Cloning full length cDNAs is a difficult task especially if mRNAs are not abundant or if tissue is only available in limited amounts. Current strategies are based on in vitro amplification of cDNAs after adding a homopolymeric tail at the 3' end of the ss-cDNA. Since subsequent amplification steps yield unspecific amplified DNA mostly due to non-specific annealing of the reverse primer containing a homopolymeric tail, we have devised a new strategy based on the ligation of single-stranded oligodeoxyribonucleotide to the 3' end of single-stranded cDNAs. The efficiency of the strategy was assessed by analyzing the 5' ends of the rat pineal gland tryptophan hydroxylase messenger. The 5' end of the least abundant messenger (0.005% of total mRNAs) could be cloned without selection. Sixty percent of the analyzed clones correspond to TPH. This technique revealed a 5-nt stretch not apparent using dG tailing strategy. The potentiality of the method for generating cDNAs libraries was tested with 10(4) PC12 cells. In this library, the abundance of tyrosine hydroxylase clones (0.03%) correlated well with the abundance of the corresponding messenger, showing that no major distortion was introduced into the construction of the library.
In a previous study, we characterized two tryptophan hydroxylase mRNAs (TPH mRNAs) in the pineal gland. However, we failed to detect these species in the raphe by Northern blot experiments. Here, we report by S1 nuclease analysis and in situ hybridization that these two TPH mRNAs, as well as a third species, are expressed both in pineal gland and in raphe. In both tissues, the three mRNAs are transcribed predominantly from the same promoter. Strikingly, from the results of S1 maping analysis, it was observed that the total level of TPH mRNA per tissue is at least 150 times lower in the raphe than in the pineal gland. In contrast, TPH antigen as quantified by immunoblot experiments is about threefold more abundant per raphe than per pineal gland. TPH mRNA from one raphe and one pineal gland yield in vitro about the same amount of TPH antigen, suggesting that the discrepancy in the ratios of TPH mRNA and TPH antigen between the raphe and the pineal gland results, at least in part, from a difference in the translation efficiency of TPH mRNAs in the two structures.
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'.
Numerous therapies and biological questions could be addressed in mammals by the application of a molecular switch that would allow physicians and/or investigators to turn individual genes on or off during the lifetime of the organism. We have constructed such a switch, composed of three elements: (i) an inducible promoter that is normally absent from mammalian genomes; (ii) a receptor that, when it is bound to an inducer drug, specifically activates transcription from the inducible promoter; and (iii) inducer drugs, such as RU486, whose pharmacological properties in humans and several mammalian species including mouse have been well studied. The molecular switch is functional in transiently and stably transfected cells. Importantly, both the total output and the induction levels of the reporter gene can be finely tuned, with induction levels of over 100-fold being readily attained. Finally, we demonstrate that the molecular switch can be used to regulate a mouse transgene using a gene therapy paradigm. The specificity of the system suggests that it should be useful in the analysis of gene function in transgenic animals and in the design of strategies for human gene therapy.
Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss-cDNAs. This strategy is referred to as SLIC for single-strand ligation to ss-cDNA (8).
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