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RNA Localization (1960-1961) Tuesday. Tissue-Specific Gene Expression III (1957-1959) 337a 1957 1958 ANTIGEN PRESENTATION AFTER DNA VACCINATION: GENERATION DIFFERENTIAL DISPLAY RT-PCR (DDRT-PCR) AS A TOOL TO STUDY OF IMMUNE RESPONSES BY EXPRESSION OF A VIRAL PROTEIN IN GENE EXPRESSION: OPTIMIZATION AND APPLICATION. ((K.R. MUSCLE CELLS IN VIVO. ((effrey B. Ulmer, R. Randall Deck, Corrille M. Luehrsenl, M.G. Brubacherl, S. Cumberledge2, A. Lloyd3, and P.E. DeWitt, John J. Donnelly, and Margaret A. Liu)) Department of Virus & Cell MayrandI)) IPerkin Elmer Corp., Applied Biosystems Division, Foster City, Biology, Merck Reearch Laboratories, West Point, PA 19486. CA 94404; 2Dept. of Biochemistry, Univ. of Massachusetts, Amherst, MA CA 94305. Transfection of muscle cells viw has been acdhieved by intramuscular 01003; 3Dept. of Biological Sciences, Stanford Univ., Stanford, injection of naked plasmid DNA (Wolff ct al, Science 247, 1465, 1990). We gene underlies the phenotypic differences between cell utilized this technique as a novel means of vaccination and generated protective Differential expression types. is a standard tool to identify cell-mediated and humoral immune responses agaist influenza in mice (Uner cDNA library construction and screening and clone the sequences of differentially expressed mRNAs. However, c al, Science 259, 1745, 1993). Tlhse results sugested that muscle cells may be in antigen presentation leading to the generation of the immune traditional cDNA libraries that rely on plasmid or phage vectors and passage involved E. from several drawbacks including the requirement of responses observed after DNA injection. However, injected DNA may have been through coli suffer libraries intralized and expessed by other cells and, moreover, muscle cells are not large amounts of polyA+ mRNA, an intensive labor commitment and considered to be antigen presenting cells. Therefore, we addressed the that are not representative of the mRNA pool. DDRT-PCR was developed by sufficiency of muscle-specific expression of a foreign antigen to generate P. Liang and A. Pardee (Science 257: 967-971 [1992]) and promises to the protecive immunity. In the present study, we demonstrated that i) valy- revolutionize cDNA library construction and screening. In the first step of infected pnmary cultured myoblasts and C2C12 myoblasts were recognized and method, a subset of mRNAs are reverse transcribed using a set of anchored lysed by influenza-specific cytotoxic (CILs) in vitro, ii) CIL poly(dT) primers. In the second step, a subset of the first-strand cDNA is T-lymphocytes The recognition and lysis were not dependent on the differentiation state of the PCR amplified using an assortment of random sequence 10-mer primers. myoblasts, dii) NP-transfected C2C12 myoblasts presented NP epitopes to CrL PCR products from each set of primers are separated by gel electrophoresis, two cell types are compared. The differentially in vitro, iv) transplantation of NP-transfected myoblasts into syngeneic mice and the cDNAs from directly are recovered and or used as for northern resulted in the genwration of NP-specific antibodies and CIL, and protective expressed cDNAs sequenced, probes small amounts of total immunity to live virus chaUllenge, and v) these immune responses were not blots or in situ hybridization. The method requires data on the generated following transplantation of lysed transfectants. Therefore, muscle RNA and can be done in a few days. We will present optimization use with Perkin Elmer thermal We will also cells appear to be capable of viral proteins in vivo, with of reaction conditions for cyclers. expressing subsequent construction cell lines of cell-mediated and humoral inimu DNA present data on DDRT-PCR library from Drosophila generation responses. and the maize uptake and expression of antigns by non-muscle cells do not seem to be reqed expressing the wingkss gene, Arabidopsis seedlings expressing to achieve the protecdve immune responses seen after DNA injection. R transcription factor. 1959 BASAL VS PHORBOL-INDUCED TRANSCRIPTION OF THE COLLAGENASE GENE DEPENDS ON MULTIPLE CIS-ACTING ELEMENTS ((L.A. White and C.E. Brinckerhoff)) Department of Biochemistry, Dartmouth Medical School, Hanover, NH, 03755. Degradation of the extracellular matrx is an integral step in normal physiology and in disease pathology, and is accomplished by the metalloproteinases. Collagenase (Matrix Metalloproteinase-l; MMP-1) is the only enzyme that degrades the interstitial collagens, the body's most abundant proteins. We used a series of 5' deletional fragments (1800 to 182 bp) of the rabbit collagenase promoter to study transcriptional regulation by phorbol myristate acetate (PMA) in primary fibroblasts. Previous data indicate an important role for an AP-1 element at -77 which binds FOS and JUN. We found that a mutation in the AP-1 site gready reduces basal/constitutive transcription of all of the fragments and, in the -182 bp fragment, mutations in this site virtually abolish PMA induction. However, mutant constructs containing more than 182 bp ofpromoter respond to PMA in a manner comparable to