The presence of an intron relieves gene repression caused by promoter-proximal four-bp specific sequences in yeast.

Introns can enhance gene expression in eukaryotic cells in a process called intron-mediated enhancement (IME). The levels of enhancement are affected not only by the intron sequence but also by coding sequences (CDSs). However, the parts of CDSs responsible for mediating IME have not yet been identified. In this study, we identified an IME-mediating sequence by analyzing three pairs of IME-sensitive and -insensitive CDSs in Saccharomyces cerevisiae. Expression of the CDSs yCLuc, yRoGLU1, and KmBGA1 was enhanced by the presence of an intron (i.e., they were IME sensitive), but the expression of each corresponding codon-changed CDS, which encoded the identical amino acid sequence, was not enhanced (i.e., they were IME insensitive). Interestingly, the IME-insensitive CDSs showed higher expression levels that were like intron-enhanced expression of IME-sensitive CDSs, suggesting that expression of IME-sensitive CDSs was repressed. A four-nucleotide sequence (TCTT) located in the promoter-proximal position of either the untranslated or coding region was found to be responsible for repression in IME-sensitive CDSs, and repression caused by the TCTT sequence was relieved by the presence of an intron. Further, it was found that the expression of intron-containing yeast-native genes, UBC4 and MPT5, was repressed by TCTT in the CDS but relieved by the introns. These results indicate that TCTT sequences in promoter-proximal positions repress gene expression and that introns play a role in relieving gene repression caused by sequences such as TCTT.

Triple gene expressions in yeast, Escherichia coli, and mammalian cells by transferring DNA fragments amplified from a mother yeast expression plasmid.

Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and research, thus various plasmid vectors have been developed. However, the vectors are designed only for a single host owing to their host-specific genetic elements such as promoters and selection markers. In this study, we developed a yeast expression plasmid that enables the expression of the same gene in E. coli and mammalian cells via the transfer of PCR products amplified from the plasmid as a template. The yeast plasmid YHp26352 was constructed to contain the following regions sequentially: yeast TDH3 promoter (TDH3p), red fluorescent protein (eEmRFP), SV40 terminator (SVpA), E. coli origin (ori), ampicillin resistant gene (AmpR), mammalian cytomegalovirus promoter (CMVp), E. coli srlA promoter (srlAp), and yeast selection marker URA3, which expressed eEmRFP in yeast. To express eEmRFP in mammalian cells, an end-promoter DNA fragment encompassing the eEmRFP-SVpA-ori-AmpR-CMVp region was amplified by PCR and directly used for transfection to mammalian culture cells, resulting in gene expression in mammalian cells through non-homologous end joining. Homologous recombination-mediated circularization was carried out for E. coli cloning and expression by attaching a short overlapping sequence to the 5'-end of a PCR primer, which was used to amplify the eEmRFP-SVpA-ori-AmpR-CMVp-srlAp fragment, after which E. coli transformation was performed. Proof-of-concept experiments were performed by expressing GFP-fused human synaptobrevin VAMP1, and wild-type and codon-changed CLuc luciferase genes in yeast, E. coli, and HEK293 cells. This is the first all-in-one plasmid applicable for expression in three host organisms.

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae.

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5'-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5'-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3'-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5'-SS and BPS sequences. Intriguingly, several deletion mutants between the 5'-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5'-SS and BPS sequences and 3'-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5'-SS and BPS play an essential role in IME.