The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

Cloning and characterization of a gene encoding pig epidermal growth factor.

A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron-exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5' end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5' end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.

Amplification of plasmid copy number by thymidine kinase expression in Saccharomyces cerevisiae.

A 2 micron circle-based chimaeric plasmid containing the yeast LEU2 and the Herpes Simplex Virus type 1 thymidine kinase (HSV-1 TK) genes was constructed. Transformants grown under selective conditions for the LEU2 gene harboured the plasmid at about 15 copies per cell whilst selection for the HSV-1 TK gene led to an increase to about 100 copies per cell. Furthermore, the plasmid copy number could be controlled by the stringency of selection for the TK gene, and the increase in TK gene dosage was reflected in an increase in intracellular thymidine kinase activity. The mitotic stability of the plasmid in "high-copy" and "low-copy" number cells was determined. "High-copy" number cells showed a greater mitotic stability. The relationship of TK expression to plasmid copy number may be useful for the isolation of plasmid copy number mutants in yeast and the control of heterologous gene expression.

The secretion and post translational modification of interferons from Saccharomyces cerevisiae.

Studies with three interferon molecules, IFN-alpha 2, IFN-beta 1, and a "hybrid" interferon, IFNX-430 are described which illustrate that both the expression and secretion characteristics of heterologous proteins in yeast cells reflect properties of the proteins themselves. Recombinant DNA techniques have also been used to demonstrate that the efficient processing of mature heterologous proteins from the yeast alpha factor secretion leader can be affected by sequences on the carboxyl side of the initial cleavage site. Secretion studies with heterologous proteins in S. cerevisiae are aimed at maximising yield, the percentage of extracellular product and correct amino terminus sequence. The results presented here show that all three factors are susceptible to currently unpredictable properties of the foreign sequence. This situation, in turn, means that heterologous proteins can be used as tools in the biochemical dissection of the yeast secretion process.

Novel modified beta-interferons: gene cloning, expression, and biological activity in bacterial extracts.

A series of novel, modified interferons based on the structure of human beta-interferon have been expressed in Escherichia coli. Modified interferon genes were constructed from sequences derived from the natural beta-interferon gene, a synthetic beta-interferon gene, or a specific combination of the two. A total of 23 out of the 25 novel interferons exhibited antiviral (AV) and antiproliferative (AP) activity which varied from 3 to 230% and 8 to 490% of the values for beta-interferon, respectively. None of the novel interferons had only AV or AP activity, although one had a much reduced ratio of AV/AP activity compared with beta-interferon. Substitution of beta-interferon amino acids 2-7 or 28-46 resulted in interferons with significantly increased AP activity on Daudi lymphoblastoid cells (four- to fivefold). All the novel interferons except two with modifications in the 82-105 region reacted with a neutralizing beta-interferon monoclonal antibody.

Secretion of mammalian polypeptides from yeast.

Yeast cells are capable of expressing and secreting foreign polypeptides into the medium. Mammalian glycoproteins are glycosylated when secreted from yeast although the exact oligosaccharide sequence is not reproduced.

Biological properties of human interferon beta 1 synthesized in recombinant bacteria.

Human fibroblast interferon, designated IFN-beta 1, has been produced in E. coli by direct expression of the cloned cDNA coding for the mature polypeptide. Bacterial lysates from recombinant cultures contain a polypeptide with an apparent molecular weight of 17,500 that corresponds in size to the unglycosylated IFN-beta 1 molecule. The latter could be specifically immunoprecipitated by antibodies to purified natural IFN-beta and could inhibit the replication of Herpes simplex virus types 1 and 2 in many different cell lines. Like the natural fibroblast IFN-beta, the bacterial IFN-beta 1 was active in many human cell lines, less active in a monkey cell line and inactive in rabbit and mouse fibroblasts. The antibody titre required to neutralise the anti-herpes activity of both IFN preparations was similar suggesting that they have the same specific activities. Similarly, the bacterial IFN-beta 1 was equally active in inhibiting the proliferation of Daudi cells grown in culture. Bacterial IFN-beta 1 was also capable of enhancing natural killer cell activity and antibody-dependent cellular cytotoxicity in vitro. Thus, IFN-beta 1 produced in recombinant bacteria displays a large range of biological properties ascribed to the natural fibroblast IFN-beta molecule.

The absence of introns within a human fibroblast interferon gene.

Experiments in which immobilised restriction fragments of genomic DNA were hybridised with a cloned human fibroblast interferon cDNA indicate that the homologous chromosomal genes exist in only one basic arrangement. This is in marked contrast to recent studies by Nagata et al. (1) showing that there are at least eight gene arrangements for human leukocyte interferon. Having isolated a chromosomal human fibroblast interferon gene from a gene bank, we conclude from nucleotide sequencing studies that there is a complete absence of introns within the RNA-coding region. In view of a similar observation recently made for a human leukocyte interferon gene (1), it would appear as if interferon genes in general are unlike the vast majority of eukaryote genes in this respect.

The complete amino acid sequence of human fibroblast interferon as deduced using synthetic oligodeoxyribonucleotide primers of reverse transcriptase.

Using synthetic oligodeoxyribonucleotides to prime the transcription of interferon mRNA and cDNA, we recently determined the mRNA sequence coding for the 47 amino-terminal amino acids of mature human fibroblast interferon (1). From this sequence, we have now synthesised an oligodeoxyribonucleotide that is homologous with the mRNA sequence coding for amino acids 42-45 and used it as a primer to selectively transcribe an interferon cDNA template. The sequence of the newly synthesised DNA predicted the sequence of amino acids 48-109 in the interferon polypeptide. By repeating this process with one more primer, we have determined the complete amino acid sequence of mature human fibroblast interferon, a polypeptide of 166 amino acids.

The amino-terminal sequence of human fibroblast interferon as deduced from reverse transcripts obtained using synthetic oligonucleotide primers.

From recently published data on the amino-terminal structures of human and mouse interferons, we have predicted and synthesised an oligonucleotide capable of priming specifically the reverse transcription of human fibroblast interferon mRNA present within a total mRNA population. From these transcripts we determined the sequence of the 5'-terminus of the mRNA and identified a putative pre-peptide signal sequence. This enabled us to predict the sequence of another primer capable of directing the synthesis of interferon double-stranded cDNA corresponding to the entire coding region of the mRNA. Further sequencing studies also enabled us to establish the identity of 47 consecutive amino acids beginning with the methionine residue at the amino-terminus of the mature protein.