Development of an Autonomously Replicating Vector for Filamentous Fungi

Robertson, James Scott (1992) Development of an Autonomously Replicating Vector for Filamentous Fungi. PhD thesis, University of Glasgow.

Abstract

The plasmid ARpl contains a sequence called amal; amal appears to confer the ability to replicate autonomously. The amal sequence is an inverted repeat of about 6kb in size and consists of two 3kb arms separated by a 345bp unique region. The unique central region and one complete arm of the amal insert has been sequenced, (Chapter 3). The amal sequence appears to consist of Aspergillus genomic DNA and rearranged pUC-like DNA. No recognisable ARS sequences were found in amal, but amal does contain A/T rich regions. Northern blots suggest that amal is transcribed not at all frequently. The involvement of the amal sequence in plasmid transformation frequency, instability and plasmid rearrangements was studied using subclones of ARpl, (Chapter 4). The results suggest that no single identifiable amal region is responsible for either enhancing transformation frequency or plasmid maintenance. The Chapter 4 results indicate that some component of pUC8 plays a role in the autonomous replication of Aspergillus plasmids. ARpl plasmid DNA isolated directly from Aspergillus transformants has been photographed using Electron Microscopy techniques, (Chapter 5). The results indicate that ARpl is present in Aspergillus in monomeric, dimeric, trimeric and tetrameric forms. A number of amal-like sequences have been identified in an Aspergillus cosmid library, constructed by Brody. These sequences have been investigated, (Chapter 6). Cotransformation experiments with some of these amal-like sequences suggest that these sequences are capable of promoting autonomous replication. The ARpl-derived amal sequence was used as a probe to isolate similar sequences from Penicillium chrvsoaenum and Cephalosporium acremonium. (Chapter 7). Three such sequences were isolated from Penicillium. These sequences were called pamla. pamlb and pam2. Transformation experiments with these sequences suggest that these sequences are capable of promoting autonomous replication in both Penicillium and Aspergillus. Similarly, ARpl-derived amal subclones give rise to autonomously replicating plasmids in Penicillium. ARpl-derived amal subclones were used to clone the argB. niaP and nirA genes from wild type Penicillium via cotransformation with fragmented genomic DNA, (Chapter 8). This technique has been called The Instant Gene Bank, (Gems, 1990). Fungal transformants grown on selective medium were obtained, but in all cases it was not possible to isolate plasmid DNA containing the genes of interest.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: John Clutterbuck
• Keywords: Genetics
• Date of Award: 1992
• Depositing User: Enlighten Team
• Unique ID: glathesis:1992-74610
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 13 Nov 2019 15:58
• Last Modified: 13 Nov 2019 15:58
• URI: https://theses.gla.ac.uk/id/eprint/74610