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TSG101 is a tumor susceptibility gene, which exhibits many biological functions including the regulation of transcription, cell growth and differentation, protein trafficking and receptor recycling. Recent studies have revealed overexpression of TSG101 in human cancers of papillary thyroid carcinoma and breast cancer, whereas downregulation in the periphery of cancer tissue. These data indicated that the amount of TSG101 gene products might be relevant to tumor formation. Understanding the detail regulation of TSG101 gene expression might advance our knowledge on the processes of neoplastic conversion. In this regard, our lab have cloned and characterized upstream sequence of TSG101 transcription start site, and defined the region of -1~-436 as proximal promoter by luciferase reporter assay. Sequence analysis of this region using NSITE program on Softberry web site has revealed several transcription factor binding sites including MAZ, Sp1, ETF and ETS-1. Using EMSA and luciferase assay, we have demonstrated MAZ and Sp1 as major transcription factors regulate TSG101 proximal promoter. In this thesis, we advance our study to further explore the role of ETF and ETS-1 sites in regulation TSG101 promoter. We first cloned the region encompassing two ETS-1 sites in ¡V190~-1 region into pGL3-basic( -190/pGL3-basic ). The luciferase reporter assay of wildtype and mutant ¡V190/pGL3-basic plasmids further demonstrated important role of these ETS-1 sites. To explore detail contribution of the above mentioned transcription factor binding sites in regulation TSG101 promotor, we have made mutant reporter constructs containing different assortment of mutations in these binding sites, and assayed the effect of mutant on TSG101 promoter activity. The results showed that in cancer cell lines (COS-1, ARO and WRO) tested, Sp1, ETF, and ETS-1 binding sites are essential and they act co-operatively in regulating the activity of TSG101 proximal promoter. The results also indicated that Sp1 and ETS-1 were positive regulators, while ETF worked as a negative regulator.

Abstract¡G TAL1 gene was originally discovered as a result of its activation by chromosome rearrangements in T cell acute lymphoblastic leukaemia(T-ALL). Further studies have shown that TAL1 expression is aberrantly activated by several mechanisms including chromosome translocations, interstitial deletion and transactivation without detectable chromosomal alteration. TAL1 gene encodes a bHLH transcription factor, that is essential for the development of all haematopoietic lineages and its expression is maintained during differentiation along erythroid, mast and megakaryocytic cell lineages, but not in normal peripheral T-lymphocytes. The bHLH motif of these protein is responsible for DNA binding and dimerization with other bHLH proteins involved in transcription regulation. TAL1 protein is able to form heterodimers with the ubiquitously expressed E2A gene products, E47 and E12, and the heterodimers bind to E-box motif with the general sequence CANNTG. But the target genes for TAL1 oncoprotein have not yet been identified. We have previously isolated TAL1/E12 heterodimer bound genomic fragments by chromatin immunoprecipitation from K562 cells, and selected 6 fragments with one to four E-box CANNTG sequences. In order to determine if these fragments could be the regulatory elements of potential target genes of TAL1 oncoprotein, we inserted these 6 DNA fragments individually into pGL3 to generate recombinant reporter plasmids. The transfection experiments indicated that K34 and K94 DNA fragments behaved as a transcriptional transactivating sequence, and TAL1 and E12 proteins are required for efficient transcriptional activity. We also showed that transfection of these two recombinant constructs into K562 cells generated positive transcriptional activity, in a level similar to that in TAL1 and E12 co-transfected COS1 cells. These results established that both K34 and K94 DNA fragments are likely to contain a promoter of potential TAL1 target genes.

