Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation.

We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5'-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.

Cloning, characterization, and expression of two alpha-amylase genes from Aspergillus niger var. awamori.

Using synthetic oligonucleotide probes, we cloned genomic DNA sequences encoding an alpha-amylase gene from Aspergillus niger var. awamori (A. awamori) on a 5.8 kb EcoRI fragment. Hybridization experiments, using a portion of this cloned fragment to probe DNA from A. awamori, suggested the presence of two alpha-amylase gene copies which were subsequently cloned as 7 kb (designated as amyA) and 4 kb (amyB) HindIII fragments. DNA sequence analysis of the amyA and amyB genes revealed the following: (1) Both genes are arranged as nine exons and eight introns; (2) The nucleotide sequences of amyA and amyB are identical throughout all but the last few nucleotides of their respective coding regions; (3) The amyA and amyB genes from A. awamori share extensive homology (greater than or equal to 98% identity) with the genes encoding Taka-amylase from A. oryzae. In order to test whether both amyA and amyB were functional in the genome, we constructed vectors containing gene fusions of either amyA and amyB to bovine prochymosin cDNA and used these vectors to transform A. awamori. Transformants which contained either the amyA- or amyB-prochymosin gene fusions produced extracellular chymosin, suggesting that both genes are functional.

A new locus (leuK) affecting the regulation of branched-chain amino acid, histidine, and tryptophan biosynthetic enzymes.

A locus (leuK) affecting regulation of the leucine operon was selected by isolating a spontaneous Ara+ derivative of an Escherichia coli B/r strain carrying an ara-leu fusion in which the arabinose operon is under leucine control. Genetic analyses by P1 transduction demonstrated that the lesion is located to the right of the galactose operon. Regulation of the biosynthetic enzymes for leucine, isoleucine-valine, histidine, and tryptophan was altered in a strain carrying leuK16. High-level gene expression in the heterozygous merodiploid strain F' leuK+/leuK16) demonstrated the dominance of the mutant allele to the wild-type allele. No apparent effect was observed in the mutant on N-acetylornithinase, a biosynthetic enzyme in the arginine pathway, nor on any of the 18 aminoacyl-tRNA synthetases examined. However, compared with that of the parent strain, the extent of the charging of leucyl-, isoleucyl-, valyl-, histidyl-, and arginyl-tRNA was decreased in the mutant.

Medical technologist supervisors: are they prepared to manage?

A group of 330 supervisory technologists in California was surveyed to determine their level of responsibility for each of 21 tasks and the type of education they had received for each task. Their perceived effectiveness as managers was determined using the responses from five questions to construct an Effectiveness Perceived Index (EPI). Two groups were analyzed--those with high task responsibility and low education and those with high task responsibility and high education. For 17 of the 21 tasks, the correlation between low education and low perceived effectiveness was significant, using a .05 significance level. Promotion systems were also explored, and it was found that promotion into supervisory positions is still based mainly on technical competence, not managerial ability. The great need for management development and more enlightened promotion policies in California's laboratories is very much supported by this survey.

Mutation in Saccharomyces cerevisiae conferring streptomycin and cold sensitivity by affecting ribosome formation and function.

A cold-sensitive, streptomycin-sensitive mutant of Saccharomyces cerevisiae accumulates a 28S ribonucleoprotein particle when grown at low temperature. This particle contains 17S ribosomal ribonculeic acid which is degraded when exposed to ribonuclease. The particle does not serve as a precursor to 60 and 40S ribosomal subunits nor is it turned over when growth is allowed to resume at the permissive temperature; rather it is only diluted by growth. That streptomycin sensitivity (allelic with cold sensitivity) is ribosomal is evidenced by the inhibition of protein synthesis in vitro by streptomycin and the binding of labeled streptomycin to the mutant but not the parental 40S ribosomal subunit.

Selection of ribosomal mutants by antibiotic suppression in yeast.

Wild-type Saccharomyces cerevisiae is highly resistant to streptomycin. A histidine auxotroph was found which could grow without histidine in the presence of high concentrations of streptomycin. Selection for derivatives of this strain which could be suppressed by much lower concentrations of streptomycin yielded streptomycin-sensitive mutants which are cold-sensitive and have altered ribosomal profiles.