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Nucleic Acids Research, 1989, Vol. 17, No. 16 6439-6448
© 1989

MOLECULAR BIOLOGY

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

Jacques Delort, Jean Baptiste Dumas, Michèle C. Darmon and Jacques Mallet*

Laboratoire de Neurobiologie Molèculaire et Cellulaire, Centre National de la Recherche Scientifique 91198 Gif-sur-Yvette, France

*To whom correspondence should be addressed

Received July 7, 1989. Accepted July 27, 1989.

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’.


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