Our effort to provide an efficient route to serine glycosides with utility in glycopeptide synthesis has led to the identification of two particularly effective promoters of O-glycosylation. Indium(III) bromide and scandium(III) triflate were shown to be superior promoters of microwave accelerated O-glycosylation utilizing peracetyl carbohydrate donors. 247, 249 These Lewis acids afforded several advantages over previously described promoters including, increased yields, tolerance to moisture, decreased environmental toxicity, ease of work up, and increased reproducibility. Both affected the microwave accelerated glycosylation of Fmoc-ser-OH with sugar peracetates providing superior yields to previously reported methods. For larger scale work the two step route involving the glycosylation of Fmoc-Ser-OBn followed by removal of the benzyl protecting group via hydrogenolysis was preferred. Of the two Lewis acids, the minimally active indium (III) bromide was preferred, as it afforded slightly higher yields and was effective in catalytic quantities. Three groups of helical DAMGO glycopeptide analogs were synthesized in order to provide a better understanding of the structure activity relationships of these opioid peptides. Although the introduction of the amphipathic helix significantly affected binding of the DAMGO message, there was no correlation between binding affinity at the individual opioid receptors and the degree of helicity. In general, addition of the helical address imparted increased affinity for the kappa receptor. The nature of the linker connecting the N-terminal DAMGO sequence and the C-terminal helical address effected binding affinity only slightly. Successive addition of positive charges to the address increased binding at all three opioid receptors until a maximum was reached at a positive two address charge. Although, the amphipathic helix was shown to moderate receptor selectivity, the native mu preference of the DAMGO message was retained Two groups of fluorescent analogs of the mixed δ / μ opioid agonist MD100 were prepared. Within the first series, the fluorescent label was attached to the interior of the address sequence employing the pNZ moiety as a secondary protecting group. The second series of analogs was based on NovaTag™ resin, and allowed for attachment of the fluorophore at the carboxy terminus. The influence on helicity imparted by fluorophore conjugation depended on the nature and point of attachment of the label. The disruption of secondary structure associated with attachment of the fluorescent correlated with decreased binding affinity at the individual opioid receptors. Preliminary in vivo results were encouraging. The least parent like of the MD100 fluorescent analogs was shown to be taken up into endothelial cells. This suggests that the labeled glycopeptides are likely to cross the blood-brain barrier.

A family of E. coli - P. ruminis shuttle-plasmids was constructed to allow the isolation and characterization of gene promoters from the rumen bacterium P. ruminis. The promoter rescue plasmid pBK was used to isolate a total of 4 genomic DNA fragments that promoted transcription in P. ruminis strains 0/10. These promoters, and an additional promoter, previously isolated from P. ruminis strain OR38 (Schoep, 1999), were identified by their ability to initiate expression of a promoterless ermAM gene in P. ruminis. Within 4 of the fragments, a total of 5 transcription start sites were identified in P. ruminis using a novel, fluorescent-primer extension analysis protocol. Comparison of promoters isolated in this and previous studies revealed a strong consensus RNA polymerase DNA-binding motif, including the well characterized –35 and –10 elements. Consensus sequences established for these elements were: TTgacA and AtAATAta respectively, where bold upper-case font, regular upper-case, and lower-case fonts represent conservation in 100%, 80%, and 70% of promoters respectively. The −10 and −35 motifs were interspaced by 16 – 18 nt. Among the newly identified promoters, the consensus for the –10 element was extended one nucleotide upstream and downstream of the standard hexamer (boxed). These motifs were similar to those recognized by eubacterial RNA polymerase containing the σ70-like factor. Promoters also contained possible UP elements, and were significantly more curved than protein-coding regions. Additional plasmid vectors were constructed, to allow the use of both the quantitative SYBR green real time PCR and ß-glucuronidase assays, to examine 4 promoters in depth. This showed a wide range of promoter strengths within the group. However, no correlation was found between the composition and context of elements within P. ruminis promoters, and promoter strength. A mutation within the –35 element of one promoter revealed that promoter strength, and the choice of transcription start site were both sensitive to single nucleotide

