Stability of DNA with sequence symmetry and the level of unrestrained negative supercoiling in different chromosomal locations within a human cell /
A 122 bp inverted repeat flanked by 6 bp direct repeats was inserted into a neo gene which was then integrated into the genome of human fibrosarcoma cells. Three cell lines, each of which contained the mutated neo gene at a different chromosomal locus, were tested for the ability to produce cells r...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1996.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739668221&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A 122 bp inverted repeat flanked by 6 bp direct repeats was inserted into a neo gene which was then integrated into the genome of human fibrosarcoma cells. Three cell lines, each of which contained the mutated neo gene at a different chromosomal locus, were tested for the ability to produce cells resistant to G-418. Calculations based on the number of independent cultures that lacked revertant cells suggested reversion rates of 2.1X10-8 to <3.1X10-9 revertants/cell/division for the three cell lines. Physical analysis of the neo genes in revertant cells showed that resistance to G-418 was always accompanied by deletion of the 122 hp inverted repeat along with one copy of the 6 bp direct repeat. In contrast, none of the three cell lines that carried an integrated neo gene containing a 122 hp insertion that was not palindromic produced G-418-resistant clones. These results suggest that this inverted repeat within the human genome has a low deletion rate. Using these same cells, superhelical tension was measured within a transcriptionally active hygromycin B phosphotransferase (hph) gene in the five transformed human fibrosarcoma cell lines. The level of unrestrained DNA supercoiling was analyzed using a Me,-psoralen photobinding assay. A single copy of the hph gene integrated into one chromosomal region in two human cell lines (122-2, F14C-8) and two locations in cell line F14C-23. All three cell lines had similar, high levels of negative superhelical tension. In another chromosomal region in cell line 122-2, a lower level of negative tension was detected. One chromosomal region in cell line F14C-4 had no detectable unrestrained superhelical tension. These results demonstrate that different levels of unrestrained negative superhelical tension can exist in the same gene sequence inserted into different chromosomal locations. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | ix, 118 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 102-116. |