Analysis of expression of the gene encoding for the nuclear autoantigen La/SS-B using reporter gene constructs.
In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was replaced with the exon 1'. Moreover, exon 1' La mRNAs were found to start at different 5'-regions. In dependence on the 5'-start the exon 1' La mRNAs encoded for up to three open reading frames upstream of the La frame, which starts in the exon 2. The exon 1' was located in the intron about 70 nts downstream of the exon 1. The exon 1' La mRNA was proposed to be the result of a promoter switch in combination with an alternative splicing mechanism. The commonly used technique to study the expression of a eucaryotic gene is to fuse a reportergene immediately downstream of the proposed regulatory elements. Due to (i) the short distance between exon 1 and exon 1', (ii) the varying 5'-starts of the exon 1' La mRNAs, and (iii) the upstream open reading frames in the exon 1' La mRNAs this technique appeared to be difficult to apply to the La gene. In order to overcome these problems a luciferase reportergene construct was cloned which started about 2500 nts upstream of the exon 1 and contained the exon 1, the intron including the exon 1', and a portion of the exon 2. Luciferase was fused into the exon 2. This construct was used to prepare 5'-deletion mutants. The constructs were transiently transfected into HeLa cells. RNAs were isolated from the transiently transfected cells and analyzed using the 5'-Rapid Amplification of cDNA End technique. The PCR products were subcloned and sequenced. This analysis showed that exon 1 and exon 1' transcripts were correctly transcribed and spliced from the La luciferase fusion construct. Moreover, the 5'-start of the respective transcript allowed to identify those genomic regions in the La gene that were most likely being involved in determining the respective transcription initiation site. In parallel to the estimation of the 5'-start of the transcripts, the luciferase activity was measured. Thereby we detected a cryptic promoter element in the intron between the exon 1 and exon 2.
Grölz
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Institut für Physiologische Chemie, Joh.-Gutenberg Universität, Mainz, Germany.
Tröster
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Semsei
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Bachmann
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D017398:Alternative Splicing; D001324:Autoantigens / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D015870:Gene Expression; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis; D012261:Ribonucleoproteins / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic; D000097585:SS-B Antigen
D001324:Autoantigens; D008156:Luciferases; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D000097585:SS-B Antigen