Regulation of restrictocin production in Aspergillus restrictus

Abstract

The production of restrictwin (a cytotoxin that specifically cleaves ribosomal RNA) by cultures of Aspergiffus restrictus grown in liquid medium was investigated. The function of restrictwin, the method of its accumulation and the mode of resistance to restrictwin in A. restrictus are unknown. Previous studies have indicated that restrictwin accumulates in the medium with culture age. These observations have been extended in this study by cloning the cDNA of the res gene and using this cDNA clone to probe the onset of messenger RNA synthesis in the cells. The results of the Northern analysis were compared to the production and accumulation of restrictwin and morphological differentiation of the cells in culture. Restrictwin was found in the medium at the same time that mRNA was detected in the cells. This suggests that the leader sequence encoded by the cDNA provides an efficient secretion system for the protein. Both the protein and the mRNA were detected coincident with the formation of differentiated cell structures. These structures develop into conidiophores with one layer of sterigmata and conidia forming from the sterigmata. These results suggest that restrictwin is either involved in the process of conidiation or is coordinately regulated with differentiation leading to conidiation.

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

“…Though restrictocin can be purified from the filtrate of A. restrictus, it is not desirable to handle the fungus in view of the fact that it is thought to be the causative agent of aspergillosis [6]. Earlier gene and cDNA coding for restrictocin have been expressed in Aspergillus nidulans and Saccharomyces cerevisiae, respectively, to study the role of leader peptide on the survival of the host [7,8]. Ribotoxins, ~-sarcin and mitogillin have been expressed earlier using ompA and pelB signal sequences but the protein levels obtained were low [9][10][11][12][13].…”

Production and localization of restrictocin in Aspergillus restrictus

“…The blot was stained with amido black and blocked with blotto (Johnson et al, 1984). The indirect, alkalinephosphatase-linked detection system described by Yang & Kenealy (1992) was used for the detection of restrictocin on this blot. The BCIP/NBT substrate system from Kirkegaard and Perry Laboratories was used for the colour formation reaction.…”

“…Each sample for restrictocin analysis was serially diluted with 1 % SDS to yield concentrations of 1 x , 0.5 x and 0.1 x . A purified restrictocin control sample (Yang & Kenealy, 1992) was also diluted to provide samples containing 500,400,300,200, 100, 75, 50, 25 and long, respectively. Experimental and control dilutions were run together on a 12% polyacrylamide SDS gel and Western blotted as described above.…”

“…A correlation between the appearance of restrictocin and the appearance of certain differentiated structures (conidiop hores) in liquid medium has previously been observed in this laboratory (Yang & Kenealy, 1992). Growing A .…”

The Therapeutic Potential of Fungal Ribotoxins

“…These mutational studies have revealed the involvement of several residues, which are conserved among the different microbial extracellular RNases, in catalysis. Thus, it is well known that His137 and Glu96 are the only residues that are essential for the cleavage reaction performed by α-sarcin [35,45,[70][71][72][73][74][75], whereas His-50, Tyr-48, Arg-121, and Leu-145 mostly contribute to the stabilization of the transition state [45][46][47][48], as stated above. Most of these residues are located in the central β-sheet and their side-chains point towards the concave face of the protein structure where the substrate is supposed to dock [29].…”