Gregory S. May

Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulincoding genes

Plasmids have been constructed in which expression of a gene can be placed under the control of the inducible promoter of the alcA gene encoding alcohol dehydrogenase I in Aspergillus nidulans. Simplified shuttle vectors carrying pyr4 which complements pyrG89 mutations have also been constructed. These are based on pUC19 and retain alpha-peptide expression. The beta-tubulin genes, tubC and benA, have been placed under the control of alcA and their expression studied. Levels of expression can be assayed phenotypically because increased synthesis of beta-tubulin inhibits vegetative growth. Sensitivity of asexual spore formation to the anti-microtubule drug benomyl provides a means of detecting very low levels of expression of the chimeric genes. Glucose almost completely represses the chimeric genes. Induction is rapid and is maximal within an hour. When a strain carrying seven copies of an alcA::tubC gene fusion was grown under inducing conditions, 6.5% of total sulfate labelled protein consisted of tubC product. Cyclopentanone was the most potent inducer of the chimeric genes on solid media but it also partially inhibited growth. Chimeric alcA::tubC and alcA::benA genes were expressed to very similar levels despite the fact that tubC utilizes many rare codons.

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

A novel phage λ replacement Cre-lox vector that has automatic subcloning capabilities

We have developed a novel phage lambda replacement cloning vector, lambda pAn. lambda pAn allows one to automatically subclone the insert as a plasmid using the Cre-loxP site-specific recombination system. This eliminates the need to subclone insert fragments and permits the rapid structural analysis of insert DNA. lambda pAn is similar to other phage lambda replacement vectors taking inserts ranging in size from 5 to 19 kb. We have placed the pyrG gene of Aspergillus nidulans on the vector as a nutritional selective marker for transformation. We have developed this vector as part of an overall plan to facilitate the cloning of dominant extragenic suppressor mutations from A. nidulans, but also know that it is a generally useful vector for the purposes of isolating genomic clones without the need to subclone from the phage lambda vector.

Developmental regulation of a conidiation specific β-tubulin in Aspergillus nidulans

A beta-tubulin gene previously suggested to participate in conidial development in the filamentous fungus Aspergillus nidulans is shown to be developmentally regulated in its expression. A quantitative S1 assay was used to show that the abundance of the tubC messenger RNA increases during conidial development relative to the benA messenger RNA. Morphological analysis of cultures, used to prepare RNA for the S1 analysis, demonstrated that the increase in tubC messenger RNA was directly correlated with the appearance of conidiating cell types and structures and that as these cell types become more prevalent in the culture, the level of the messenger RNA increases. Two-dimensional gel electrophoresis of labeled proteins showed that the relative increase in tubC messenger RNA was reflected in an increase in the tubC protein beta 3. RNA blot hybridization analysis and in vitro translation of total RNA from mycelia and conidia were used to demonstrate that both benA and tubC beta-tubulin messenger RNAs are absent from conidia.

Isolation of the Aspergillus nidulans sudD gene and its human homologue

We have been studying the heat-sensitive bimD6 mutation of Aspergillus nidulans. At a restrictive temperature, the chromosomes of bimD6 mutant strains fail to attach properly to the spindle microtubules, and the mutant also displays a high rate of chromosome loss. We previously cloned the sudA gene, an extragenic suppressor of the heat-sensitive bimD6 mutation and showed that it coded for a DA-box or SMC protein. SMC proteins have been demonstrated to function in chromosome condensation, segregation and global gene regulation. We have now cloned the sudD gene, another of the extragenic suppressor genes of the bimD6 mutation. The predicted SUDD protein is the founding member of a widely expressed protein family. Similar proteins are found in sequence databases for Saccharomyces cerevisiae, Caenorhabditis elegans, mammals and four species of archaebacteria. We have also cloned and sequenced a human cDNA that encodes the human homologue of SUDD and mapped the gene to 18q11.2. The predicted SUDD proteins from A. nidulans, Homo sapiens and S. cerevisiae all share a variety of features. The predicted proteins are approximately 60000Da in mass and have a serine-plus-threonine content of about 11%. The evolutionary conservation of the proteins suggests an ancient origin and conserved function for these proteins.

The unique histone H2A gene of Aspergillus nidulans contains three introns

The histone H2A gene of the filamentous fungus Aspergillus nidulans has been cloned and sequenced. There is a single H2A gene in the genome of A. nidulans, and it contains three introns. The introns are 51 nucleotides (nt), 56 nt and 50 nt in length and split codons for amino acids (aa) 18, 48 and 116 of the predicted protein. The transcriptional start and termination points have been determined using an S1 nuclease protection assay. The predicted protein is 132 aa residues in length and surprisingly has a threonine after the initiator methionine instead of the usual serine. The sequence of the predicted histone H2A protein is compared to histone H2A proteins from Schizosaccharomyces pombe, Saccharomyces cerevisiae and calf thymus. Comparison of the amino acid sequence to these other H2A proteins shows that the divergence of amino acid sequences between H2A proteins is found in two clustered sites.

The uvsF gene region in Aspergillus nidulans codes for a protein with homology to DNA replication factor C

The UV-sensitive mutant uvsF201 of Aspergillus nidulans shows increased spontaneous and UV-induced mutation and generally resembles mutants defective in nucleotide excision repair (NER). Fully-complementing uvsF clones were isolated from cosmid and cDNA libraries for sequencing. The uvsF gene is approximately 3.75 kb long and codes for a predicted polypeptide of 1092 amino acids (aa). Three small introns are clustered early in the coding region of the protein. A major part of the sequence shows homology to human, mouse and yeast RFC1 genes which code for the large subunit of the DNA replication factor C. The uvsF gene product may therefore function primarily in general DNA replication but in addition be required for the replication step of DNA repair. Extended sequencing of the uvsF gene region identified a second closely adjacent gene of unknown function which is divergently transcribed from a small (0.2 kb) intergenic promoter region.