Although it was previously reported that Dictyostelium discoideum possessed a single ras gene (Ddras) that was maximally expressed during the pseudoplasmodial stage of development, a second ras gene (DdrasG), has been isolated and characterized. It encodes a protein that is similar to the protein encoded by Ddras and the human ras proteins. However, in contrast to Ddras, the DdrasG gene was only expressed during growth and early development. The two ras proteins may fulfill different functions: the DdrasG protein having a role during cell growth and the Ddras protein having a role in signal transduction during multicellular development. However, the expression of the DdrasG gene throughout development did not appear to have a detrimental effect on differentiation. Although other eukaryotic organisms possess more than one ras gene, D. discoideum is thus far unique in expressing different ras genes at different stages of development. Ras genes are members of a large ras-related multigene family that has been found in a wide variety of organisms. A ras-related gene was isolated from D. discoideum that hybridized to both the Ddras and DdrasG genes under low, but not under high stringency conditions. The predicted amino acid sequence shows a high degree of sequence identity with the human rap proteins and thus has been designated Ddrapl. During vegetative and early development a single 1.1 kb mRNA was present, but by aggregation this transcript was no longer detected and two new transcripts of 1.0 and 1.3 kb were observed and were present throughout the remainder of development. The maximum levels of the Ddrapl specific mRNAs appeared during aggregation and culmination, developmental stages where the levels of DdrasG and Ddras messages were declining. The reciprocal nature of the Ddrapl gene expression with respect to that of the two ras genes suggests the possibility that the ras and rap gene products in D. discoideum have antagonistic roles. Antibodies that are specific for the Ddras, DdrasG and Ddrapl proteins have been generated and can be used to help elucidate the biological functions of the individual proteins. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A digoxigenin-labeled nonradioactive detection system was used to screen a zebrafish cDNA library for p53-like and ras-like genes. One clone was isolated and identified as an incomplete p53-like gene. The insert size of this clone is 1777 bp, which encodes part of evolutionarily conserved region II and all of regions III, IV, and V. A magnetically enriched whole zebrafish cDNA library was constructed to enhance possible recovery of ras-like genes in zebrafish. One clone, termed Zras-Bl, carried an insert of 2592 bp with an open reading frame encoding a 188 amino acid residue ras p21 protein. Based on total protein sequence, this expressed zebrafish ras p21 is most closely related to human N-ras (91% homology), with lesser homology to Ha-ras (84%) and Ki-ras (85%). Preliminary partial sequence data obtained by genomic and reverase transcriptasepolymerase chain reaction (RT-PCR) screening indicate the presence of at least one additional expressed ras gene in zebrafish. The tumorigenicity and Ki-ras mutational properties of dietary 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DBP) were compared in rainbow trout. Both chemicals elicited predominantly 12(1)G->A and 12(2)G->T mutations in trout liver tumors. Two {12(1)G->T and 12(2)G->T} and one {12(1)G->A and 12(2)G->T} double mutation were also observed in DBP livers tumors, but not in DMBA liver tumors. Some stomach tumors from both chemicals exhibited so much DNA degradation that routine PCR amplification was not possible. Among sixteen DMBA stomach tumors with intact DNA, no Ki-ras mutations were found. Of sixteen DBP stomach tumors examined, one had 12(1)G->A and two had 13(1)G->C mutations. The observed G->T transversions are compatible with apurinic mutagenesis driven by unstable DNA adducts arising from one-electron oxidation, but this is not true for the major G->A transitions or G->C transversions and rare double mutations found in this study. The low sensitivity of direct sequencing may limit the frequency of ras mutant detection in this study. / Graduation date: 1996

