Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

The diptericidal $\textit{Bacillus thuringiensis}$ (Bt) ssp. $\textit{fukuokaensis}$ strains 84-I and 17A were investigated for the presence of novel Cry proteins. N-terminal amino acid, immunological and PCR analysis indicated that both strains contain a novel set of $\delta$-endotoxins. N-terminal amino acid sequence analysis indicated that the larger proteins from each strain (90 and 72-kDa of 84-I and 70 and 65-kDa of 17A) were related to the Cry proteins of Bt ssp. $\textit{israelensis}$(Bti). Immunoblotting experiments confirmed that Cry10A-type proteins were present in both strains although subsequent PCR did not give a positive reaction for either strain using $\textit{cry10A}$ specific primers indicating that the Cry10-types were indeed novel. To further investigate the 65-kDa protein of 17A, the gene encoding it was cloned from a size-enriched plasmid DNA library. Unsuccessful attempts were also made to clone the 90-kDa protein of 84-I. Sequence alignments of the deduced protein product of the 17A gene ($\textit{am1}$) showed it to represent the second identification of a natural C-terminal truncate of a Cry4-type protein, the first being Cry10A. The missing C-terminal region of AMl appears to be encoded as a complete Orf ($\textit{am2}$) immediately downstream of the first protein gene. When DNA containing both the $\textit{am1}$ and $\textit{am2}$ genes was subcloned into the pSVP27A expression vector high levels of expression of both proteins were observed in acrystalliferous Bt. The protein was deposited in inclusion bodies which were found to be toxic to $\textit{Dacus oleae}$. Extensive phylogenetic analysis was carried out to determine the relationship between, and possible evolutionary origins of, AMl, the Cry proteins of Bti and two further Cry10A-type $\delta$-endotoxins (Cry19A from Bt ssp. $\textit{jegathesan}$ and Cry20A from 84-I) identified in other laboratories during the course of this project. Based on the amino acid sequence alignment, all seven proteins appear to have evolved from a common ancestor to form three distinct groups which mirror the structural organisation of the genes. Based on these groupings and a previous hypothesis of Dervyn $\textit{et al.}$ (1995), a hypothesis was proposed as to the evolution of the 130-kDa Cry4-type proteins from a 70-kDa Cry2-type ancestor. The above hypothesis is based on the assumption that transfer of $\delta$-endotoxin genes between subspecies has occurred at some point in evolutionary history. Evidence for this transfer was found when the genetic context of the $\textit{am1}$ gene was investigated. Two novel insertion sequences (Tl) and (T2) were identified with sequence similarity to IS$\textit{240A}$ from Bti and an insertion sequence associated with the $\textit{Orf1}$ gene of 84-I. The identification of a further incomplete reading frame with similarity to integrase/recombinase proteins involved in Class II transposition raises the possibility that T1 and T2 form part of a novel Class II transposon. A novel $\alpha$/$\beta$-type small, acid soluble protein (SASP) gene was also discovered. This gene, which may be plasmid encoded, showed considerable sequence similarity to $\alpha$/$\beta$-type SASP from $\textit{Bacillus megaterium}$. The discovery of this gene raises new questions about taxonomic relations between the $\textit{Bacilli}$.

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No description available.

