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Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae
In the title the 'I' in 'UASI' is subscript.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2012
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| _version_ | 1867613807534145537 |
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| access_status_str | Open Access |
| author | Fourie, Magdelena Gertruida |
| author2 | Van Vuuren, H. J. J. |
| author_browse | Fourie, Magdelena Gertruida Van Vuuren, H. J. J. |
| author_facet | Van Vuuren, H. J. J. Fourie, Magdelena Gertruida |
| author_sort | Fourie, Magdelena Gertruida |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | In the title the 'I' in 'UASI' is subscript. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/69693 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:42:01.161Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2012 |
| publishDateRange | 2012 |
| publishDateSort | 2012 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/69693 Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae Fourie, Magdelena Gertruida Van Vuuren, H. J. J. Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics and Institute of Plant Biotechnology. Saccharomyces cerevisiae Mutation (Biology) Yeast -- Genetics Dissertations -- Microbiology In the title the 'I' in 'UASI' is subscript. Thesis (MScAgric) -- University of Stellenbosch, 1992. The CAR1 gene of Saccharomyces cerevisiae encodes arginase, the first enzyme in the arginine degradation pathway. The level of arginase responds to both induction and nitrogen catabolite repression and it was shown that these processes are transcriptionally regulated. Seven different regulatory proteins are possibly involved in CAR1 regulation. Dissection of the CAR1 5'-flanking region identified multiple positive and negative cis-acting elements required for CAR1 expression including two TATA elements, an upstream repression sequence (URS) and three upstream activation sequences (UAS₍C1₎, UAS₍C2₎ and UASᵢ). UAS₍C1₎ and UAS₍C2₎ support inducer-independent transcriptional activation while UASᵢ functions only when inducer is present. In the absence of inducer the URS element represses the operation of the two UAS elements, thus maintaining a low level of gene expression. In the presence of inducer, inducer-dependent UASᵢ-mediated activation and its synergistic enhancement of UAS₍C2₎-mediated activation overcome the action of the URS. It was recently shown that UASᵢ consists of three roughly homologous sequences (A, B and C). Although these elements function at varying efficacies, any of them in combination with UAS₍C2₎ are sufficient to support gene expression. However, each of the three UASᵢ sequences contain putative binding sites for some of the CAR1 regulatory proteins. The exact location of UASᵢC and the nucleotides within this element critical for function of the gene, was determined by saturation mutagenesis. Furthermore, the UASᵢB area was subjected to transversion mutations to identify those nucleotides required for response to inducer. Synthetic oligonucleotides containing these mutated elements were cloned into the lacZ expression vector, pNG15. Yeast transformants were assayed for β-galactosidase activity. In order to avoid problems that might result from a variation in copy number, 25% of the UASᵢC inserts were also cloned into a CENIV based vector pHP41. The same pattern of activity was observed for the two types of vectors. The UASᵢC consisted of a 8 bp palindromic sequence, 5'-AAAGTGAA-3'. Only the A at position -170 and the G at -167 required a unique nucleotide and for three others only one substitution was permissible. Transversion mutagenesis of UASᵢB identified nucleotides at positions -203 to -200 and -198 as the critical for transcriptional activation. In contrast to the DAL7 UIS, both these UASᵢ elements showed significant sequence flexibility, probably caused by suppression. Masters 2012-08-27T12:27:13Z 2012-08-27T12:27:13Z 1992 Thesis http://hdl.handle.net/10019.1/69693 en Stellenbosch University 115 pages application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Saccharomyces cerevisiae Mutation (Biology) Yeast -- Genetics Dissertations -- Microbiology Fourie, Magdelena Gertruida Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title | Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title_full | Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title_fullStr | Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title_full_unstemmed | Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title_short | Mutational analysis of the CAR1 UASI responsible for induced expression of the arginase gene in Saccharomyces cerevisiae |
| title_sort | mutational analysis of the car1 uasi responsible for induced expression of the arginase gene in saccharomyces cerevisiae |
| topic | Saccharomyces cerevisiae Mutation (Biology) Yeast -- Genetics Dissertations -- Microbiology |
| url | http://hdl.handle.net/10019.1/69693 |
| work_keys_str_mv | AT fouriemagdelenagertruida mutationalanalysisofthecar1uasiresponsibleforinducedexpressionofthearginasegeneinsaccharomycescerevisiae |