DNA-dependent RNA polymerases from the fungus Aspergillus nidulans

The aim of the work presented here was the isolation and characterization of the DNA-dependent RNA polymerases from the fungus Aspergillus nidulans, which was a part of a project concerning the regulation of gene expression in this lower eukaryote.The transcription of a genome and the regulation mechanisms involved are basic steps in the development and differentiation of an organism. The regulation mechanisms necessary for the development of a single fertilized egg cell into an organism like men, must be very precise and complicated, if one considers that the organism consists of dozens of different cell types, each having a specific function as part of the whole. The signals, which trigger a cell to develop into a highly specialized blood or brain cell, are largely unknown at the moment. Because the developmental and differentiation process in higher eukaryotes is so complex, relatively simple differentiating organisms, like Aspergillus nidulans may be more suitable for the study of the molecular mechanisms underlying the developmental regulation of gene expression. The limited knowledge of the biochemical organization of Aspergillus, as compared to a lower eukaryote like yeast, is certainly a disadvantage, but at the same time a challenge for the investigator. On the other hand, the genetics of Aspergillus has been extensively studied and this can be of great use in biochemical and developmental studies of this organism.The transcription of coding sequences of the DNA into RNA, is one of the first processes of a complex chain of events underlying the expression of genetic information. The specific mechanisms involved in the regulation of transcription are largely unknown, but they must directly or indirectly affect the activity of the DNA-dependent RNA polymerases, responsible for the differential transcription of genetic information. Apart from the regulation at the cellular level of the enzyme, other mechanisms must be responsible for the differential transcription of specific classes of genes, that are transcribed by a common enzyme. Structural modification of the chromatin could influence the accessibility of specific genes and hence their ability to be transcribed by an RNA polymerase. Transcription may also be controlled directly by regulators, altering the interaction between the enzyme and a specific gene or genes. It is therefore important to purify eukaryotic nuclear RNA polymerases and to study their structure and function. For understanding the actual transcription mechanism, the development and study of cell-free systems, supplemented with purified RNA polymerases and well characterized templates, will be required.This thesis describes how the DNA-dependent RNA polymerases I and II from Aspergillus nidulans can be successfully purified and subsequently characterized with respect to their catalytic properties and subunit composition (Chapters 2 and 3). Preparation of protoplasts from Aspergillus (Chapter 4) was initally thought to be necessary for the isolation of the RNA polymerases, because desintegration of the rigid cell wall of Aspergillus was the first difficulty encountered. Although large amounts of protoplasts could be prepared, the procedure appeared to be rather timeconsuming and impractical as a standard, large-scale procedure for the isolation of the RNA polymerases. Protoplasts, however, can be very useful when micro-assays or a gentle treatment to break open the cell wall are required. This is demonstrated in Chapter 5, where the effect of inhibitors of RNA synthesis has been studied in vivo in metabolically active protoplasts. The isolation and characterization of RNA polymerase III could not be achieved within the limited time available for the project.