We have shown earlier that a mitochondrial presequence peptide can form an amphiphilic helix. However, the importance of amphiphilicity for mitochondrial presequence function became doubtful when an artificial presequence, designed to be non‐amphiphilic, proved to be active as a mitochondrial import signal. We now show experimentally that this ‘non‐amphiphilic’ presequence peptide is, in fact, highly amphiphilic as measured by its ability to insert into phospholipid monolayers and to disrupt phospholipid vesicles. This result, and similar tests on three additional artificial presequences (two functionally active and one inactive), revealed that all active presequences were amphiphilic whereas the inactive presequence was non‐amphiphilic. One of the active presequence peptides was non‐helical in solution and in the presence of detergent micelles. We conclude that amphiphilicity is necessary for mitochondrial presequence function whereas a helical structure may not be essential.

The SUP4‐o gene of Saccharomyces cerevisiae codes for an altered tRNATyr capable of suppressing ochre mutations. We constructed mutant SUP4‐o genes with deletions in the 3′‐flanking sequence and tested each for its ability to suppress ochre mutations in transformed yeast cells. The effects of the different 3′ deletions on various aspects of in vitro transcription and RNA processing were also determined, using a yeast cell‐free extract. Deletions that leave five or fewer consecutive T residues in the 3′‐flanking sequence of SUP4‐o were found to result in decreased efficiency of transcription termination, both in vitro and in vivo. Unexpectedly, the suppression strength of each mutant SUP4‐o gene is highly correlated with the relative extent of transcription termination at the 3′ end of the gene. This result indicates that SUP4‐o readthrough transcripts are not efficiently processed to functional suppressor tRNA in yeast cells. Deletions that extend into the T cluster in the 3′‐flanking sequence also significantly decrease the ability of SUP4‐o to compete for a transcription factor that is limiting in our extracts. This latter finding implies that the 3′‐flanking sequence of SUP4 plays a role in transcription factor binding.

Synthetic oligonucleotides were used to construct artificial mitochondrial presequences that contained, besides the initiator methionine, only arginine, serine, and leucine. The ratio of these three amino acids was adjusted to match that of basic, hydroxylated, and hydrophobic residues in natural mitochondrial presequences. When these sequences were fused to the N terminus of yeast cytochrome oxidase subunit IV lacking its own presequence, they directed the attached subunit IV to its correct intramitochondrial location in vivo. They also mediated import of subunit IV into isolated yeast mitochondria. In contrast, artificial sequences containing glutamine, arginine, and serine residues following the initiator methionine were inactive. Thus, the targeting function of mitochondrial presequences does not depend on specific amino acid sequences but may instead depend on the overall balance between basic, hydrophobic, and hydroxylated amino acids.

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High-level expression of a recombinant protein in Chinese hamster ovary (CHO) cells typically requires the laborious and time-consuming procedure of stepwise gene amplification. We hypothesized that use of transcription control regions from a highly expressed gene in CHO cells to drive expression of a gene of interest might reduce the requirement for gene amplification. To this end, we cloned a 19 kb DNA fragment containing the Chinese hamster elongation factor-1alpha (EF-1alpha) gene, as well as 12 kb of 5' flanking sequence and 4 kb of 3' flanking sequence. Expression vectors containing 5' and 3' flanking sequences from the Chinese hamster EF-1alpha (CHEF1) gene were constructed and, after insertion of six different reporter genes, transfected into CHO cells. For comparison, CHO cells were also transfected with the same six reporter genes inserted into commercial vectors utilizing either the immediate early promoter from cytomegalovirus (CMV) or the human EF-1alpha promoter. The striking result from these studies was that average expression levels from pooled, stable transfectants of CHEF1 vectors were 6- to 35-fold higher than expression levels from commercial vectors that utilize the CMV or the human EF-1alpha promoters. We also used a CHEF1 vector to express a secreted and a membrane-bound protein in stably transfected non-CHO cell lines. CHEF1-driven expression of secreted alkaline phosphatase (SEAP) in three of four cell lines tested (HEK 293, K562, L1.2, and HCT 116) was 13- to 280-fold greater than that from a commercial vector employing the CMV promoter. After transfection of four different cell lines of hematopoietic origin (K562, L1.2, JY, and Jurkat), the CHEF1 vector was found to express the chemokine receptor CCR4 at >10-fold higher levels than that driven from a commercial vector utilizing the CMV promoter. Results from these experiments suggest that the CHEF1 vectors will be useful for high-level protein expression not only in CHO cells, but also in a variety of other mammalian cell lines.