Journal of Plant Biotechnology

The Korean Society of Plant Biotechnology (한국식물생명공학회)
권호 표지

Unusual Orientation of cDNAs Found in a cDNA Library

  • Lee Jeongyeo (Genome Research Institute, Chungnam National University) ;
  • Song Hayoung (Genome Research Institute, Chungnam National University) ;
  • Lim Yong-Pyo (Genome Research Institute, Chungnam National University) ;
  • Hur Yoonkang (Genome Research Institute, Chungnam National University)
  • Published : 2005.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Many cloning vectors in which cDNAs can be inserted to the sense orientation have been developed. Uni-ZAP XR vector (Stratagene) should contain clones that are oriented to sense direction with respect to T3 RNA polymerase primer. Unexpectedly, large portions of cDNAs in Chinese cabbage cDNA library showed unusual insertions, antisense orientation and a hybrid of two different clones. Using two clones, 4H03 and 53-B10, derived from different cDNA libraries, we proposed and demonstrated the possibility of unusual-construct formation by in vitro translation and northern blot analysis. The 4H03 clone was inserted with inverse direction, and its transcript and translation product could be produced by T7 RNA polymerase, indicating that this clone is definitely inserted into inverse orientation. The 53-B10 that contains two independent genes was turned out to be a hybrid in which two genes are inserted to opposite direction each other. All unusual constructs might be due to the presence of small fragments of DNA, like adapter. However, the mechanism underlined the formation of unusual constructs is still remain to be solved.

Keywords

References

  1. Baltimore D (1970) RNA-dependent DNA polymerase in virions of RNA tumour viruses. Nature 226: 1209-1211 https://doi.org/10.1038/2261209a0
  2. Bank A, Terada M, Metafora S, Dow L, Marks PA (1972) In vitro synthesis of DNA components of human genes for globins. Nat New Biol 235: 167-169 https://doi.org/10.1038/235167a0
  3. Kretz PL, Reid CH, Greener A, Short JM (1989) Effect of lambda packaging extract mer restriction activity on DNA cloning. Nucleic Acids Res 17: 5409 https://doi.org/10.1093/nar/17.13.5409
  4. Krug MS, Berger SL (1987) First-strand cDNA synthesis primed with oligo (dT). Method Enzymol 152: 316-325 https://doi.org/10.1016/0076-6879(87)52036-5
  5. Ross J, Aviv H, Scolnick E, Leder P (1972) In vitro synthesis of DNA complementary to purified rabbit globin mRNA (RNA-dependent DNA polymerase-reticulocyte-hemoglobindensity grdient centrifugation-oligo (dT) primer). Proc Natl Acad Sci 69: 264-268 https://doi.org/10.1073/pnas.69.1.264
  6. Rabbius TH (1976) Bacterial cloning of plasm ids carrying copies of rabbit globin messenger RNA. Nature 260: 221-225 https://doi.org/10.1038/260221a0
  7. Rougeon F, Kourilsky P, Mach B (1975) Insertion of a rabbit ${\beta}$ -globin gene sequence into an E. coli plasmid. Nucleic Acids Res 2: 2365-2378 https://doi.org/10.1093/nar/2.12.2365
  8. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, New York
  9. Short JM, Fernandez JM, Sorge JA, Huse ED (1988) ${\lambda}$ ZAP: a bacteriophage ${\lambda}$expression vector with in vivo excision properties. Nucleic Acids Res 16: 7583-7600 https://doi.org/10.1093/nar/16.15.7583
  10. Temin HM, Mizutani S (1970) RNA-dependent DNA polymerase in virions of Rous sarcoma virus. Nature 226: 1211-1213 https://doi.org/10.1038/2261211a0
  11. Verma IM, Temple GF, Fan H, Baltimore D (1972) In vitro synthesis of DNA complementary to rabbit reticulocyte 10S RNA. Nat New Biol 235: 163-167 https://doi.org/10.1038/235163a0