Thomas D. Ingolia

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Active bacterial luciferase from a fused gene: expression of a Vibrio harveyi luxAB translational fusion in bacteria, yeast and plant cells.

The luxA and luxB genes encoding the luciferase from Vibrio harvey were fused by site-directed mutagenesis so that one polypeptide was encoded by the fused gene. The fused gene facilitated light production in Escherichia coli, Saccharomyces cerevisiae, and Nicotiana plumbaginifolia when the substrates decanal and reduced flavin mononucleotide were present. The specific activity of the encoded enzyme is not known. In E. coli and S. cerevisiae cells the light emission could be measured in viable, intact cells. The luxAB fusion provides a simple reporter gene for in vivo measurement of promoter strength in these species and may be useful in other systems as well.

Organization of the 5.8S, 16-18S, and 23-28S ribosomal RNA genes of Cephalosporium acremonium

SummaryA 9.2 kb Pst1 restriction fragment, repeated tandemly head-to-tail in the genome, contains the 5.8S, 16–18S, and 23–28S ribosomal RNA (rRNA) genes of Cephalosporium acremonium, a filamentous fungus used at the industrial scale for production of cephalosporin antibiotics. These rRNA genes were located in Pst1 digests of C. acremonium genomic DNA using a hybridization probe that contained the 5.8S, 18S, and 25S rRNA genes from the yeast Saccharomyces cerevisiae. This probe was also used in screening a recombinant lambda library to identify phage carrying rRNA genes of C. acremonium. Yeast rRNA genes contained separately on individual 32P-labeled plasmids were used to demonstrate that a cloned 7.2 kb C. acremonium sequence, represented in the repeated 9.2 kb Pst1 fragment, contained DNA from the C. acremonium 5.8S, 16–18S, and 23–28S rRNA genes. These genes were ordered with the 5.8S gene located between the 16–18S and 23–28S rRNA genes. The order of the 16–18S, 5.8S, and 23–28S rRNA genes observed in C. acremonium is the same as that observed in rRNA repeats of many other lower eucaryotes, e.g. S. cerevisiae, Aspergillus nidulans, and Neurospora crassa.

Plasmid-mediated complementation of a Δ-aminolevulinic-acid-requiring Saccharomyces cerevisiae mutant

Recombinant plasmids able to complement the Saccharomyces cerevisiae ole3 mutation were isolated. The nucleotide sequence responsible for complementation was localized to a 3.5-kb region. The level of delta-aminolevulinate (ALV) synthase activity in wild-type cells was six-fold lower than in plasmid-transformed ole3 mutant cells. Certain clones secreted a compound that supported growth of a lawn of adjacent ole3 mutant cells.