Expression of a prokaryotic metF gene in eukaryotic cells /
| Main Author: | |
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| Other Authors: | , |
| Format: | Thesis Book |
| Language: | English |
| Published: |
1989.
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| Subjects: | |
| Online Access: | Link to OAKTrust copy |
| Abstract: | E. coli W122 (metF) constructed from E. coli JJ122 by P1 phage transduction was an efficient recipient for plasmid transformation. The large T and small t antigens were removed from the pSV3-neo and replaced with the E. coli metF gene in such a way that transcription of the gene is controlled by the SV40 early promoter. The Tn5 sequence between the metF translational start site and the 3' terminus of the SV40 early promoter in pSV3-neo-F was digested by Bal 31 deletion. Two derivatives, pSV3-neo-FD1 and pSV3-neo-FD2, with varied deletions were analyzed by restriction enzyme mapping and nucleotide sequence analysis. Introduction of the constructed pSV3-neo-F and its derivative plasmids into mammalian cells by transfection showed that expression of the neo gene could be obtained either transiently or continuously in CV-1 cells. No metF transcript was detected from the RNA extracts of transient transformants, pooled transformants or stable transformants transfected by pSV3-neo-F. Low expression of metF gene was obtained from the pooled transformants and stable transformants transfected by pSV3-neo-FD1 and pSV3-neo-FD2. Only one of the each 4 stable transformants transfected by pSV3-neo-F derivatives showed the signal of metF transcripts. It was estimated that the lengths of these transcripts were about 2200 bases and 2000 bases respectively. However, slot blot analysis showed the neo and metF expression in pooled transformants and stable individual transformants transfected by pSV3-neo-F and 2 derivative plasmid DNA. Northern blot analysis indicated that the neo gene could be expressed transiently in WG cells, but no metF mRNA was detected. Methods which employed either calcium phosphate or DEAE-dextran as a carrier to facilitate transfection of DNA into cells were tested. However, no permanent G418 resistant transformants were obtained, indicating that WG 355 cells were very refractory to permanent, stable transfection by the constructed plasmids. |
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| Item Description: | Typescript (photocopy). Vita. "Major subject: Microbiology." |
| Physical Description: | x, 108 leaves : illustrations ; 29 cm |
| Bibliography: | Includes bibliographical references. |