2_1997 - Molecular Biology 2nd Exam - spring 199‘ ALL...

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Unformatted text preview: Molecular Biology 2nd Exam - spring 199‘ ALL EXAMS ARE DUE AT 8:30 am ON MONDAY APRIL 7. There is no time limit on this test. You may find it easier to take this test over several days, though if you are confident in your molecular skills, you could wait until Sunday night. However, I predict it will take many of you a bit longer to think of all the answers (just some friendly advice). You are not allowed to use your notes, any books or journals, nor are you allowed to discuss the test with anyone until all exams are turned in at 9:30 am on Monday, April 7. You may use a calculator and/or a ruler and graph paper. The answers to the questions must be typed, though you may want to supplement your text with hand drawn figures (write neatly for any labels in your figures). -3 pts if you do not follow this direction. Please do not write your name on any page other than this cover page. Staple ally (INCLUDING THE TEST PAGES) together when finished with the exam. Name (please print): our pages Write out the full pledge and sign: How long did this exam take you to complete (excluding typing)? 10 pts. 1) As you can see from this first abstract (figure 1), a lot of scientists find it difficult to write in plain English. Please summarize this abstract in your own words using as little jargon as possible. Your summary should NOT be longer than the original summary. 10 pts. 2) Figure 2 shows some DNA footprint analysis. CC+20 is a particular sequence of DNA. CRP is a protein, as is RNA polymerase. Notice that the top line of + or — is indicating the presence or absence of CRP; the bottom line indicates the presence or absence of RNA polymerase. The numbers to the right of the data are intended to be used as a yard stick for your convenience. Please interpret these data as completely as possible. 10 pts. 3) Figure 3 is examining the role of different alleles of p53 in tumor formation. Note that Giesma stain lables DNA and thus what you see are dark spots of foci; these spots are evidence of rapid cellular proliferation. Please interpret these data. Note that p53dl has no functional p53. 10 pts. 4) Figure four shows a band shift assay in which the promoter DNA is radioactive (or “hot”) and every lane has purified transcription factor E2F added to it. A “—” indicates that there is no addition of identical promoter DNA sequence that is non—radioactively labeled (no “cold” DNA added), while “+” indicates that there was addition of identical promoter DNA sequence that is non-radioactively labeled (“cold” DNA added). The molecular weight of E2F plus promoter is indicated by the arrow. The labels at the top indicate the source of additional cell extracts. For example, L means that the cell line “L” was homogenized and then these proteins were added to the tube for the pair of lanes (— and +) before the band shift gel was run. a) Explain the data you see. b) What is the purpose of the “+” lanes? 5) Here is another band shift assay (figure 5) . This time we will compare three different binding proteins (HMG—l, MATHZO, and MATHlO) and two different promoters (SAR and SV40). Make the assumption that all these lanes were analyzed on a single get so there is no variation due to differences in different gels. Note that the first lanes in panels A and C are negative signs and not the number one; these lanes had no protein added to the assay. Interpret these data 10 pts. 6) Figure 6 is a set of results from CAT assays. NF-kB, IRF—1, and ATF2/cJun are three transcription factors. The two different reporter plasmids vary in their promoter sequences such that the DNA helix has been added to by half a rotation (panel B) compared to the promoter in panel A. As a result, all the sequences on one side of the double helix that were pointing up (panel A) are now pointing down (panel B). What can you conclude from these data? 10 pts. 7) Figure 7C shows hydropathy plots for two related molecules (FRLl and FRLZ). Assuming that their structures are well conserved and that differences in amino acid sequence have no major impact on overall structure, what can you deduce about the structures/topology of FRLl and FRLZ? 10 pts. 8) Given the data in figure 8A — 8C, what can you conclude about FRLl and FRL2 expression? 10 pts. 9) Determine the chromosomal location of XRCC. (It must be nice to have the data for an entire figure donated to you by another researcher!) 