| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 88 === Abstract
Part I
DNA replicons are found very near or within a transcription unit in both prokaryotic and eucaryotic cells. To study the relationship between DNA replication and transcription, we examined a strong promoter/enhancer effect on simian virus 40 (SV40) origin-based DNA replication. The human cytomegalovirus (CMV) immediate early gene enhancer/promoter has been found to exhibit a strong inhibition on SV40 origin activity in transfected Cos-1 cells in a position- and dose- dependent effect. Deletion mapping analysis showed that the CMV enhancer had a strong suppressing effect on DNA replication. Several mechanisms were tested. The mechanism of one-to-one interaction between regulatory elements SV40 origin and CMV enhancer was excluded because the inhibition by CMV regulatory element was only partially alleviated in the presence of extra copies of SV40 origin. Nucleosome phase reorganization on the SV40 origin using micrococcal nuclease for analysis did not support nucleosome phase reorganization as the cause of CMV enhancer-mediated inhibition. We also excluded the possibility that inhibition was due to DNA chain elongation being blocked by CMV regulatory sequence, using two-dimensional gel electrophoresis for analysis.
P1 nuclease analysis of nuclease-hypersensitive sites in transfected plasmid revealed that only one regulatory sequence was cleaved per chromatin even in cases where many regulatory elements were present in the plasmid, suggesting that only one nuclease-hypersensitive site existed per chromatin. A positive correlation was observed between the degree of inhibition of SV40 origin-based DNA replication and the decrease of P1 cleavage frequency at the SV40 origin. This result suggests that competition for the formation of active chromatin conformation is the mechanism for the inhibition of SV40 origin by CMV enhancer. This interpretation is further supported by the fact that CMV promoter/enhancer initiated chloramphenicol acetyltransferase expression was found to be inhibited by CMV regulatory element in a dosage-dependent manner.
Part II
In the second part of the thesis we investigated the interactions between SV40 origins in plasmids containing either tandem copies or separated copies of SV40 origin. In the case of tandem copies the core origin adjacent to the 72-21 repeats of the downstream origin has poor replication activity as shown by differential inactivation of one of the origins. That the 72-21 bp repeats exerted a negative effect on the early side of the origin. P1 nuclease analysis also suggested different chromatin conformation of the two origins indicating that nearby sequence could exert influence on the chromatin conformation of SV40 origin. When the two origins were separated by 1.6 kb, each has equivalent activity as shown by differential mutation of one of the origins. However, two dimensional gel analysis of replication intermediates of plasmids containing two separated origins demonstrated a complex replication pattern suggesting influence of replication initiated by one origin by the second origin. As far as we know this is the first case demonstrating alteration of DNA replication pattern by additional origin. Furthermore analysis is needed to study the molecular basis for the observed phenomenon.
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