Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination.

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination.

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme.

Kinetics of mRNA and protein synthesis of genes controlled by the 1,4-beta-endoxylanase A promoter in controlled fermentations of Aspergillus awamori.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-beta-endoxylanase A (EXLA)-overproducing strain.

An expression system based on the promoter region of the Aspergillus awamori 1,4-beta-endoxylanase A gene.

A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression in regulated at the transcriptional level. Using a beta-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an efficient inducer of the exlA promoter, whereas sucrose or maltodextrin were not.

The effect of pre- and pro-sequences and multicopy integration on heterologous expression of the Fusarium solani pisi cutinase gene in Aspergillus awamori.

A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus.

Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus; a novel pectinolytic enzyme.

Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da.

Construction and heterologous expression of a synthetic copy of the cutinase cDNA from Fusarium solani pisi.

A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.

A novel strategy for the isolation of defined pyrG mutants and the development of a site-specific integration system for Aspergillus awamori.

A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi.

Replacement of the L11 binding region within E.coli 23S ribosomal RNA with its homologue from yeast: in vivo and in vitro analysis of hybrid ribosomes altered in the GTPase centre.

Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes.

Cloning and disruption of the LEU2 gene of Kluyveromyces marxianus CBS 6556.

The LEU2 gene, coding for beta-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38 kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities.

The conserved GTPase center and variable region V9 from Saccharomyces cerevisiae 26S rRNA can be replaced by their equivalents from other prokaryotes or eukaryotes without detectable loss of ribosomal function.

Using the "tagged" rRNA gene system, which allows in vivo mutational analysis of Saccharomyces cerevisiae rRNA, we studied the role of two distinct structural elements of 26S rRNA in ribosome biogenesis and function--namely, the evolutionarily highly conserved "GTPase center" located in domain II and the eukaroyote-specific variable region V9 in domain III. Replacement of the S. cerevisiae GTPase center with its counterpart from Escherichia coli did not affect the assembly of the mutant 26S rRNA into functional (as judged by their polysomal distribution) 60S subunits, indicating that the E.

Functional analysis of transcribed spacers of yeast ribosomal DNA.

Making use of an rDNA unit, containing oligonucleotide tags in both the 17S and 26S rRNA gene, we have analyzed the effect of various deletions in the External Transcribed Spacer (ETS) and in one of the Internal Transcribed Spacers 1 (ITS1) on the process of ribosome formation in yeast. By following the fate of the tagged transcripts of this rDNA unit in vivo by Northern hybridization we found that deleting various parts of the ETS prevents the accumulation of tagged 17S rRNA and its assembly into 40S subunits, but not the formation of 60S subunits. Deleting the central region of ITS1, including a processing site that is used in an early stage of the maturation process, was also found to prevent the accumulation of functional 49 S subunits, whereas no effect on the formation of 60S subunits was detected.

Linker scanning of the yeast RNA polymerase I promoter.

To define the RNA polymerase I promoter in the rDNA of Saccharomyces cerevisiae more precisely, we have constructed a series of 5'- and 3'-deletion mutants in a novel, plasmid-borne rDNA minigene, that also contains the transcriptional enhancer. Our data show that the Pol I promoter, in this context, extends from position -155 to +27, with 5'-deletions up to -134 and 3'-deletions up to -2 removing essential sequence information. To investigate the internal organization of the yeast Pol I promoter, linker scanning mutants were constructed, that traverse the Pol I promoter region and comprise between 5 and 12 clustered point mutations.

A system for the analysis of yeast ribosomal DNA mutations.

To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits.

Deletion mapping of the yeast Pol I promoter.

Deletions in the promoter region of the 37S pre-rRNA operon in yeast were constructed and analysed in vivo using an artificial ribosomal minigene present on an extrachromosomal yeast vector. Sequences required for correct transcription initiation were found to be located between positions -192 and +15 relative to the start; a 5'-deletion down to position -133 reduces the transcription yield of the minigene at least five-fold. To allow detection of transcription of the minigene in isolated nuclei of yeast transformed with a minigene-bearing plasmid we attempted to increase the minigene copy number.

Transcription of an artificial ribosomal RNA gene in yeast.

We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain. The artificial gene comprised a fragment containing the sequence from position -207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre-rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position -36 to +101 relative to the 3' end of the 26S rRNA gene. The resulting construct was cloned into the yeast-Escherichia coli shuttle vector pJDB207.

Production and characterization of antibodies specific for domains of human IgM.

A method of preparing antibodies against c mu 3 and c mu 4 domains of human IgM is described. c mu 3- and c mu 4-binding antibody fractions were isolated by affinity chromatography from IgG fractions of antisera raised against Fc5 mu and Fc mu' fragments. c mu 3 and c mu 4 fragments had been prepared from human IgM kappa (Key) by hot trypsin digestion.