10 pts. 10) And the final question... Figure 10 shows a series of immunofluorescence micrographs using NIH3T3 cells (which is generic cell line that many researches grow in culture) that have been infected/transfected as described in the legend. HA stands for Hemagglutinin which is used as an epitope tag similar to the c—myc tag. The anti-p19 antibody was generated by injecting a mouse with a (cognate) peptide from the full—length p19. Panels D and B have the exact same NIH 3T3 cells grown in the presence of a modified form of dUTP (called BrdU in panel E) which can be incorporated into growing DNA strands in place of dTTP, and can be detected by a monoclonal antibody. Please interpret panels A, B and C, and then tell me the conclusion from panels D and E. #1 A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans- Golgi network, followed by budding and granule for- mation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule mem— branes. CPE specifically bound regulated secretory pathway proteins, including prohorrnones, but not constitutiver secreted proteins. We show that in the Cpe’“ mutant mouse lacking CPE, the pituitary prohor- mone, pro-opiomelanocortin, was missorted to the i constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting recep- tor, leads to multiple endocrine disorders in these ge- netically defective mice, including hyperproinsulin- ) emia and infertility. l Transformation by __my_c__ plus ros #2 p53 vol-I35 Figure I. Effect of p53val135 on Transformation by myc + ras at Different Temperatures A combination of plasmids encoding myc (1 pg) and res (1.5 pg) was cotransfected into REFS together with a plasmid encoding p53va|135 (1.5 pg) or with a control plasmid (pLTRp53dl. 1.5 pg). Cells were main- tained at 375°C for 9 days or at 32 5°C for 13 days, after which cultures were stained with Giemsa stain. 4‘ v : if? 5‘ xi? a? «3? 0 <3? v K o e b s s Q F—flF—Tl—T‘fir—‘Tr—WF—fil li——‘| — + — + — + — + — + — + — + - + .A‘QQQOOQO-o—n-ha in: l as e .... Figur Heterogeneous E2F Binding Activity in ExtractsVof Various Cell Lines N A HMG-I B MATH20 C MATH‘IO -32233333222232 -53323232 A new“: -uo lFN-pCA’r an o . o-.. - - a O .. - -. O C...'.9.'. 9. 9"91’9” Vector + NF-KB 4 4 I [RF-l ~ A A __ ATFflcJun A A ____ B Reporter: -ll0]FN-BCAT(I/ll6) .9999909 0 99909 099 yum $211561l910l1113 NE“; A "(F-I A’l‘l-‘Z/cJun 7-}C m m 2‘ :§ 3 23 .D o 1 ‘ l 12 .C - 01 o. 0 rv-wv i " _ 0 LL g; "V “in 1‘ EJ -3-2 100 FRL-1 ‘ A FigurefMATH Proteins Bind SAR DNA with Great Specificity The 310 bp SAR subfragment (SAR), derived from the Drosophila histone SAR (Mirkovitch et al., 1984), and the 223 bp non-SAR fragment (SV) containing the SV40 promoter region (Zhao et al., 1993) were end-labeled and ana- lyzed by band shift with an increasing amount of HMG-l (A), MATH20 (B), and MATH1O (C) in nanograms, as indicated. Figureé Synergistic Activation of the IFNB Gene Enhancer in Cultured Mammalian Cells Mouse embryonal P19 cells were cotrans- iected with the wild-type IFNB reporter plasmid (A) or the helical mutated reporter (B). along with increasing amounts of expression vectors directing the synthesis of k 6 ATP-2 plus c-Jun, and lFlF-1. Lane 1, 9 pg of empty expression vector. Lanes 2-5. increasing amounts of an equimolar mixture of expression plasmids (100 ng, 300 ng, 1 pg, and 3 ug). Lanes 6—9, inereasing amounts of [RF-1 expression plasmid (100 ng. 300 ng, 1 pg, and 3 pg). Lanes 10—13, increasing amounts of an equimolar mixture of ATF-2 and c—jun expression plasmids (100 ng, 300 ng, 1 pg, and 3 pg). Lanes 14—17. increasing amounts (100 ng, 300 ng, 1 ug, and 3 pg) of all the expression vectors. Lanes 18-21, in- creasing amounts (3 ng, 10 ng, 30 ng, and 100 ng) of all the expression vectors (A)/’ ' ,1,_'.o-ruu> *‘6 A eggt01214161820263138 FRL-1 399101214161820232628313538 FRL-2 as one B Em Dr Vn Vg An C FRL“ gscfin s» EFla M- A 7 Figure 8. The Temporal and Spatial Expression of FFlL1 and FFlL2 Genes during Embryogenesis (A) Temporal expression. RNA was isolated from unfertilized eggs and 1 2 3 Size (Kb) the indicated stages of embryos. The expression of the FRL1 and FRL2 genes was assayed by RT-PCR. Ornithine decarboxylase (CDC) is 3 A present as an internal control. f; S v (8) Spatial expression. Stage 10 embryos were dissected into the dor- ‘i sal half (Dr) and the ventral half (Vn), or into the vegetal half including ‘ _ 2 3 the marginal zone (V9) and the animal half (An), and RNA was isolated “ '9'4 from these tissues or from stage 10 embryos (Em). The expression A 6 of the FRL1 gene was assayed by RT—PCR. goosecoid (gsc) is known 5 .- ‘6 to be expressed in the dorsal marginal zone (Cho et al., 1991). EF1a _> Q - V _ 4. 4 is a ubquitously expressed control‘(Krieg et al., 1989). (C) Whole-mount m srtu hybridization In the taIlbud stage embryo of m FFlL2 RNA. Purple staining indicates the localization of the antisense g FRL2 probe. - 2 . 3 "" “‘1 - 2. O ‘1‘ Figure“ Chromosomal Localization of Human XRCC4 Gene (A) Genomic DNA from indicated cell lines (Lien et al., 1991) was digested with Taql and assayed by Southern blotting methods for hy- bridization to the human XRCC4 probe. Lane 1, human DNA; lane 2, DNA from a CHO—human hybrid cell line with a single human chromo- some 5 missing the region 5q11.2-13.3; lane 3, DNA from a CHO- human hybrid cell line with a normal human chromosome 5 as the sole human component. Arrow points to the endogenous hamster XFiCC4 gene in the CHO—human hybrids. This blot was provided by Dr. L. Kunkel. A»)-‘u\%£_n1.,v.>‘ ‘ ... wwmufix U, Anti-p19 Anti-p19 + Peptide D E w Anti-p19 Anti-BrdU Figure 9: - Localization of p194” Cytospin preparations of NIH 3T3 cells infected for 48 hr with a vector encoding HA-tagged p19“F were fixed and stained with antiserum to p19ARF (A), anti-p19"RF plus cognate peptide (8), or anti-HA serumCC) slaw-n ...
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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_1997 - Molecular Biology 2nd Exam - spring 199‘ ALL...

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