Characterization of the 5' flanking region of rainbow trout ki-ras gene was begun with the cloning and sequencing of this region by the inverse PCR technique and dideoxynucleotide chain termination method. In total, a nucleotide sequence of 1080 bp upstream from the first coding ATG was sequenced. Although this region showed certain promoter elements, it does not share common features with other mammalian ras promoters, which lack the TATA and contain multiple GC boxes with Spl binding activities. In contrast, this region in trout ras contains typical TATA and CCAAT boxes. This structural difference of the trout ki-ras promoter from that of other mammalian ras genes may suggest that different transcriptional regulation mechanisms of the ras ger.e are used at various levels in evolution. The chromatin structure of the trout ki-ras gene was studied by probing invivo for DNase I hypersensitive sites. To overcome the difficulties of using the traditional indirect end labeling method for a single-copy gene, the technique of ligation-mediated PCR was applied. No hypersensitive sites were observed at or near the codon 12 region of the gene, either in normal (protooncogene) or tumor (oncogene) tissue from the liver. This result suggests that the local chromatin structure of trout ki-ras gene may not be an important factor for codon 12 mutations induced by genotoxins, and that changes of chromatin structure are unlikely to be promoted after tumor formation. Studies by micrococcal nuclease demonstrate that this ras gene, in the region around 12, lacks ordered nucleosome positioning or may be even free of nucleosomes. Such an irregular organization of ras oncogenic chromatin would resemble that of many other "normal", highly active eukaryotic genes. The intrinsic affinity of trout ki-ras gene for aflatoxin B₁ was determined by in vitro alkylation experiments. Exon 1 of the gene was synthesized and labeled at the 5'end of the coding strand by the PCR technique. Taking advantage of the selective cleavage of AFB1-DNA adducts by piperidine under alkali conditions, the frequency of AFB 1 attack to each guanyl site was determined by densitometric scans after the cleaved fragments were electrophoresed on sequencing gels. The results demonstrated that two guanyl sites of codon 12 had differential affinity to AFBl, the more 5' G was relatively inaccessible but the more 3' G was accessible, indicating that the sequence selectivity of AFB I may contribute to the preference of the initial adduction in vivo. / Graduation date: 1993

Thesis (Ph. D. in Cell and Developmental Biology)--Vanderbilt University, Aug. 2006. / Title from title screen. Includes bibliographical references.

Thesis (Ph.D.)--Indiana University, 2006. / Title from screen (viewed on Apr. 27, 2007) Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI) Includes vita. Includes bibliographical references (leaves 181-239)

