A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames.

A one-step (all reactants added simultaneously) reverse transcription and polymerase chain reaction (RT-PCR) procedure for amplification of full-length open reading frames (ORFs) of relatively rare transcripts was developed. It was applied for cloning rat luteinizing hormone/chorionic gonadotropin receptor cDNA isoforms larger than two kb. In the procedure developed, manual work is minimized, thus large numbers of samples can be handled, since after denaturation of template RNA and the primers and addition of other reagents, no further manual steps are needed. No inhibitory effect of avian myeloblastosis virus (AMV) reverse transcriptase (RT) on Thermus aquaticus (Taq) DNA Polymerase was found. This was because, under the conditions described, Taq DNA Polymerase effectively amplified picogram amounts of plasmid DNA or template reverse transcribed from nanograms of total ovarian RNA in the presence of AMV-RT. Even a large excess of AMV-RT did not inhibit Taq DNA Polymerase. Thus, our coupled one-step RT-PCR procedure amplifies fast and reproducibly full-length ORFs from nanogram amounts of total RNA.

Human chorionic gonadotrophin (CG)-induced down-regulation of the rat luteal LH/CG receptor results in part from the down-regulation of its synthesis, involving increased alternative processing of the primary transcript.

To elucidate the molecular mechanisms involved in the homologous regulation of LH/chorionic gonadotrophin (CG) receptors, the receptor and its mRNA levels were analysed in the same pseudopregnant rat ovarian samples after human (h)CG-induced down-regulation using a binding assay, ligand blotting, immunoblotting and Northern blotting together with the polymerase chain reaction (PCR). Treatment of the animals with 500 IU hCG resulted in a loss of 125I-labelled hCG binding and the 90 kDa receptor on the ligand and immunoblots within 12 and 24 h respectively, followed by a transient partial recovery on days 4 and 5, while a distinct decline occurred only on day 7 in the controls. Northern blots of total ovarian RNA, as probed with a 293 bp AvaI/HindIII fragment from the extracellular domain of PCR-generated full-length rat LH/CG receptor cDNA, revealed six major mRNAs of 7.0, 4.2, 2.8, 2.0, 1.4 and 1.1 kb. The 4.2 kb mRNA, which was the most abundant, possibly encodes the 90 kDa receptor, while the smaller species probably represent alternatively spliced forms of the LH/CG receptor pre-mRNA, as also supported by the finding that PCR produced three cDNA bands of 2.1, 2.0 and 1.8 kb when oligomers derived from the N and C termini of rat LH/CG receptor cDNA were used as primers and rat ovarian total RNA as a template. Treatment with hCG led to the down-regulation of all six mRNAs in a fashion parallel to the changes in receptor protein. No smaller receptor components capable of binding radiolabelled hCG or receptor antibody appeared on the ligand or immunoblots prior to or during down-regulation or the subsequent transient period of up-regulation, suggesting that the smaller mRNA species are translated in minute amounts in vivo or are not translated at all. Laser densitometric analysis of the Northern blots revealed that the amounts of the four smallest mRNA species increased during the period of down-regulation in relation to the 4.2 kb mRNA, and correspondingly decreased during the subsequent period of up-regulation, indicating changes in the alternative splicing of the primary transcript. The data suggest that hCG-induced transient down-regulation of the LH/CG receptor results in part from down-regulation of its mRNA levels, and that changes in alternative processing of the receptor pre-mRNA may play a regulatory role in the expression of functional LH/CG receptor during down- and up-regulation.

Expression of the LH/CG receptor gene in rat ovarian tissue is regulated by an extensive alternative splicing of the primary transcript.

Luteinizing hormone/chorionic gonadotropin (LH/CG) receptor complementary DNA (cDNA) isoforms were amplified using pseudopregnant rat ovarian total RNA as a template and the primers reaching over the coding regions at both ends in a reverse transcriptase-polymerase chain reaction (RT-PCR). Agarose gel electrophoresis of the PCR products revealed three bands corresponding to about 2.1, 2.0 and 1.8 kilobases (kb). Subcloning of pooled PCR products into EcoRI site of pUCBM20 resulted in 167 clones, from which five different restriction patterns were obtained by digestion with EcoRI and HaeIII. One clone of each was further characterized. It could be predicted from the nucleotide sequences that the clone rLHR2100 encoded a full-length receptor (a 674 amino acid mature protein), the clone rLHR2075 lacked part of exon IX (nucleotides 693-717) and encoded a truncated 225 amino acid mature protein, the clone rLHR1950 lacked exons III and IV (nucleotides 246-395) and encoded a nearly full-length protein (a 624 amino acid mature protein), and the clones rLHR1834 and rLHR1759 lacked the same part of exon XI (nucleotides 960-1225), with exon V (nucleotides 396-470) also absent in the latter, the deletion in exon XI leading both these clones to premature termination. The clone rLHR1834 encoded a 316 amino acid mature protein and rLHR1759 a 291 amino acid mature protein, respectively. The sequence data suggest that all of these isoforms contain the putative signal sequence and are derived from a single copy gene via alternative splicing. These results point further to the fact that the expression of the 90 kDa LH/CG receptor is regulated via an extensive alternative splicing of the receptor gene primary transcript.