Yeast is unable to excise foreign intervening sequences from hybrid gene transcripts.

Abstract

To investigate whether transcripts from foreign split genes are correctly processed in yeast cells we have constructed two hybrid genes by inserting into the split yeast actin gene an intron-containing fragment from either the Acanthamoeba actin I gene or the duck aD-globin gene. The hybrid genes were inserted into the autonomously replicating yeast plasmid YRp7, which was then used to transform yeast cells. It was found that the yeast but not the foreign intervening sequences were excised from the chimeric transcripts. This indicates that the recognition of intervening sequences or the splicing mechanism of RNA polymerase II transcripts is not universal.Introns in all eukaryotic genes transcribed by RNA polymerase II have two common structural features: their sequences begin with the dinucleotide 5'-G-T-3' and end with the dinucleotide 5'-A-G-3' (1). Besides these invariant nucleotides, there is limited structural similarity at and around the intron-exon junctions. Consensus sequences of about 10 nucleotides for the 5' and 3' splice sites have been derived from a comparison ofmore than 100 splice junctions (2, 3).Because of the similarity of splice junction sequences in species as distant as yeast and man, it has been speculated that splicing may not be species specific. Experiments supporting this notion have shown that, for instance, mouse and rat genes inserted into simian virus 40 (SV40) vectors are transcribed in monkey cells and their transcripts are properly spliced (4, 5) and that transcripts ofrabbit and chicken genes are correctly spliced in mouse cells (6, 7). Even a hybrid intervening sequence with the 5' splice region from the mouse ,3-globin gene was properly excised from a chimeric transcript in monkey cells (8), although the splice junction sequences of the two donor genes do not exhibit obvious similarities, except for the conserved G-T and A-G dinucleotides. All these studies were undertaken with genes and cells from higher eukaryotes and it is likely that, at least in vertebrates, the splicing machinery for RNA polymerase II transcripts is interchangeable.The actin gene of the unicellular eukaryote Saccharomyces cerevisiae contains an intron with 5' and 3' intron-exon junction sequences resembling the eukaryotic consensus sequences (9, 10). An indication that the RNA splicing mechanism in yeast might nevertheless differ from that in higher eukaryotes came from a study in which the mouse -globin gene was introduced into yeast cells viaa self-replicating recombinant plasmid (11). In this case the first P-globin intervening sequence was not removed, but this could also be related to the incorrect initiation and termination observed with this gene transcript.We report here on the construction of two hybrid genes in which either an ameba actin or a duck globin gene fragment containing an intron has been inserted into the middle of the yeast actin gene. We show that in transformed yeast cells the splicing of the chimeric transcripts is selective in the sense that the yeast intervening sequ...

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

“…Differences exist in features thought to be involved in gene expression in S. cerei'isiae and higher eucaryotes. This is perhaps best demonstrated by the inability of S. cerevisiae to process heterologous intervening sequences (4,29). Species specificity, and evolution, of control elements is not unexpected.…”

Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

“…The GT:AG rule (3), the two-step pathway of lariat formation and exon ligation (7,28,34,43), and spliceosome requirement define pre-mRNA splicing myces pombe and Saccharomyces cerevisiae (23). These observations help explain why S. cerevisiae cannot splice most higher eucaryotic pre-mRNAs (2,20).…”

Engineering of gentiopicroside-producing yeast strain using low-energy ion implantation mediated synthetic biology

“…However, previous research on heterologous gene expression in yeasts has shown that yeasts are incapable of correctly removing intervening sequences from transcripts of plants due to the differences between plant and yeast intron splicing. [22] Interestingly, in the circumstances of ion beam-induced exogenous DNA transfer, the relevant genes, or gene clusters, involved in the biosynthesis of gentiopicroside in plants must have been successfully expressed in H. polymorpha. This phenomenon might be explained either by yeast alternative splicing, [23] or by the biological effects induced by ion implantation, including energy absorption, mass deposition and charge transfer of energetic ions in organisms.…”