1887

Abstract

We succeeded in producing human gamma interferon (IFN-γ) in tobacco protoplasts in quantity using genetically engineered brome mosaic virus (BMV strain ATCC66). This strain of BMV produces two types of coat protein, a full-length coat protein (20K) and a truncated coat protein (19K) which are translated from the first and second initiation codons, respectively. We replaced the truncated coat protein gene with the IFN-γ gene and synthesized BMV–IFN-γ chimera RNAs using an transcription system. The BMV–IFN-γ chimera RNAs were used to inoculate tobacco protoplasts together with BMV RNA 1 and RNA 2 and produced IFN-γ to a level of 5 to 10% of total extracted proteins per infected protoplast after 24 h of incubation. The efficient production of IFN-γ was attributed to the high translation activity of the BMV–IFN-γ chimera RNA. We demonstrate that 24 nucleotides coding for the N-terminal amino acids of the full-length coat protein were probably involved in the high translation activity of the BMV–IFN-γ chimera RNA.

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1993-07-01
2024-04-19
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References

  1. Bradford MM. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 168:48–53
    [Google Scholar]
  2. Chomczynski P, Sacchi N. 1987; Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162:156–159
    [Google Scholar]
  3. Donson J, Kearney CM, Hilf ME, Dawson WO. 1991; Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector. Proceedings of the National Academy of Sciences, U.S.A. 88:7204–7208
    [Google Scholar]
  4. French R, Ahlquist P. 1987; Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA 3. Journal of Virology 61:1457–1465
    [Google Scholar]
  5. French R, Ahlquist P. 1988; Characterization and engineering of sequences controlling in vivo synthesis of brome mosaic virus subgenomic RNA. Journal of Virology 62:2411–2420
    [Google Scholar]
  6. French R, Janda M, Ahlquist P. 1986; Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyl-edonous plant cells. Science 231:1294–1297
    [Google Scholar]
  7. Joshi RT, Joshi V, Ow DW. 1990; BSMV genome mediated expression of a foreign gene in dicot and monocot plant cells. EMBO Journal 9:2663–2669
    [Google Scholar]
  8. Kiberstis PA, Loesch-Fries LS, Hall TC. 1981; Viral protein synthesis in barley protoplasts inoculated with native and fractionated brome mosaic virus RNA. Virology 112:804–808
    [Google Scholar]
  9. Kozak M. 1991; A short leader sequence impairs the fidelity of initiation by eukaryotic ribosome. Gene Expression 1:111–115
    [Google Scholar]
  10. Kunkel TA, Roberts JD, Zakour RA. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154:367–382
    [Google Scholar]
  11. Laemmli UK. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227:680–685
    [Google Scholar]
  12. Lane LC. 1981; Bromoviruses. In Handbook of Plant Virus Infection: Comparative Diagnosis pp. 333–376Edited by Kurstak E. Amsterdam: Elsevier;
    [Google Scholar]
  13. Marsh LE, Dreher TW, Hall TC. 1988; Mutational analysis of the core and modulator sequences of BMV RNA3 subgenomic promoter. Nucleic Acids Research 16:981–995
    [Google Scholar]
  14. Marsh LE, Pogue GP, Szybiak U, Connell JP, Hall TC. 1991; Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication. Journal of General Virology 72:2367–2374
    [Google Scholar]
  15. Miller WA, Dreher TW, Hall TC. 1985; Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on ( —)-sense genomic RNA. Nature, London 313:68–72
    [Google Scholar]
  16. Mise K, Tuge S, Nagao K, Okuno T, Furusawa I. 1992; Nucleotide sequence responsible for the synthesis of a truncated coat protein of brome mosaic virus strain ATCC66. Journal of General Virology 73:2543–2551
    [Google Scholar]
  17. Miyata K, Yamamoto Y, Ueda M, Kawade Y, Matsumoto K, Kubota I. 1986; Purification of natural human interferongamma by antibody affinity chromatography: analysis of constituent protein species in the dimers. Journal of Biochemistry 99:1681–1688
    [Google Scholar]
  18. Mori M, Mis K, Kobayashi K, Okuno T, Furusawa I. 1991; Infectivity of plasmids containing brome mosaic virus cDNA linked to the cauliflower mosaic virus 35S RNA promoter. Journal of General Virology 72:243–246
    [Google Scholar]
  19. Mori M, Mis K, Okuno T, Kobayashi K, Furusawa I. 1992; Expression of brome mosaic virus-encoded replicase genes in transgenic tobacco plants. Journal of General Virology 73:169–172
    [Google Scholar]
  20. Sacher R, Ahlquist P. 1989; Effects of deletion in the N-terminal basic arm of brome mosaic virus coat protein on RNA packaging and systemic infection. Journal of Virology 63:4545–4552
    [Google Scholar]
  21. Takamatsu N, Ishikawa M, Meshi T, Okada Y. 1987; Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA. EMBO Journal 6:307–311
    [Google Scholar]
  22. Towbin H, Staehelin T, Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, U.S.A. 76:4350–4354
    [Google Scholar]
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