Synthesis

Transcriptional regulation of genes is vital to cell success making it an important aspect of research. Transcriptional regulation can occur in many ways; transcription factors bind to the promoter region and block transcription, disrupt an activator protein, or interact with histones to lead to higher order chromatin. Plant HomeoDomain can recognize and bind to different methylation states of histone tails. PHD proteins use other functional regions to carry out functions. Two associated domains having DNA-binding capacity were characterized in this study; the ARID domains of JARID1A and JARID1C and the DDT domains of BAZ1A, BAZ1B and BAZ2A. These genes are important because of their roles in various diseases such as cancer. The consensus sequences for BAZ1A-DDT is GGACGGRnnGG, GnGAGRGCRnnGGnG, RAGGGGGRnG and CRYCGGT. Consensus sequences for BAZ1B-DDT were CGnCCAnCTTnTGGG and YGCCCCTCCCCnR. Consensus sequences for BAZ2A-DDT were TACnnAGCnY and CnnCCRGCnRTGnYY. Consensus sequence for JARID1A-ARID was GnYnGCGYRCYnCnG. Consensus sequences for JARID1C-ARID was RGGRGCCRGGY.

Longitudinals lacking gene (LOLA) is a transcription factor that is involved in a variety of axon guidance decisions in Drosophila melanogaster nervous system. Besides having a role as an epigenetic silencer and in the programmed cell death in Drosophila's ovary, this gene is also an example of complex transcription unit. LOLA is a transcription repressor and can generate 17 DNA - binding isoforms, through alternative splicing, each containing distinct zinc-finger proteins. This unique DNAbinding binding sequence to which LOLA-ZFP binds has been determined for four of the lola isoforms F, J, P and K. Also, bioinformatics' tool approach has been taken to identify the target genes that are regulated by these four LOLA splice variants. Future work will be done for the five other LOLA isoforms to categorize their putative DNA-binding sequences and subsequently their protein interactions.

There are approximately 20,000 genes in the human genome. Around 2% of these genes code for transcriptional repressors known as KRAB-ZFPs. It is already known that Zinc-Finger Proteins contain two main functional domains at either end of the polypeptide. In today's database, you will find a KRAB (Kruppell-associated Box) domain at one end and a tandem array of Zinc-finger repeats at the other end. The carboxyl terminal tandem Zinc-finger repeats function as sequence-specific DNA-binding domains. The amino terminal KRAB domain serves as a repressor domain, which will recruit a co-repressor termed KAP-1 (KRAB Associated Protein-1). Located in between these two domains is a region of uncharacterized DNA referred to as the "Linker Region". This thesis will explore the DNA-binding domains of 6 known KRAB-ZFPs, as well as utilize the linker regions to derive an evolutionary history for this superfamily.

The conversion of alkynyl carbonyls to allenyl carbonyls via manganese mediated coordination followed by a base-catalyzed isomerization was carried out using a range of chiral and achiral amine bases. Chiral amidine and chiral DBU derivatives were synthesized to carry out the isomerization enantioselectively. We employed HPLC equipped with a chiral column to determine the enantiomeric excess. We also proved that the mechanism of that the manganese-coordinated alkyne/allene rearrangement reaction involved an intermediate cumenolate. It was also confirmed that amine base with pKa lower than that of DBU (pKa = 13.6) would not carry out the isomerization. Alkoxy base were also used in isomerization and the mechanism was also investigated.

The zinc finger associated domain (ZAD) containing family of transcription factors is not well described in the literature, in part because it is very difficult to study by mutagenesis. We used in vitro-binding techniques to identify characteristics of the ZAD family, by constructing glutathione Stransferase (GST)-ZAD domain chimeric proteins for use in protein binding assays, and GST-Zinc finger array domain chimera for binding site selections. Protein binding assays indicated a possible shared cofactor, as seen in the analogous KRAB system in mammals. DNA binding assays have provided a consensus binding sequence for five of the ZAD proteins, consistent with previously reported work on ZAD and unpublished work on mammalian transcription factors. Research is ongoing with an additional ~50 ZAD proteins to more fully map the binding characters of ZAD proteins.

Carbohydrate Active Enzyme family 6 (CA6) glycosyltransferases (GTs) are type II transmembrane proteins localized in the Golgi apparatus. CA6 GTs have a GT-A fold, a type of structure that resembles the Rossman fold and catalyze the transfer either galactose (Gal) or N-acetylgalactosamine (GalNAc) from the UDP nucleotide sugar to an non-reducing terminal Gal or GalNAc on an acceptor via an a-1,3 linkage. In this reaction, the anomeric configuration of the sugar moiety of the donor is retained in the product. CA6 GTs includes the histo-blood group A and B GTs, a-galactosyltransferase (a3GT), Forssman glycolipid synthase (FS), isogloboside 3 synthase (iGb3) in mammals. a3GT and its products (a-Gal epitode) are present in most mammals but are absent in humans and old world primates because of inactivating mutations. The absence of a3GT and its products results in the production of anti-a-Gal epitope natural antibodies in these species.

Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions.

RNA damage occurring under oxidative stress has been shown to cause RNA dysfunction and must be detrimental to cells and organisms. We propose that damaged RNA can be removed by specific RNA surveillance activities. In this work, we investigated the role of polynucleotide phosphorylase (PNPase), a 3'->5' exoribonuclease, in protecting the cells against oxidative stress and eliminating oxidatively-damaged RNA. Previously, it was reported that E. coli PNPase has a higher affinity to poly(8-oxoG:A). We further confirmed that E. coli PNPase can specifically bind to an oxidized RNA with a high affinity. An E. coli strain deficient in PNPase (pnp) is hypersensitive to hydrogen peroxide (H2O2). Importantly, the level of H2O2-induced RNA damage, measured by the content of 8-hydroxyguanosine, increases significantly in the pnp mutant cells. Consistent with the notion that PNPase plays a direct role in these processes, introduction of the pnp gene encoding E. coli PNPase can restore the viability and RNA oxidation level of the pnp mutant cells in response to H2O2 treatment. Interestingly, degradosome-association is not required for PNPase to protect cell against oxidative stress. PNPase is evolutionary conserved in most of organisms of all domains of life. The human polynucleotide phosphorylase (hPNPase) localizes mainly in mitochondria and plays pleiotropic roles in cell differentiation and has been previously shown to bind 8- oxoG-RNA with a high affinity. Here we show that similar to E. coli PNPase, hPNPase plays an indispensable role in protecting HeLa cells against oxidative stress. The viability in HeLa cell and 8-oxoG levels in RNA are inversely correlated in response to H2O2- treatment. After removal of oxidative challenge, the elevated level of 8-oxoG in RNA decreases, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA.