ANALYSIS OF EXPRESSION OF THE GENE ENCODING FOR THE NUCLEAR AUTOANTIGEN LA SS-B USING REPORTERGENE CONSTRUCTS/

In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was repl aced 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 f rame, which starts in the exon 2. The exon 1' was located in the intro n about 70 nts downstream of the exon 1. The exon 1' La mRNA was propo sed to be the result of a promoter switch in combination with an alter native splicing mechanism. The commonly used technique to study the ex pression of a eucaryotic gene is to fuse a reportergene immediately do wnstream of the proposed regulatory elements. Due to (i) the short dis tance between exon 1 and exon 1', (ii) the varying 5'-starts of the ex on 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 L a 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 p ortion of the exon 2. Luciferase was fused into the exon 2. This const ruct was used to prepare 5'-deletion mutants. The constructs were tran siently transfected into HeLa cells. RNAs were isolated from the trans iently 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 correct ly transcribed and spliced from the La luciferase fusion construct. Mo reover, the 5'-start of the respective transcript allowed to identify those genomic regions in the La gene that were most likely being invol ved in determining the respective transcription initiation site. In pa rallel to the estimation of the 5'-start of the transcripts, the lucif erase activity was measured. Thereby we detected a cryptic promoter el ement in the intron between the exon 1 and exon 2. (C) 1998 Elsevier S cience B.V.