Lau Yee Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-157). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / List of Figures --- p.viii / List of Tables --- p.xi / Contents --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.1 --- Background of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.1.2 --- Etiology of HCC --- p.2 / Chapter 1.1.3 --- Relationship between HCC and HBV --- p.3 / Chapter 1.1.4 --- Differential gene expression under induction of HBx protein by microarray analysis --- p.5 / Chapter 1.1.5 --- Confirmation of candidate genes --- p.6 / Chapter 1.2 --- Ras-Oncogene --- p.8 / Chapter 1.2.1 --- Ras superfamily --- p.8 / Chapter 1.2.1.1 --- Rho family --- p.9 / Chapter 1.2.1.2 --- Rab family --- p.10 / Chapter 1.2.2 --- Functional mechanism of small GTPase --- p.11 / Chapter 1.2.3 --- Possible functions of Rho and Rab family members --- p.14 / Chapter 1.3 --- RhoC --- p.16 / Chapter 1.3.1 --- The genomic and protein structures of RhoC --- p.16 / Chapter 1.3.2 --- Relationship between RhoC and tumours --- p.19 / Chapter 1.4 --- Rabl4 --- p.20 / Chapter 1.4.1 --- The genomic and protein structures of Rabl4 --- p.20 / Chapter 1.4.2 --- Relationship between Rabl4 and tumours --- p.23 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Cell lines --- p.25 / Chapter 2.1.2 --- Cell culture reagents --- p.26 / Chapter 2.1.3 --- Reagents for total RNA isolation --- p.29 / Chapter 2.1.4 --- Reagents for reverse transcription polymerase chain reaction (RT-PCR) --- p.30 / Chapter 2.1.5 --- Reagents and buffers for Western blot analysis --- p.31 / Chapter 2.1.6 --- Vectors for cloning --- p.39 / Chapter 2.1.7 --- Reagents for polymerase chain reaction (PCR) --- p.39 / Chapter 2.1.8 --- Restriction digestion reagents --- p.42 / Chapter 2.1.9 --- Reagents for agarose gel electrophoresis --- p.42 / Chapter 2.1.10 --- Ligation reagents --- p.44 / Chapter 2.1.11 --- Bacterial culture medium --- p.44 / Chapter 2.1.12 --- Dyes and reagents for fluorescent microscope --- p.46 / Chapter 2.1.13 --- Reagents for flow cytometry --- p.48 / Chapter 2.1.14 --- Detection of apoptosis --- p.48 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Identification of gene expression of candidate genes in HCC --- p.50 / Chapter 2.2.1.1 --- cDNA preparation --- p.50 / Chapter (1) --- Cell culture of HepG2 and WRL-68 cell lines --- p.50 / Chapter (2) --- Total RNA isolation --- p.50 / Chapter (3) --- First-strand cDNA synthesis --- p.51 / Chapter 2.2.1.2 --- RT-PCR of candidate genes --- p.52 / Chapter 2.2.1.3 --- Western blotting --- p.53 / Chapter (1) --- Cell culture --- p.53 / Chapter (2) --- Protein extraction --- p.53 / Chapter (3) --- Quantification of proteins --- p.53 / Chapter (4) --- Detection of RhoC and Rabl4 protein by western blot analysis --- p.54 / Chapter (5) --- Western blotting luminol detection --- p.56 / Chapter 2.2.2 --- Cloning protocol --- p.57 / Chapter 2.2.2.1 --- Amplification of RhoC and Rabl4 genes --- p.57 / Chapter 2.2.2.2 --- Purification of PCR product --- p.58 / Chapter 2.2.2.3 --- Restriction enzymes digestion --- p.53 / Chapter 2.2.2.4 --- Insert/vector ligation --- p.59 / Chapter 2.2.2.5 --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.60 / Chapter 2.2.2.6 --- Transformation of ligation product into chemically competent bacterial cells --- p.61 / Chapter 2.2.2.7 --- Small-scale preparation of bacterial plasmid DNA --- p.61 / Chapter 2.2.2.8 --- Screening for recombinant clones --- p.62 / Chapter 2.2.2.9 --- DNA sequencing of cloned plasmid DNA --- p.63 / Chapter 2.2.2.10 --- Midi-scale preparation of recombinant plasmid DNA --- p.64 / Chapter 2.2.3 --- Visualization of the subcellular localization patterns --- p.66 / Chapter 2.2.3.1 --- Cell culture of AML12 and HepG2 cell lines --- p.66 / Chapter 2.2.3.2 --- Transfection of GFP fusion constructs into cells --- p.66 / Chapter 2.2.3.3 --- DAPI staining --- p.67 / Chapter 2.2.3.4 --- ER-Tracker´ёØ Blue-White DPX staining --- p.68 / Chapter 2.2.3.5 --- Subcellular localization study using Epi-fluorescence microscopy --- p.68 / Chapter 2.2.4 --- Analysis of cell cycle --- p.69 / Chapter 2.2.4.1 --- Transfection of GFP vectors / GFP-tagged proteins into cells --- p.69 / Chapter 2.2.4.2 --- Analysis of cell cycle by flow cytometry --- p.69 / Chapter 2.2.5 --- Detection of apoptosis --- p.70 / Chapter 2.2.5.1 --- Transfection --- p.70 / Chapter 2.2.5.2 --- Detection of DNA fragmentation --- p.70 / Chapter 2.2.6 --- Reorganization of Actin cytoskeleton by RhoC --- p.71 / Chapter 2.2.6.1 --- Transfection of GFP vectors/GFP-tagged proteins into cells --- p.71 / Chapter 2.2.6.2 --- Rhodamine phalloidin (RP) staining --- p.71 / Chapter 2.2.6.3 --- Epi-fluorescence microscopy --- p.72 / Chapter 2.2.7 --- Analysis of cell invasion under induction of RhoC --- p.72 / Chapter 2.2.7.1 --- "Sub-cloning of human RhoC gene into a mammalian expression vector, pHM6" --- p.72 / Chapter 2.2.7.2 --- Transfection of pHM6-RhoC --- p.73 / Chapter 2.2.7.3 --- Cell invasion assay --- p.73 / Chapter 2.2.8 --- Analysis of downstream effectors in RhoC-mediated pathway --- p.75 / Chapter 2.2.8.1 --- RT-PCR --- p.75 / Chapter 2.2.8.2 --- Western blotting --- p.75 / Chapter 2.2.9 --- Analysis of role of Rabl4 in membrane trafficking --- p.76 / Chapter 2.2.9.1 --- Cloning and transfection --- p.76 / Chapter 2.2.9.2 --- Alexa 594 transferrin conjugate staining --- p.76 / Chapter 2.2.9.3 --- Epi-fluorescence microscopy --- p.77 / Chapter 2.2.10 --- Statistics --- p.77 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of RhoC and Rabl4 in hepatoma cells --- p.78 / Chapter 3.1.1 --- RT-PCR --- p.78 / Chapter 3.1.2 --- Western blotting --- p.81 / Chapter 3.2 --- Subcellular localization of RhoC and Rab 14 --- p.85 / Chapter 3.3 --- Characterization of RhoC --- p.93 / Chapter 3.3.1 --- Cell cycle analysis --- p.93 / Chapter 3.3.2 --- Apoptosis --- p.95 / Chapter 3.3.3 --- Actin cytoskeleton reorganization --- p.97 / Chapter 3.3.4 --- Cell invasion ability --- p.99 / Chapter 3.3.5 --- Downstream effectors of RhoC in cytoskeletal reorganization --- p.102 / Chapter 3.4 --- Characterization of Rabl4 --- p.107 / Chapter 3.4.1 --- Cell cycle analysis --- p.107 / Chapter 3.4.2 --- Apoptosis --- p.109 / Chapter 3.4.3 --- Roles in intracellular transportation --- p.111 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Strong expression of RhoC and Rabl4 in hepatoma cells --- p.117 / Chapter 4.2 --- Subcellular localization of RhoC and Rabl4 --- p.119 / Chapter 4.3 --- The effects of RhoC in normal liver cells --- p.122 / Chapter 4.3.1 --- Cell cycle progression by RhoC through regulating of G1 to S phase transition --- p.122 / Chapter 4.3.2 --- RhoC shows no apoptotic effect in normal liver cell systems --- p.123 / Chapter 4.3.3 --- Formation of actin filaments and stress fibers --- p.124 / Chapter 4.3.4 --- Induction of cell invasion in RhoC-expressing cells --- p.125 / Chapter 4.3.5 --- Downstream effectors in signaling pathway of RhoC in actin filment reorganization and cell invasion --- p.126 / Chapter 4.4 --- The effects of Rabl4 in normal liver cells --- p.132 / Chapter 4.4.1 --- Cell proliferation effects of Rabl4 by increasing percentage of cells in S phase for DNA synthesis --- p.132 / Chapter 4.4.2 --- Rabl4 has no apoptotic effects --- p.133 / Chapter 4.4.3 --- Roles of Rabl4 in vesicular transport --- p.134 / Chapter 4.5 --- Conclusion --- p.138 / Chapter 4.6 --- Future prospects --- p.140 / Appendix --- p.143 / References --- p.147

