Previous research performed in 1999 by Harris et al. has shown that many varieties of ruminants serve as the host species for Mycobacterium avium subspecies paratuberculosis (MparaTb) infections. Gene sequencing has supported the contention that organisms isolated from different hosts harbor different gene sequences; this has been exemplified by Amonsin et al. in 2004 with the sequencing of the mfd (transcription-repair coupling factor) and by Motiwala et al. in 2005 through sequence analysis of phosphatidylethanolaminebinding proteins which reveal a host-specific correlation of isolates. Some contradicting reports from Bannantine et al. from 2003 have further claimed that MparaTb is a monogenic organism based upon sequence data from regions flanking the origin of replication and the 16s rRNA. One of the drawbacks to the techniques implemented in these reports is the extremely restricted region of the bacterial genome that was analyzed; furthermore, only a select number of isolates were analyzed. In the present studies, amplified fragment length polymorphism (AFLP) was used as a tool for a genome scale comparison of MparaTb isolates from differing isolation types as well as a comparison of MparaTb isolates to the genetically similar yet avirulent Mycobacterium avium subspecies avium isolates. AFLP data reveals the MparaTb genome to be much more plastic and polymorphic than previously thought. These polymorphic regions were identified and characterized and are shown to be unique to the organism when compared to an array of Mycobacterial isolates of differing species. These polymorphic regions were also utilized in polymerase chain reaction (PCR) based diagnostic as well as epidemiologic tests. Furthermore, AFLP comparative analysis of intracellular and fecal MparaTb isolates reveals polymorphic regions unique to each isolate type. While these genomic differences are not based upon differences in the genetic code, they are based upon epigenetic modifications such as DNA methylation. These DNA methylation patterns are unique to intracellular MparaTb isolates as opposed to isolates from fecal material. Furthermore, AFLP comparisons of fecal MparaTb isolates that were passaged through the bovine ileum revealed banding pattern differences as compared to the original inoculum.

Meiosis, the core engine of sexual reproduction, is a complex process that results in the production of recombinant haploid genomes. In the meioses of Neurospora, worms and mice, gene expression from DNA that lacks a pairing partner is silenced. We posit that this is a two-step process. First, a process called meiotic trans-sensing compares the chromosomes from each parent and identifies significant differences as unpaired DNA. Second, if unpaired DNA is identified, a process called meiotic silencing inhibits expression of genes within the unpaired region and regions sharing sequence identity. Meiotic silencing is mechanistically most likely related to RNAi in other eukaryotes. We used a combination of forward and reverse genetic strategies aimed at understanding the mechanisms of meiotic trans-sensing and meiotic silencing. Here, we present genetic evidence that arguably differentiates the meiotic transsensing step from meiotic silencing, by demonstrating that DNA methylation affects sensing of specific allele-types without interfering with silencing in general. We also determined that DNA sequence is an important parameter scrutinized during meiotic trans-sensing. This, and other observations, led us to hypothesize meiotic recombination as the mechanism for meiotic trans-sensing. However, we find that mutants of key genes required for recombination and chromosome pairing are not required for locus-specific meiotic silencing. We conclude that two interesting possibilities remain: meiotic trans-sensing occurs through a previously uncharacterized recombination pathway or chromosomal regions are carefully compared in the absence of recombination. Finally, forward genetics revealed a novel component of meiotic silencing, Sms-4, encoding the Neurospora ortholog of mammalian mRNP component ELG protein. Unlike previous loss-of-function mutants that abate meiotic silencing by unpaired DNA, Sms-4 is not required for successful meiosis, showing that meiosis and meiotic silencing are distinct, yet overlapping, phenomena. Intriguingly, SMS-4 is the first component to be localized with bulk chromatin in the nucleus, presumably the site of trans-sensing. Finally, we carried out a critical examination of the current evidence in the field and present alternative models for meiotic trans-sensing and meiotic silencing in Neurospora.

The 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.

Defects in mismatch repair genes, particularly the hMLH1 and hMSH2 genes, are associated with pathogenesis and prognosis of some cancers. The lack of correlation between replication error phenotype and mutations in hMLH1 in sporadic human cancers suggested that inactivation of the hMLH1 gene may be associated with promoter hypermethylation. This study was to investigate the association of hMLH1 promoter hypermethylation and hMLH1 protein expression in oral cancer. Our results indicated that all 75 cases (100%) were without any methylation of hMLH1 promoter by use of methylation-specific PCR (MSP). Nineteen of 99 cases (19.2%) were partial methylation by HpaII-based PCR. In addition, 24 (26.1%) of 92 cases of OSCC had reduced levels of hMLH1 protein. The concordance analysis showed that the expression level of hMLH1 protein was not correlated with methylation of hMLH1 promoter. Furthermore, the prognosis significance of hMLH1 or hMSH2 proteins on OSCC was also investigated. We analyzed the association of hMLH1 and hMSH2 protein expression with clinicopathological data of 92 cases of OSCC at KSVGH. We found that 24 (26.1%) of 92 cases of OSCC had reduced levels of hMLH1 protein, however only 10 cases (10.9%) had reduced hMSH2 by use of IHC. In addition, the reduced expression of hMLH1 correlated with the tumor differentiation and N classification. However, none of these clinical and pathological characteristics of the OSCC patients were associated with the extent of hMSH2 expression. Finally, previous studies reports that the hMLH1 and Aurora-A are directly involved in the prognosis of several cancers. The expression levels of hMLH1 and Aurora-A protein were investigated in the 138 tumor samples for consecutive patients with pathological confirmed primary buccal carcinoma (BC). Then the association of the protein expression with clinicopathological data and survival were also evaluated. The loss of hMLH1 protein was found in 15 (10.9%) of 138 tumor sections by IHC. In addition, loss of hMLH1 protein expression was not any correlated with clinical features and patients¡¦ prognosis. The up-regulation of Aurora-A protein was found in 118 (85.5%) of 138 tumor sections by IHC. In addition, the up-regulation of Aurora-A protein expression was correlated with the pathological stage and T classification, but Aurora-A protein up-regulation was not correlated with prognosis. In conclusion, promoter methylation of hMLH1 might not play a potent role in the gene expression in oral cancer. Defective expression of hMLH1 but not hMSH2 was associated with the development of OSCC. In addition, the Aurora-A protein expression but not hMLH1 may affect the malignant behavior of BC. However, the hMLH1 and Aurora-A protein expression might be not the prognostic factors for BC patients.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1963. / Includes bibliographical references (p. 65-69).

Thesis (Ph. D.)--Institute of Paper Chemistry, 1940. / Includes bibliographical references (p. 96-97).

Multiple myeloma (MM) is an incurable haematological malignancy. It is characterized clinically by an asymptomatic precursor stage, known as monoclonal gammopathy of undetermined significance (MGUS), which will transform into symptomatic MM at a rate of 1% per year. Gene promoter hypermethylation by catalytic conversion of cytosine into 5?methylcytosine at promoter?associated CpG island is an alternative mechanism of gene inactivation. MicroRNA (miRNA) is a class of short, single?stranded, non?coding RNA molecules, which will repress the expression of target protein by sequence?specific binding to the three prime untranslated region of the corresponding messenger RNA. In carcinogenesis, miRNA can be either oncogenic when tumour suppressor genes are targeted, or tumour suppressive when oncogenes are targeted. Despite reports of hypermethylation of multiple protein?coding tumour suppressor genes, little is known about DNA methylation of non?coding tumour suppressor miRNA genes in MM. This thesis aimed to investigate the role of promoter hypermethylation of tumour suppressor miRNA genes in MM using a candidate miRNA approach. Moreover, the prognostic significance of tumour suppressor miRNA hypermethylation was studied in a uniformly?treated cohort of MM patients. The role of DNA methylation at the promoter of miR?203, miR?34a, miR?34b/c, miR?124?1, miR?129?2 and miR?224 were studied in MM. The tumour suppressor role of miR?34b/c, miR?124?1, miR?203 and miR?224 were demonstrated in human myeloma cell lines (HMCLs). In particular, restoration of miR?203 in MM cells was shown to inhibit cellular proliferation via targeting and hence direct downregulation of a proto?oncogene, cyclic AMP responsive element binding protein. There are several observations in primary MM samples. First, there was frequent methylation of miR?129?2, miR?203 and miR?224 but infrequent methylation of miR?34a, miR?34b/c and miR?124?1 in MM at diagnosis. Second, tumour?specific hypermethylation of each of the miR?203 and miR?224 promoters was detected at comparable frequencies in MGUS, diagnostic and relapsed/progressed MM, and hence implicated as an early event in myelomagenesis. Thirdly, miR?129?2 methylation was more frequent in diagnostic MM than MGUS, and hence implicated in MGUS progression to MM. On the other hand, despite rare miR?34b/c methylation at diagnosis, miR?34b/c methylation was frequent at relapse/progression, thereby implicating miR?34b/c methylation in MM relapse/progression. Fourthly, despite frequent miR?124?1 methylation in HMCLs, miR?124?1 methylation was rare in both diagnostic and relapsed MM marrow samples, suggesting that miR?124?1 methylation was acquired during in vitro cell culture. Finally, the prognostic significance of methylation of a panel of tumour suppressor miRNAs was studied in a uniformly?treated cohort of MM patients, which revealed that miR?224 hypermethylation as an independent favourable prognostic factor for survival. In conclusion, hypermethylation of tumour suppressor miRNAs is implicated in the pathogenesis (miR?203, miR?129?2, miR?224), progression (miR?34b/c), and prognostification (miR?224) of MM. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The Wilms’ tumor gene 1 (WT1) was first reported as a tumor suppressor gene in Wilms’ tumor. However, later studies have shown the oncogenic properties of WT1 in a variety of tumors. It was recently proposed that WT1 was a chameleon gene, due to its dual functions in tumorigenesis. We aimed to investigate the clinical significance of WT1 as biomarker in acute myeloid leukemia (AML) and clear cell renal cell carcinoma (ccRCC) and to elucidate the function of WT1 as an oncogene in squamous cell carcinoma of head and neck (SCCHN). In AML, it was suggested that WT1 expression was an applicable marker of minimal residual disease (MRD). In adult patients with AML, we found a good correlation between WT1 expression levels normalized to two control genes, β-actin and ABL. Outcome could be predicted by a reduction in WT1 expression in bone marrow (≥ 1-log) detected less than 1 month after diagnosis, when β-actin was used as control. Also, irrespective of the control gene used, outcome could be predicted by a reduction in WT1 expression in peripheral blood (≥ 2-log) detected between 1 and 6 months after treatment initiation. Previous studies in RCC demonstrated that WT1 acted as a tumor suppressor. Thus, we tested whether single nucleotide polymorphisms (SNPs) or mutations in WT1 might be associated with WT1 expression and clinical outcome in patients with ccRCC. We performed sequencing analysis on 10 exons of the WT1 gene in a total of 182 patient samples, and we identified six different SNPs in the WT1 gene. We found that at least one or two copies of the minor allele were present in 61% of ccRCC tumor samples. However, no correlation was observed between WT1 SNP genotypes and RNA expression levels. Moreover, none of the previously reported WT1 mutations were found in ccRCC. Nevertheless, we found that a favorable outcome was associated the homozygous minor allele for WT1 SNP. We then further investigated whether WT1 methylation was related to WT1 expression and its clinical significance. Methylation array and pyrosequencing analyses showed that the WT1 promoter region CpG site, cg22975913, was the most frequently hypermethylated CpG site. We found a trend that showed nearly significant correlation between WT1 mRNA levels and hypermethylation in the 5’-untranslated region. Hypermethylation in the WT1 CpG site, cg22975913, was found to be associated with patient age and a worse prognosis. One previous study reported that WT1 was overexpressed in SCCHN. That finding suggested that WT1 might play a role in oncogenesis. We found that both WT1 and p63 could promote cell proliferation. A positive correlation between WT1 and p63 expression was observed, and we identified p63 as a WT1 target gene. Furthermore, several known WT1 and p63 target genes were affected by knocking down WT1. Also, co-immunoprecipitation analyses demonstrated a protein interaction between WT1 and p53. In summary, WT1 gene expression can provide useful information for MRD detection during treatment of patients with AML. In RCC, our results suggested that the prognostic impact of WT1 SNPs was limited to the subgroup of patients that were homozygous for the minor allele, and that WT1 promoter hypermethylation could be used as a prognostic biomarker. In SCCHN, WT1 and p63 acted as oncogenes by affecting multiple genes involved in cancer cell growth.

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

Genomic imprinting, the parent-of-origin specific silencing of alleles, plays an important role in phenotypic plasticity and consequently evolution. The leading explanation for genomic imprinting is Haig's conflict theory, which suggests that alleles from each parent have evolved under different selectional pressures, resulting in the differential expression of patrigenes and matrigenes. Previous studies have mainly used mammals and flowering plants to test Haig’s theory. However, there is a lack of independent evidence to support the theory. My PhD thesis attempts to conduct an independent test of Haig’s conflict theory using buff tailed bumblebee Bombus terrestris. A methylation system to facilitate genomic imprinting has not been found in this species. Therefore the first aim of the study was to establish the presence of a functional methylation system in B. terrestris before testing Haig's conflict theory using worker reproduction in queen-less colonies. The initial finding is that a methylation system exists in B. terrestris. The next study, investigating the presence of methylated genes, revealed differential methylation patterns in caste and life stages. Finally, genes involved with worker reproduction in a range of social insects were identified, but distinguishing the matrigene and the patrigene for each gene was unsuccessful. Therefore the final study investigating the presence of imprinted genes in B. terrestris and whether they conform to the expression patterns hypothesised by Haig’s conflict theory could not be analysed. Although this study did not provide conclusive evidence to support Haig’s conflict theory, the presence of methylation in genes involved with worker reproduction in reproducing and non-reproducing B. terrestris workers suggests that further analysis is needed. With adequate evidence, proving Haig’s conflict theory will not only expand our knowledge of invertebrate methylation, but also our understanding of conflict within social insect societies and our knowledge of how genomic imprinting affects phenotypic plasticity.