BTBD7 is a newly identified candidate gene for HCC using a high-throughput cDNA/EST microassay. This gene encodes for a protein of 410 amino acid residues. This protein was previously named as the function unknown protein 1 (FUP1) because the biological function of this protein was unknown at that time. Bioinformatics analysis revealed that this protein contains two bric-a-brac, tramtrack, broad-complex (BTB) domains located at amino acid positions 143 to 230 and 274 to 342. In order to reflect its structure and functions, and to be consistent with the GeneBank database (Accession No. NM_018167), we rename it as BTBD7 (BTB domain containing 7). / In conclusion, our study demonstrated that BTBD7 is a novel oncogene, which is associated with hepatocellular carcinoma and is essential for the inhibition of cell growth and tumorigenesis. To our knowledge, BTBD7 is the first identified regulator of p16INK4A through inhibiting the promoter activity of p16INK4A. BTBD7 may thus serve as a new tumor marker or as a potential target of treating hepatocellular carcinoma. / In previous studies, the expression of BTBD7 was shown to be tissue-specific as demonstrated by Northern blot. Furthermore, we collected 18-paired HCC samples to further reveal the correlation of BTBD7 gene expression profiles with tumorigenesis. Our data showed that BTBD7 was significantly elevated in 44.4% of the HCC samples. Compared with immortalized hepatocyte cell lines MIHA or LO2, both mRNA level and protein level of BTBD7 were also elevated in the hepatoma cell lines HepG2, BEL7404, Hep3B and Huh7. This gave a due that the expression of BTBD7 may be correlated with carcinogenesis of liver cells. / In the present study, the function of BTBD7 was investigated. We used RNAi approach to silence BTBD7. Compared with the control, siBTBD7 induced cell cycle arrest at G1 phase and later caused obvious cell death. The cell death was further demonstrated to be apoptosis through activation of caspase 3. Furthermore, we carried out candidate gene search using knockdown of BTBD7. The mRNA level of tumor suppresser p16INK4A was upregulated and hTERT was downregulated in BTBD7 knocked down cells. The other key genes involved in cell growth, cell cycle control, cell death and survival (c-myc, c-fos, c-jun, p21CIP1, p27KIP1, p53, Survivin, E2F, NF-kappaB, Bax, p14ARF, p16INK4A and hTERT) did not respond to the reduced BTBD7 levels. On the other hand, double knockdown of p16INK4A and BTBD7 markedly reduced the effects of cell cycle arrest and the death ratio caused by dysfunction of BTBD7 or overexpression of p16INK4A, suggesting that p16 INK4A is a downstream target of BTBD7. We further adopted a dominant negative approach to confirm these results. / Liu, Zheng. / Advisers: C. H. K. Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3449. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Kou Ying Chuck. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 186-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Methionine dependence, the inability of cells to grow when the amino acid methionine is replaced in culture medium by its metabolic precursor homocysteine, is characteristic of many cancer cell lines. Most cells proliferate normally under these conditions. The methionine dependent tumorigenic human melanoma cell line MeWo-LC1 was derived from the methionine independent non-tumorigenic line MeWo. The MeWo-LC1 cell line has been shown to have a cellular phenotype similar to that of cells from patients with the cblC inborn error of cobalamin metabolism, with decreased synthesis of cobalamin coenzymes and decreased activity of the cobalamin dependent enzymes methionine synthase and methylmalonyl-CoA mutase. Inability of cblC cells to complement the defect in cobalamin metabolism in MeWo-LC1 suggested that the defect was caused by decreased activity of the MMACHC gene product. However, no potentially disease causing mutations could be detected in the coding sequence of MMACHC in MeWo-LC1. No MMACHC expression could be detected in MeWo-LC1, and there was virtually complete methylation of a CpG island at the 5' end of the MMACHC gene in MeWo-LC1, consistent with inactivation of the gene by methylation; the CpG island was partially methylated in MeWo and only lightly methylated in control fibroblasts. Transfection of MeWo-LC1 with wild type MMACHC with a constitutive promoter resulted in correction of the defect in cobalamin metabolism and restoration of the ability of cells to grow in medium containing homocysteine. We conclude that epigenetic inactivation of the MMACHC gene is responsible for methionine dependence in MeWo-LC1.

In mammals, circadian rhythms are generated by transcriptional-translational feedback loops consisting of a set of clock genes and their protein products. Among them, Bmal1 is a critical clock gene in generating and maintaining circadian rhythms. Moreover, orphan nuclear receptors REV-ERBs and RORs were known to respectively repress and activate Bmal1 transcription. In our study, we further demonstrated that: (1) REV-ERBalpha might be the main regulator in maintaining Bmal1 oscillation in thymus. (2) Rorgamma mRNA is constant in muscle and testis, and rhythmic in liver, while Rorgammat mRNA is only expressed in thymus, at constant levels. Moreover, the expressions of these two Rorgamma isoforms are affected in Clock mutant mice in a distinct way. (3) RORgamma and RORgammat can activate Bmal1 transcription at a similar level. (4) Rorgamma is a clock-controlled gene. Altogether, our results suggest that the crucial role of REV-ERBs and RORs in peripheral clocks. Furthermore, our work highlights functional differences among mammalian peripheral clocks, which provides important insights into the complexity of the circadian system.

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No description available.