Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Aug 25;17(16):6439–6448. doi: 10.1093/nar/17.16.6439

An efficient strategy for cloning 5' extremities of rare transcripts permits isolation of multiple 5'-untranslated regions of rat tryptophan hydroxylase mRNA.

J Delort 1, J B Dumas 1, M C Darmon 1, J Mallet 1
PMCID: PMC318339  PMID: 2780283

Abstract

The 5' end mapping of rat tryptophan hydroxylase (TPH) mRNA indicated a diversity in 5'-untranslated regions. Corresponding sequences were isolated by a variant of the Polymerase Chain Reaction, recently designated as 'anchor PCR', and a 'cRNA enrichment' procedure. The latter circumvents the limitations of 'anchor PCR', which failed to yield minor TPH sequences: this novel strategy allows purification of specific DNA fragments by elimination of the unspecific products, generated by the PCR, which prevent further amplification. Analysis of TPH sequences strongly suggests that TPH mRNAs are synthesized from at least two promoters, the proximal one exhibiting two 'CCAAT homologies'.

Full text

PDF
6439

Images in this article

6442Image
on p.6442
6444Image
on p.6444

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albrecht G., Krowczynska A., Brawerman G. Configuration of beta-globin messenger RNA in rabbit reticulocytes. Identification of sites exposed to endogenous and exogenous nucleases. J Mol Biol. 1984 Oct 5;178(4):881–896. doi: 10.1016/0022-2836(84)90317-6. [DOI] [PubMed] [Google Scholar]
  2. Aziz N., Munro H. N. Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8478–8482. doi: 10.1073/pnas.84.23.8478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Civelli O., Birnberg N., Herbert E. Detection and quantitation of pro-opiomelanocortin mRNA in pituitary and brain tissues from different species. J Biol Chem. 1982 Jun 25;257(12):6783–6787. [PubMed] [Google Scholar]
  5. Connelly S., Manley J. L. A CCAAT box sequence in the adenovirus major late promoter functions as part of an RNA polymerase II termination signal. Cell. 1989 May 19;57(4):561–571. doi: 10.1016/0092-8674(89)90126-8. [DOI] [PubMed] [Google Scholar]
  6. Darmon M. C., Grima B., Cash C. D., Maitre M., Mallet J. Isolation of a rat pineal gland cDNA clone homologous to tyrosine and phenylalanine hydroxylases. FEBS Lett. 1986 Sep 29;206(1):43–46. doi: 10.1016/0014-5793(86)81337-0. [DOI] [PubMed] [Google Scholar]
  7. Darmon M. C., Guibert B., Leviel V., Ehret M., Maitre M., Mallet J. Sequence of two mRNAs encoding active rat tryptophan hydroxylase. J Neurochem. 1988 Jul;51(1):312–316. doi: 10.1111/j.1471-4159.1988.tb04871.x. [DOI] [PubMed] [Google Scholar]
  8. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gorski K., Roch J. M., Prentki P., Krisch H. M. The stability of bacteriophage T4 gene 32 mRNA: a 5' leader sequence that can stabilize mRNA transcripts. Cell. 1985 Dec;43(2 Pt 1):461–469. doi: 10.1016/0092-8674(85)90176-x. [DOI] [PubMed] [Google Scholar]
  10. Grenett H. E., Ledley F. D., Reed L. L., Woo S. L. Full-length cDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5530–5534. doi: 10.1073/pnas.84.16.5530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jeffreys A. J., Wilson V., Neumann R., Keyte J. Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells. Nucleic Acids Res. 1988 Dec 9;16(23):10953–10971. doi: 10.1093/nar/16.23.10953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kim H. S., Smithies O. Recombinant fragment assay for gene targetting based on the polymerase chain reaction. Nucleic Acids Res. 1988 Sep 26;16(18):8887–8903. doi: 10.1093/nar/16.18.8887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kimmel A. R., Berger S. L. Preparation of cDNA and the generation of cDNA libraries: overview. Methods Enzymol. 1987;152:307–316. doi: 10.1016/0076-6879(87)52035-3. [DOI] [PubMed] [Google Scholar]
  14. Kozak M. A profusion of controls. J Cell Biol. 1988 Jul;107(1):1–7. doi: 10.1083/jcb.107.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Loh E. Y., Elliott J. F., Cwirla S., Lanier L. L., Davis M. M. Polymerase chain reaction with single-sided specificity: analysis of T cell receptor delta chain. Science. 1989 Jan 13;243(4888):217–220. doi: 10.1126/science.2463672. [DOI] [PubMed] [Google Scholar]
  16. Maniatis T., Goodbourn S., Fischer J. A. Regulation of inducible and tissue-specific gene expression. Science. 1987 Jun 5;236(4806):1237–1245. doi: 10.1126/science.3296191. [DOI] [PubMed] [Google Scholar]
  17. McKnight S., Tjian R. Transcriptional selectivity of viral genes in mammalian cells. Cell. 1986 Sep 12;46(6):795–805. doi: 10.1016/0092-8674(86)90061-9. [DOI] [PubMed] [Google Scholar]
  18. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  19. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  20. Stoflet E. S., Koeberl D. D., Sarkar G., Sommer S. S. Genomic amplification with transcript sequencing. Science. 1988 Jan 29;239(4839):491–494. doi: 10.1126/science.3340835. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES