DNA methylation of the Dc8 gene in carrot (Daucus carota L.) and its association with development /
The Dc8 gene in carrot (Daucus carota L.) expresses
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1996.
|
| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=743274471&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The Dc8 gene in carrot (Daucus carota L.) expresses differentially. The methylation pattern in its promoter during somatic embryogenesis was investigated and the association of Dc8 methylation with its expression was studied by bombarding a Dc8-GUS construct with different methylation patterns into carrot calli and somatic embryos followed by GUS transient assay. Dc8-GUS expression can be enhanced by 5-azacytidine (5-azaC) treatment of carrot calli and somatic embryos prior to bombardment and decreased by in vitro enzymatic methylation of the plasmid. The plasmid bearing Dc8-GUS obtained from 5-azaC treated E. coli also has a slightly higher expression, which is associated with decreased level of methylation. GUS expression, increased by the 5-azaC treatment, is enhanced by ABA treatment of both calli and somatic embryos and was more prominent in the latter. In vitro methylation of Dc8-GUS by methylases generally resulted in a lower frequency of GUS expression. Sssl methylase completely inhibited GUS expression. Methylation patterns of a segment covering +120 to -446 of Dc8 allele 6 were investigated by modifying DNA with sodium bisulfite. While the 5' to 3' strand was almost completely nonmethylated, the 3' to 5' strand showed variation in methylation patterns. Although there was an obvious phenomenon of demethylation during embryogenesis, methylated cytosines were clustered in specific regions. In addition, most of the methylated cytosines were not limited to CpG or CpNpG sequences. The obvious difference in the extents of methylation between the two complementary strands implies the existence of a mechanism for strand specific methylation and that typical maintenance methylase activity is lacking. Digestion of DNA with Maell, Hpall and Tagl followed by PCR also revealed differences in methylation status at different sites during somatic embryogenesis. The Maell sites, which are closer to the transcriptional start point, showed greater loss of methylcytosine associated with Dc8 expression than the more distal Hpall and Tagl sites. Two improvements were made to the sodium bisulfite technique. The methylation status can be detected by just one, rather than four types of sequencing termination reactions. The strand origin of the cloned PCR insert derived from bisulfite-modified DNA can be determined through the types of base conversions of the sequencing data. |
|---|---|
| Item Description: | Vita. "Major Subject: Plant Physiology". In title, symbols and numerals are used. |
| Physical Description: | xi, 102 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |