For this question, I want you to show off your computer skills that might be required of you for a job as a lab tech. You may use the web to figure out how to do this, including this site <http://www.bio.davidson.edu/Biology/Courses/Molbio/NIHsearch.htm>, but you may choose your favorite way. A. You work in a lab that studies Okazaki fragments. Find the RasMol image for Taq DNA polymerase and put it on your web click on it from your main page. B. You work for Monsanto and have obtained a peptide sequence protein. Search Genbank and tell me two things. 1) What is the name of the full—length protein and what species did it come from? 2 page so that I can (IEESQFAIVVFSENY) from a plant 2) Are there any proteins from other species that have a high degree of sequence similarity. Explain your answer. (You might want to cut and paste some of your Genbank information to support your answer.) 10 pts. 8) Figure 7 deals with a protein called Fng that is involved in the growth of hair. A mutant strain of mice exists that has had this gene mutated so that normal F gf5 protein is not made (recessive mutation). A. Interpret the results from figure 7. B. Given what you have learned in figure 7, hypothesize how the mutant phenotype can be very long hair compared to wild—type. Questions 9 - 11 are related questions on the same topic. You might want to look at all 3 questions before answering any of them. 8 pts. 9) Figure shows two immunofluorescence micrographs from a familiar lab (see title and authors). Interpret the results from figure 8 if you know that p58 is a protein that is located in the salvage compartment. 8 pts. 10) Figure 9 shows some experiments with lysozyme with modified carboxyl-termini. Three constructs were made that terminate in either KDEL, HDEL, or DDEL. Interpret these results. 8 pts. 11) What do we learn new about ERD2 from figure 10 if you know that GalT is a protein that is found in the Golgi compartment. (By the way, did you realize that the Golgi body was discovered 100 years ago in 1898?!) 9 pts. 12) Briefly answer these lab questions: A) Tell me how you would make a 0.5% agarose gel that has a volume of 60 n11, is made‘of 0.5X TBE (you have 1 L of 5X TBE) and the MW of agarose is 89. B) What is calf intestine alkaline phosphatase used for (outside a calf, that is)? C) What is the function of phenol/chloroform in a DNA prep? 2 pts. 13) What unit of measure is used for the molecular weight of proteins? (This is a give—me question to bring the total up to 100 pts.) Permeabilized 37C for 60 min Non-permeabilized Figure 1. Binding and 'ntomalization 04 IqG Computes by COS Coils Transiected mm FcRIl-81 and Fem-82 Twenty bur hours anal lranstocfion with Fchl- 81 o: -82 cDNAs. transienin exptassing COS—1 coils were inCubaled wnh 20 uglml 596 com plmsbtzmaxt‘c Afterwashmgmthoold PBS—yucca! (5 mM). the cultures were fixed using parabrmaldehydo-Iyune—periodate (A andC)0twannod1037°va1hnowowon- doqmsis pm In fixation (8 and D), Fixed cells were permeabihzod mm saponin and then stained with fluorescein-oonjugalod goat anti- rabbfl lgG b localize sum-haunt! andIot In- Iamalized Iigand. A: FcRII-BZ. 2 m 4°C. 3: Fchl—BZ. 1 hr 37°C; C; FchLBL 2 h! 4°C; 0: Fchl-BL 1 hr 37°C. gal Figuro 2. Immunofluorascence Determination 01 Ligand Iniemalimion by CHO Cod Linea Stabiy Explessing Fowl-81. 82 o: ail-minus Permanen! GHQ 09" lines translocted with FcRILBZ. -81. ot the Fchl-uiHninus mutant w. W with 20 uglml IgG oomplms WZNumAWMMmMmm Pesducou (5 mM). muted u 370 for 1 mwfindumombmpomumwimm Iomwdohydo-tyuno-pomdam To muslin both surface-bound and intraceflular Bound. fixed coax m Wad with uponin prim In adding 1 WWW“ god uni-«abbot 390 Wan-had) To vusuafizo ligand mmng on me plasma membrlm W.WWCUNUM¢VIXMMM incubated enemy mm fluorescent second anti- body «about detergent coda-on (non-perm.- abmzod). A: Fowl—82. Mum. 8: Fchl- 82.noo—permblhzod.C:FcRII~81.pormoabi- lizod. D: Fchl-B1.nongermoabdmed; E: FcRIL taiHninus. pormoatxhzod. F‘ FchluILmunus. nompemieabs‘lizod. F51 MiSsense Mutations in the Adhalin Gene Linked to Autosomal Recessive Muscular Dystrophy A x9 \ 9" ¢ £9 ~\ 0 5 \° 0° Q ‘~ \9’ ° (1‘ 9 9 o 0 4° 0 6° 0 £96 Q‘ \9x} o‘éq" 9.5’ 7.5> ‘.4’ 2.4. 14». 