This is the first study to explore the ability of an enzyme to recognize and repair spontaneous age-dependent damage to its own sequence. Protein (D-aspartyl/L-isoaspartyl) carboxyl methyltransferase (PCM) is known to repair damage that arises from a spontaneous isomerization of aspartyl and asparaginyl residues in other proteins during aging. As PCM contains several conserved aspartyl and asparaginyl residues, this dissertation tested whether PCM can serve as a methyl acceptor in its own methylation reaction. In investigating the ability of PCM to automethylate, it was discovered that PCM is damaged. The mechanism of this automethylation reaction was determined to be an intermolecular, high affinity, slow turnover reaction and was limited to a subpopulation of damaged PCM molecules, termed ��PCM. / Graduation date: 1996

Bladder cancer is the second most common genitourinary tumor and is a significant cause of morbidity and mortality. To find the noninvasive tumor marker for bladder cancer is still a challenge. Recently, the circulating cell-free DNA had been used for detection of some tumors. However, most of these studies selected the blood sample as the free DNA source. The purpose of this thesis is to develop and evaluate the more noninvasive method and tumor marker for bladder cancer detection, especially for the cell-free DNA. Three directions were addressed as followed: 1). urine cell-free DNA makers such as creatinine-adjusted cell-free DNA concentration, 2). DNA integrity (DNA length) and 3). Promoter hypermethylation for DNA repair genes. First, creatinine-adjusted urine cell-free DNA concentration was quantified via real time PCR. The real time PCR-based detection is based on detection large fragment cell-free DNA. Real time PCR-based urine cell-free DNA concentration of bladder cancer patients was higher than controls. Mean concentrations of 36 bladder cancer patients and 93 controls which detected by real time PCR were 14.98 and 1.07 pg/

Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos. Bovine preimplantation embryos derived from nuclear transfer experiments exhibit a global state of genomic hypermethylation that likely account for the large number of developmental abnormalities observed to date. The central hypotheses of this work is that the genomic hypermethylation and improper epigenetic reprogramming reported in studies of bovine nuclear transfer, are in large part due to abnormal expression and regulation of the DNA methyltransferase proteins. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2γ and Dnmt3a4 represent previously unreported sequence combinations. Investigation of bovine DNA methyltransferase expression in the bovine oocyte and early preimplantation development has revealed an intricate system divergent from observations previously reported in the mouse. Specifically, the somatic version of Dnmt1 along with Dnmt2, 3a and 3b are all expressed during these initial stages of bovine development. Further, real time analyses of the Dnmt transcripts in cloned and in vitro produced embryos reveal significant differences in the mRNA expression levels of Dnmt1 and 2 but not Dnmt3a and 3b suggesting that the de novo methyltransferases may be functioning normally while Dnmt1 and Dnmt2 are aberrantly methylating the genome during a critical time when methylation levels should be receding. Real time PCR analysis of the Dnmt transcripts in fetal and adult tissues has revealed a developmental and tissue specific expression pattern suggesting that proper expression and function of these enzymes is a key element in the process of differentiation. These results are further supported by studies of Dnmt expression in aging bovine fibroblast cultures, which suggest that the Dnmts may play some as yet unidentified role in cellular senescence. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.

Allopolyploids are formed by the combination of evolutionarily-diverged genomes, the union of which leads to dynamic changes in gene expression and genome organization. Expression patterns of orthologous genes are rapidly and stochastically established in newly created allotetraploids, where gene silencing is maintained by microRNAs, DNA methylation, and other chromatin modifications. Among them, DNA methylation has been known as an important mechanism of epigenetic regulation of gene expression and chromatin structure. However, it is unclear how DNA methylation affects genome-wide expression of homoeologous genes in the natural polyploid Arabidopsis suecica that contains genome of both A. thaliana and A. arenosa. To understand the role that DNA methylation plays in the polyploidization process, a comparative analysis was performed comparing up- or down-regulated genes in met1-RNAi A. suecica lines with the non-additively expressed genes in the synthetic allotetraploids, i.e., different from the mid-parent value. The previous studies indicated that decreased DNA methylation in A. suecica induces A. arenosa-specific demethylation in centromere regions and differentially alters expression of >200 genes encoding many transposons, unknown proteins and some other functional proteins that are located along chromosomes, whereas >1,300 non-additively expressed genes in the synthetic allotetraploids are distributed randomly along the chromosomes and encode various proteins in metabolism, energy, cellular biogenesis, cell defense and aging, and hormonal regulation. The origins of the progenitors of the genes whose expressions are altered in both met1-RNAi A. suecica and resynthesized allotetraploid were analyzed with single strand conformation polymorphism (SSCP) analysis. Reactivated genes in met1-RNAi A. suecica lines were predominately derived from the A. thaliana genome in euchromatic regions, whereas the suppressed genes were mainly derived from the A. Arenosa genome, indicating that changes in DNA methylation are genome-sensitive. The data suggest that allotetraploids incidentally display chromosome-specific changes and genomedependent regulation of homoeologous genes in response to DNA methylation perturbations.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 71-78).

abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

No description available.

No description available.

When introduced into nutrient medium under air, the asexual sporangiospores of Mucor racemosus germinated within 7 to 8 hours ending in the emergence of germ tubes. Sadenosylmethionine synthetase (SAM synthetase) activity was found to be present in the dormant spore, and increased at a rapid rate after 3 hours. The inhibition of enzyme activity by cycloleucine early in the germination process completely inhibited germ tube formation and only rounded, swollen spores were seen at the end of 7.5 hours. De novo protein synthesis was found to be necessary for SAM synthetase activity throughout the germination process. De novo RNA synthesis was found to be unnecessary for the increase in enzyme activity during the first 3 hours. This suggests that SAM synthetase is coded for in the stored mRNA of the spore. General protein methylation was found to increase through the first 4.5 hours of germination. Autoradiographs of post-translationally methylated proteins subjected to polyacrylamide gel electrophoresis revealed approximately 8 bands which appear to change significantly in the degree of methylation during the 7.5 hour germination process. None of the bands were detectable until the 4.5hour period, which corresponds to the rapid increase in enzyme activity seen after 3 hours. / Department of Biology