Browsing by Subject "gene silencing"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Characterization of AtSUVR3 functions in Arabidopsis thaliana using RNA interference(2009-05-15) Wang, TaoVariability of transgene expression levels resulting from gene silencing is considered as ahindrance to the successful application of plant genetic engineering. Towards alleviatinggene silencing, I decided to screen for novel genes involved in transgene silencing and toinvestigate how these genes regulate plant development. Genes encoding putative chromatinremodeling factors, especially those including a SET domain, were selected as candidatetargets. A bioinformatic analysis of the Arabidopsis SET genes (AtSET) was performed andthese genes were classified into 6 groups based on the domain architecture. RNA interference (RNAi) vectors were constructed for ~ 20 AtSET genes and wereintroduced into both wild type lines and transgenic lines silenced for a GFP reporter gene.Surprisingly, altered developmental phenotypes were only observed for three constructs,raising questions as to the effectiveness of the RNAi approach for the chosen Arabidopsissystem. To assess this situation, I targeted a phytoene desaturase (PDS) gene using the sameRNAi approach. Inactivation of PDS renders plant a readily identifiable phenotype. Whereasthe RNAi penetrance in Arabidopsis can be very high, the expressivity of RNAi in varioustissues and among different plants can vary dramatically. Contradictory to previous reports,I found that there is correlation between transcript level and silencing phenotype. Possiblereasons for this discrepancy are discussed. No apparent correlation between transgene copynumber and RNAi phenotypes was observed. Among the three RNAi constructs that caused an abnormal development inArabidopsis, K-23 which targets SuvR3 has the highest expressivity and could reactivate asilenced GFP locus. SuvR3 RNAi lines were selfed for six generations and were screenedfor morphological phenotypes. Abnormal number of flower organs, loss of viability of malegametophytes, and decreased seedling germination percentage were found in SuvR3 RNAilines. A progressive increase in both severity and frequency of abnormal phenotypes wereseen in subsequent generations, suggesting an epigenetic regulatory mechanism involvedwith SuvR3. Alternative splicing of SuvR3 was also observed in most of Arabidopsis tissues.One of the protein isoforms, SuvR3, lacks 16 amino acids within the highly conserved SETdomain. Possible effects of isoform interaction are proposed.Item STUDY OF GENE SILENCING IN RICE: A ROOT PREFERENTIAL GENE RCG2(2010-07-14) Shi, XiangyuThe RCg2 promoter was identified in a search for root-specific genes to combat the rice water weevil (RWW) but expressed at low frequency (~10%). Spatial expression of RCg2 was investigated using two reporter constructs YXA (RCg2-gus-ocs) and YXB (RCg2-gus-RCg2) that included 1.6 kb of the RCg2 5' sequence fused to the ?-glucuronidase (gus) coding region. YXB plants were generated via Agrobacterium-mediated transformation but only 8 of 158 plants analyzed showed strong GUS activity despite the presence of an intact construct. Reactivation of RCg2 gene in rice was investigated by treatment of R0 and R1 of YXB transgenic plants with 5-azacytidine. Reactivation of RCg2-gus was observed in some transgenic plants indicating different mechanisms involved in the gene silencing of the YXB lines. DNA methylation analysis, northern blotting, RT-PCR and small RNA analysis supported the conclusion that PTGS and TGS are present in the silenced plants. Promoter analysis in silico and using promoter deletion assays predicted that the RCg2 promoter contains a complex region that includes miRNA homologs, MITEs and repetitive sequences. The high frequency of promoter-related silencing suggests functional interactions of these elements of the transgene and the homologous endogenous gene. To identify key elements contributing to the root-preferential expression of RCg2 and the high frequency of silencing observed in transgenic (YXB) lines, several RCg2 promoter deletion constructs were designed. These include 5' deletions MC1, MC2, MC4, MC7 and MC8 and internal deletions MC5, MC11, MC12 and MC13. The frequency with which silencing was encountered in populations of the deletion mutants was used to characterize the effects of various promoter elements. Deletion of the region from -406 to -208 (compared MC11 to YXB, and MC13 to MC1) revealed that region contains a negative element. Among 36 independent transformants, 33% with MC11 expressed GUS and 85% with MC13 showed GUS expression. Comparing MC7 transgenic plants to MC1 revealed that the region ?888 to ?729 is another negative regulatory element, and comparing MC11 to MC12, the proportion of expression of transgenic plants indicated the region ?729 to ?406 is a positive regulatory element.Item The Role of Chromatin Structure and Histone Modifications in Gene Silencing at the Ribosomal DNA Locus in Saccharomyces cerevisiae(2012-07-16) Williamson, Kelly M.One of the fundamental questions in science is how chromatin transitions from actively transcribed euchromatin to silent heterochromatin, and what factors affect this transition. One area of my research has focused on understanding the differences in the chromatin structure of active and silent regions in the ribosomal DNA locus (rDNA), a heterochromatin region in S. cerevisiae. Secondly, I have focused on understanding a histone methyltransferase Set1, which is involved in both euchromatin and heterochromatin regions. To distinguish actively transcribed open regions of chromatin from silent and closed regions of chromatin, we have expressed a DNA methyltransferase M.CviPI in vivo to utilize its accessibility to GpC sites. We have used this technique to study changes in nucleosome positioning within the NTS2 region of the rDNA in two cases: as a result of a silencing defect caused by the loss of Sir2, a histone deacetylase involved in silencing at the rDNA, and as an indicator of active transcription by RNA Pol I. Using this technique, we observed differences between open and closed chromatin structure by changes in nucleosome positioning within NTS2. Additionally, we have observed the presence of bound factors within the 35S rRNA gene promoter that are unique to actively transcribed genes. The second area of my research focused on the protein methyltransferase Set1 that mono-, di-, and trimethylates lysine 4 of histone H3 (H3K4) utilizing the methyl group from S-adenosyl methionine (SAM). Set1 is part of a multi protein complex called COMPASS (Complex associated with Set1), and is associated with both actively transcribed and silent regions. Thirty mutants of Set1 were made within the SET domain to learn more about the catalytic mechanism of Set1. The crystal structures of human SET domain proteins, as well as sequence alignments and a random mutagenesis of yeast Set1, were used to identify conserved amino acids in the SET domain of Set1. Mutants were analyzed for their effect on histone methylation in vivo, silencing of RNA Pol II transcription within the rDNA, suppression of ipl1-2, and COMPASS complex formation. Our results show that trimethylated H3K4 is required for silencing of RNA Pol II transcription at the rDNA. Overall, we have shown the importance of tyrosine residues in SET domain proteins. To summarize, my research has strived to understand chromatin structure and the factors that affect the transition between euchromatin and heterochromatin.