A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor al leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.Human preproinsulin consists of a prepeptide of 24 amino acid residues followed by proinsulin containing 86 amino acid residues in the configuration: prepeptide-B-Arg-Arg-C-LysArg-A in which C is the C peptide of 31 amino acid residues (1), and A and B are the A and B chain of human proinsulin, respectively. The prepeptide is removed during transport of the nascent polypeptide into the endoplasmic reticulum. By the time it reaches the Golgi, the disulfide bridges of proinsulin (2)

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By the use of glucoamylase‐specific synthetic oligodeoxyribonucleotides and molecular cloning of cDNA synthesized from Aspergillus niger total poly(A) + RNA, the primary structure of the glucoamylase G1 mRNA was determined. Glucoamylase G1 is synthesized as a precursor of 640 amino acid residues containing a putative signal peptide of 18 residues, a short propeptide of six residues and the 616 residues long mature enzyme. In vitro translations of mRNA and immunoprecipitations with glucoamylase‐specific antisera showed that two glucoamylase polypeptides are synthesized. The larger form with an apparent mol. wt. of 71 000 corresponds to the precursor of glucoamylase G1, and the shorter form with an apparent mol. wt. of 61 000 corresponds to the precursor of glucoamylase G2. From the nucleotide sequencing data of several glucoamylase‐specific cDNA recombinants it is shown that the G1 mRNA contains a 169 bp long intervening sequence that can be spliced out to generate a G2 mRNA. Only the 3′ part of the G1 mRNA is modified by this splicing event. This kind of differential mRNA processing to give different protein products from one primary transcript has previously only been demonstrated in higher eukaryotes.

Cultures were grown with reciprocal shaking at 370 in either phosphate-buffered medium M9 (10) or in Tris medium (11) with 0.2% glucose (w/v) as energy source. These growth media contained 20 pg/ml of -proline, 20 pg/ml of zAeucine, 10 pg/ml of uracil, and 50 pg/ml of all other growth requirements. To achieve rapid amino-acid starvation, cultures were shifted by membrane filtration to growth medium lacking amino acids. For shift-down experiments, cultures were grown in medium containing 0.01% glucose and 0.2%o sodium acetate. Large cultures for ribosome preparation (5 liters or more) were grown at 370 in L-broth (12) plus 1% glucose with vigorous aeration to A4W = 2.0 (about 5 X 108 cells per ml). The cultures were cooled rapidly with distilled-water ice and harvested in a cooled Sharples continuous-flow centrifuge. Transductions were carried out in Lbroth as described by Lennox (12).Radioactive Labeling. Protein and RNA synthesis were measured by adding ["4Cjproline (0.5 p&Ci/ml) and [5-8H]uracil (5 pCi/ml) (New England Nuclear Corp., Boston, Mass.) to a growing culture at the appropriate time. Samples (100 pl each) were pipetted into 2 ml of ice-cold 5% trichloroacetic acid; samples were prepared and the radioactivity was counted as described (11). Guanosine tetraphosphate measurements were made on cultures growing in Tris medium (0.35 mM phosphate) to which 20-50 ;pCi/ml of ['2PJortho-phosphate had been added at least one-half generation before the first sampling. Samples of 50 pul were pipetted at intervals into ice-cold tubes containing 5 ;pl of 13 M formic acid and were chromatographed as described (11). Tests for stringency or relaxedness in suspected mutants or in recombinants were most reliably done as follows: colonies were picked from agar plates, resuspended in separate tubes containing 0.25 ml of the appropriate selective (minimal) growth medium containing 20,ug/ml of jAeucine and 10 pg/ml'of uracil, and grown overnight with shaking. Next day each culture was diluted about 50-fold into 0.5 ml of the same growth medium and incubated with shaking at 370 for 2-4 hr. Then 1 drop of a mixture containing 500 pg/ml of 5-methyl tryptophan (or

A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.