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

For ras gene mutation analysis in the rainbow trout (Oncorhynchus mykiss) model system, a partial trout ras sequence was identified using the polymerase chain reaction (PCR). Two synthetic oligonucleotides based on rat K-ras gene sequence were used as primers for the PCR procedure. A 90 base pair (bp) sequence, referred to as the trout K-ras, was amplified from trout genomic DNA and cDNA. Cloned 90 by PCR products from several normal liver tissues were sequenced resulting in the same sequence. Large-sized PCR products, 111 and 237 bp, were also cloned and sequenced indicating that these fragments included the 90 by sequence information expressed in mRNA. This 5'-terminal partial trout K-ras nucleotide sequence was 88% homologous to that of the goldfish ras gene, and less homologous to those of mammalian ras genes. Based on the partial sequence information of two trout ras genes, K-ras and H-ras, DNA from trout tumors induced by chemical carcinogens, aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidene (MNNG), were analyzed for the presence of point mutations. Using the PCR and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated liver tumor DNA indicated evidence for ras point mutations. Of the 10 mutant ras genotypes, seven were probed as G to T transversions at the second position of codon 12, two were G to T transversions at the second position of codon 13, and one was a G to A transition at the first position of codon 12. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive mutation evidence in each case, which seemed to occur in only a fraction of the neoplastic cells. However, no mutations were detected in exon 1 of the trout K-ras gene, nor in DNA from trout normal livers. Results indicated that the hepatocarcinogen AFB1 induced similar ras gene mutations in trout as in rat liver tumors. By comparison, the mutation specificity of MNNG in trout liver tumors was for G to A transitions, but no ras mutations were detected in trout kidney tumors. This investigation was the initial study of experimentally induced ras gene point mutations in a lower vertebrate fish model. / Graduation date: 1991

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.