Previous research has shown varying degrees of renal damage on exposure to equal amounts of cadmium in occupationally exposed mining and factory workers. Further work has shown that in vitro exposure of human peripheral lymphocytes to the same cadmium levels resulted in significant variation in Metallothionein (MT) mRNA transcriptional induction over basal MT mRNA expression in a series of individuals. This variation could account for the differences in renal Cd toxicities identified previously. In this study, the human MT-IIₐ gene was cloned from 12 individuals, and the 5'promoter region was sequenced for each to determine the extent of promoter nucleotide variation. This is of interest since such an analysis has not been done in the past. No study has been done to look at the degree of polymorphism in a particular promoter region. Thus, there are no data on the degree of nucleotide drift or change which can occur in promoter regulatory elements. Such a study could provide insight into whether promoter changes could result in the type of variation described above. It could also give some insight into the degree of variation in sequences in the literature. The results obtained indicated that the human MT-IIₐ promoter region is highly conserved, with only one polymorphic site identified at position 557, between the glucocorticoid responsive element and the fourth metal regulatory element sequences. This suggests that promoter variation is not likely a significant yfactor in MT mRNA induction variability, although further analysis would be needed to show this since only 12 people were analyzed. The results were compared against a study of nucleotide polymorphism in Drosophila melanogaster, which is the only other data on nucleotide variation specifically (Kreitman, 1983; Kreitman and Hudson, 1991). As well, a number of discrepancies were noted from the original published sequences in the literature, suggesting that errors are likely published in genomic sequence which are never identified, except through trial and error. This has potential repercussions when considering the use of such sequence in cloning and sequencing projects, like the sequencing of the human genome, since this would depend on the accuracy of previously published data. / Thesis / Master of Science (MS)

PEA3 is a member of the expanding Ets family of transcription factors. In the adult mouse, PEA3 mRNA is expressed at highest levels in the brain, epididymis and at lower levels in the mammary gland, testes, ovary and uterus. PEA3 mRNA is expressed differentially during mouse embryogenesis and is down-regulated following retinoic acid induced differentiation in mouse embryonal carcinoma cell lines. PEA3 is overexpressed at the transcriptional level in 93% of all HER2/neu positive human breast tumors. The molecular basis for differential transcription of the PEA3 gene is not known. Sequence analysis revealed that the upstream region of the PEA3 gene has characteristics of a CpG island and does not possess a recognizable "TATA" element. Rapid amplification of 5' eDNA ends (5'RACE) reveals that transcription initiates from multiple sites, consistent with the absence of TATA elements. To localize cis-acting sequences required for PEA3 expression, deletions of the putative promoter were placed upstream of a luciferase reporter gene and tested for activity in the FM3A cell line. FM3A cells express substantial levels of PEA3 mRNA and protein, which suggests that all of the factors required for transcription are present in the cells. Transient transfections of 5' and 3' deletion mutants of the PEA3 promoter indicated that the efficiency of the PEA3 promoter depended on both negative and positive cis-elements, located upstream and downstream of the transcription start sites. A DNA fragment containing a region from -3 to +676, relative to the major start site of transcription, was sufficient for maximal promoter activity. Luciferase reporter plasmids containing more 5' flanking sequence had lower activity indicating the presence of silencer elements. To aid the identification of critical sequence elements within the minimal PEA3 promoter, we cloned and sequenced the putative human PEA3 promoter. Comparison of the mouse and human PEA3 DNAs revealed that sequences required for maximal promoter activity in the mouse were highly conserved in the human gene. Furthermore, these conserved sequences corresponded to a variety of consensus binding sites: 6 Sp1, 8 c-ets-1, 3 PEA3, 3 AP-2, 3 MZF-1, 2 MyoD, 2 Ik-1, 2 c/EBPB, 2 oEF-1/USF, 2 HSFI and one of each of the following: AP-4, Ik-2, SRY, CP2, HEN-I, CREB andE47. / Thesis / Master of Science (MS)

The filamentous cyanobacteria Nostoc punctiforme ATCC 29133 is a model species fordevelopment of sustainable production methods of numerous compounds. One of its uniquefeatures is the anaerobic environment of the strains nitrogen fixing heterocyst cells. To be ableto properly utilize this environment, more knowledge regarding what regulates cell specificexpression is required. In this study, three motifs of the NsiR I promoter of Anabaena sp.PCC 7120 was studied in this system utilizing YFP-fluorescence as a reporter to determinetheir impact on spatial expression pattern. Investigations were performed on immobilizedcells with the use of confocal microscopy and results point towards sigma factor regulation.

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