their wild-type counterparts. These data support a role for AP-1 in regulating basal transcription, and implicate upstream regions in phorbol responsiveness. At -186 there is an AP-1 site (J. Cell Biochem 52:337, 1993). We find that a T to G alteration in this site in 320 bp of promoter DNA does not affect basal transcription but reduces PMA inducibility by 50%. Mobility shifts with oligonucleotide (-212/-177) and extracts from PMA treated cells show specific binding that can be competed by an AP-1 oligonucleotide. Antibodies to Jun and Fos supershift the complex. Further 5' deletion between 320 and 182 bp of promoter uncover a 30 bp region from -212 to -182, which when deleted gives a 75% loss ofPMA induction. Our data indicate that basal and PMA-induced transcription are controlled in part by different mechanisms andupstream regions of the collagenase promoter, including a novel AP-1 site, contribute to PMA inducibilty. RNA Localization (1960-1961) 1960 1961 DETECTION OF TRINUCLEOTIDE REPEAT SEQUENCES IN HYBRIDIZATION OF POLY (dT) OLIGONUCLEOTIDES TO POLY (A) MYOTONIC DYSTROPHY PATIENT CELLS USING A SINGLE RNA IN LIVING CELLS. ((J.C. Politz, KL. Taneja and R.H. Singer)) Dept. OLIGONUCLEOTIDE PROBE. ((KL. Taneja, M. McCurrach*, M. of Cell Biology, Univ. of Mass. Med. Center, Worcester, MA 01655 Schalling*, D. Housman* and R.H. Singer)). Dept. of Cell Biology, University of Mass. Med. Center, Worcester, MA and *Dept. of Fundamental technology has been developed to evaluate the hybridization Biology, Cancer center, MIT. reaction between a synthetic antisense oligonucleotide (oligo) and its target mRNA in live muscle cells. Poly (A) RNA was used as an intracellular Myotonic Dystrophy (DM) is an autosomal dominant neuromuscular target for antisense oligo (dT) probes in vivo. Two approaches inherited human genetic disease with multisystem effects. DM is hybridization were used to detect this hybridization. First, exposure of cells to oligo (dT) in associated with the expansion of an unstable trinucleotide repeat to situ. This result would sequence (CTG)n, present at the 3'-UTR of protein kinase (Mt-Pk) vivo reduced subsequent hybridization oligo (dT) in mRNA.We have used a single oligonucleotide probe to the CIG repeat, be expected if the oligo hybridized to poly (A) RNA in vivo and blocked conjugated to either digoxigenin or fluorochromes, to detect the CTG subsequent hybridization in situ after fixation of the cell. Second, it was found repeat transcripts in the nucleus and in the cytoplasm of fibroblast that oligos taken up by cells can act as primers for reverse tanscription in situ cells and muscle biopsies of DM patients. The mRNA contining the (IST). Because reverse transcriptase elongates a DNA primer hybridized to repeat was found in a perinuclear region of the cytoplasm. It was found RNA, only hybridized oligos support incorporation of labelled nucleotides into that the transcript containing CIG repeat was present in the nucleus as a new DNA to give measurable signal in situ. Oligos intermalized by the cell but number (2-10) of discrete foci. No foci or other signal were found not hybridized to a target are not detected. Thus hybridization is evaluated as it in normal cells, or with the sense probe to the affected cells. Probe to the occurred within the living cell. Intemalization of fluorescently labelled poly 5'-end of Mt-Pk mRNA labelled directly with red fluorochrome (dT) oligo by live cells and subsequent intracellular hybridization, measured colocalized with the signal to the CIG repeat labelled with green using silver-gold enhancement after IST, were quantitated as a function of time fluorochrome indicating that the foci were from a single transcipt. In and Both normal cells, signal as two tofour spots could be detected probably at concentration using digital imaging microscopic analysis. the site of transcription. The foci therefore represents fluorochrome conjugated phosphodiester poly (dT) oligos and oligo (dT) released, post-tanscriptional RNA, and did not colocalize with SC-35 phosphorotisoates primed elongation by reverse transcriptase. These results "speckles" or poly(A) patches, therefore suggesting a separate demonstate directly that exogenously introduced antisense DNA hybridizes to compartment in the nucleoplasm for these released twanscripts. A single RNA in living cells and provide a rigorous method for evaluation of oligonucleotide probe can thus be used as a diagnostic test for DM hybridization efficiency as a ftmction of a variety of parameters. This expresson. methodology is applicable to visualization of RNA in living cells using fluorochrome conjugated oligos. 338a RNA Localization (1962-1967). Tuesday 1962 1963 ANNEIN II IS ASSOCIATED WITH CYTOSKELETAL-BOUND POLY- INTRON-DEPENDENT LOCALIZATION OF RNA IN THE SOMES IN KREBS II ASCIIES CELLS. ((A. Vedeler and V. Gerke*)) CELL NUCLEUS. ((S. Huang, S. Gunnery, M.B. Mathews and D.L. Department of Biochemistry and Molcular Biology, University of Bergen, Spector)) Cold Spnng Harbor Laboratory, Cold Spring Harbor, New York N-5009 Bergen, Norway and *Department of Biochemistry, Max-Planck- 11724.
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