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M Broekhuijsen

Publications and source records attributed to M Broekhuijsen.

3 recordsLinked to original sources
PubMed1989

A gene transfer system based on the homologous pyrG gene and efficient expression of bacterial genes in Aspergillus oryzae.

A homologous transformation system for Aspergillus oryzae is described. The system is based on an A. oryzae strain deficient in orotidine-5'-phosphate decarboxylase (pyrG) and the vector pAO4-2, which contains a functional A. oryzae pyrG gene as selection marker. Transformation of the A. oryzae pyrG mutant with circular pAO4-2 resulted in the appearance of Pyr+ transformants at a frequency of up to 20 per micrograms of DNA, whereas with linear pAO4-2 up to 200 transformants per micrograms DNA were obtained. In 75% of the Pyr+ transformants recombination events had occurred at the pyrG locus, most of which (90%) resulted in insertion of one or two copies of the vector and the others (10%) in a replacement of the mutant allele by the wild-type allele. Vector pAO4-2 is also capable of transforming a corresponding mutant of Aspergillus niger. This transformation system was used to introduce into A. oryzae the heterologous and non-selectable bacterial genes lacZ, encoding beta-galactosidase, and uidA, encoding beta-glucuronidase. Using the Aspergillus nidulans gpdA promoter to drive bacterial gene expression in A. oryzae, relatively high levels of activity, as well as protein per se, as judged by western blot analyses, were obtained.

Aspergillus↗
PubMed1982

Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells.

The expression of the middle-component (M) RNA of cowpea mosaic virus was studied by means of in vitro translation. In both the wheat germ extract and the rabbit reticulocyte lysate, M RNA was translated into two overlapping polypeptides of 95 and 105 kilodaltons. Incubation of these polypeptides with 30,000 x g supernatant fractions from cowpea mesophyll protoplasts inoculated with complete virus or with separate bottom (B) components alone resulted in extensive processing, yielding polypeptides of 60, 58, 48, and 47 kilodaltons. Similar proteolytic activity was found associated with the in vitro translation products from the bottom-component RNA, demonstrating that the protease present in infected cells is encoded by B RNA. Using antisera raised against the separate capsid proteins VP23 and VP37, it was shown that the 60-kilodalton cleavage product is the precursor to both capsid proteins. Cleavage of nascent 95- and 105- kilodalton polypeptides by the in vivo protease demonstrated that this capsid protein precursor is located C terminally within both polypeptides and that the synthesis of these two overlapping polypeptides is the result of two initiation sites on middle-component RNA. In addition, a second virus-induced proteolytic activity, capable of releasing VP23 from the 95- and 105-kilodalton polypeptides, was detected in leaves of infected plants, but not in infected mesophyll protoplasts. A model for the expression of the middle-component RNA is presented.

Journal Article↗