ABSTRACT
The expression of a cloned trout protamine gene transfected into mammalian cells in culture has been studied. This small intronless gene has a consensus TATA-box, a classical AATAAA sequence and the cap and polyadenylation sites are separated by only 228 base pairs (Gregory et al., ref 10). When 1kb of cloned trout genomic DNA containing this sequence was introduced into HeLa cells, S1-mapping showed that transcripts of the protamine gene were accurately initiated at the in vivo cap site but were not polyadenylated at the authentic 3'-site. Replacement of the 3'-end of the protamine transcription unit with a fragment of SV40 containing the small-t intron and early polyadenylation site resulted in only a modest increase in transcript levels over the wild-type gene in HeLa cells. However, transcripts of a fusion gene in which the 5'-end of the protamine gene was replaced by the SV40 early promoter were present at extremely low levels in transfected COS cells. The data are discussed in the context of the involvement of RNA processing events in the stabilisation of eukaryotic gene transcripts.
Subject(s)
Poly A/genetics , Protamines/genetics , RNA Processing, Post-Transcriptional , Transcription, Genetic , Animals , Chromosome Mapping , Female , Gene Expression Regulation , Genes , HeLa Cells , Humans , Plasmids , RNA, Messenger/genetics , Transfection , TroutABSTRACT
The mRNA start site of a cloned rainbow trout protamine gene (TPG-3) has been localised using S1-nuclease mapping and primer extension of in vivo synthesised trout testis poly A+-RNA. The presumptive cap site occurs within an AT-rich region, only 14 nucleotides from the start of the protein-coding sequence. Transcription of this protamine gene in vitro, using the Hela whole-cell extract system, generates products initiated at the same nucleotide as that used in vivo. In vitro transcription is abolished by deletion of sequences between -20 and -48, within which is a canonical TATA-box having an llbp homology with the strong chick conalbumin and Adenovirus-2 major late promoters (CTATAAAAGGG).