0.24> B 96 #0 cg ‘0‘ bco‘x Q9 6 0’ \> '9 95> 7.5> 41> 21’ g 3 02‘> ‘3 ‘5 Figure !. Tissue-Specific Expression ol Adhalin mRNA in Adult and Fetal Human Tissues (A) An RNA blot (Clonetech) containing 2 ug of polytA)‘ RNA from each man hr. ol eight human tissues. as indicated. was hybridized with a hu- adhalin cDNA probe. The autoradiograph was exposed for 48 (8) An RNA blot (Clonetech) containing 2 .19 ct poly(A)' RNA from each at five letal human tissues, as indicated. was hybridized with a human adhalin cDNA probe The autoradiograph was exposed for 6 days. i STOMACH PANCREAS THYMUS MUSCLE 2 III In u! D- (I) KIDNEY LIVER TESTES 9.5 — 7.5 "‘ 4.4 - 2.4 '- 14 — LUNG HEART BRAIN EYE B AILTC and rent: ‘00 m )D’.‘ .9: 5' ‘ ’ ’ Figure WASP cDNA Sequence. Predicted Polypeptide Sequence. and Hydropathy Plot (a) K quence ot WASP. The hydropathy plots were obtained by standard comma-assisted analysis. using the algonthm and hydropathy val~ ues at Kyte and Doolittle (1982i The positions at the putative nuclear localization signal (1), the highly basic region (2) and the acidic C-terminus (3) are indicated. ”\ Figure‘ Expression of WASP in Human Tissues RNA was derived lrom tissues from a 20-week- old human male letus. M5.5 was used as a probe THYROID PROSTATE ll! (data not shovm). The size of RNA markers is given in kilobases. end a control hybridization with B-actin is illustrated below. F5 Ll Eton 1 probe Intmn probe Hirile Hindi" HindHI Xhol go C 8 go C go C 8 30 C 8 ‘ -~ — ‘ ‘ — - . “MW \' s ‘3’5 3“ 1_____E.E‘ED_§______.J—_——+H — — we Figure 6. Identification and Segregat‘ h. _._A, N... MILL, . .7 nut .A-. F10“ 2/3 probe . L: ion of Three Independe (C) Restrict igests 01 PCR products identity the specific mutat heterOzygoustemele; A V t .._M‘ m, ‘ i, MnlI utations in WAS Families Figure I. Analysis of the Fng Gene in 90 Mice The autoradiographs show Southern blots at DNA isolated lrom 90/90 and (+I+) (BALE/c [B] and CS78U6 [0]) mice. digested with Hindlll or Xhol. and hybridized with three different probes as indicated Below the photographs is a restriction map ol the wildtype Fgrs gene with the coding pertion only of exon 1 (ex 1). illustrated as a- box. Exon 1 and intron 1 sequences used as probes are represented by horizontal bars; the axon 2/3 probe also used in this study was derived trom a cDNA clone containing the full—length Fgf5 coding se- quence (Hobart et at. 1990). It. - ~ A?! ‘ w " "fr-:13: .. , _ - av; win J? 0' ions. Open sauare. control ” VV' V nderlined) and leads male; closed square. aflected male; circle with dot, . Human KDEL REceptor from the Golgi Complex to the Endoplasmic Reticulum Michael J. Louis and Hugh R. B. Pelham MRO Laboralory oi Malawian Biology Hills Road Cambridge 082 am p58 ’ hERD2 Figureg Expression ol hEFlDZ in Mouse Cells double label immunofluotesoance using rabbit anti-p58 (an intermediate Stably lranslormed cells expiessing c-myc-laggod hERDZ were ptepared 10! - ti-myc monoclonal antibody 9E10. ' ‘ ' % companmem marks!) and the an bar co responds lo 25 um. -hERD2 +hERD2 KDEL HDEL DDEL Figuret. Immunofluorescent Staining oi Lysozyme in Typical Cells 1“ The C—terminal sequences 01 the lysozyme constructs are indicated; cells in the right-hand column also expressed hERDZ. while those on the left did not. The lysozyme eonstmcts that were not retained in the ER were ooncenirated in the Golgi and also in lyeoeomee. Lysosomal staining was seen with all unrelained constructs. whether or not hERDZ was present. but in this figure it is prominent only in the cell expressing hERDZ and lysozyme—HDEL. The width of each panel corresponds to approximately 140 um. KDEL Figure . COS as“: expressing "rye—tagged hERD2 and iysozyme—MRL 0f -KDEL (as indicated) were stained with anti-my: (left) and anti—galactosw Worm antibodies (right). To preserve the immunoveactivity of tho galaciosyl transferase. the as“: were fixed wim momma and acetone. which doosnotprmERstmurewofl.’ .. - - ’ , ...
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This note was uploaded on 04/11/2011 for the course BIOL 3800 taught by Professor Gross during the Spring '03 term at North Texas.

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2_1998 - Spring 1998 Molecular Biology Exam #2 - Real Data...

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