The basic helix-loop-helix (bHbH) family-of transcription factors is important in many developmental and regulatory pathways such as cellular proliferation and differentiation, lineage commitment, sex determination, neurogenesis, myogenesis, hematopoeisis and pancreatic development. The free-living nematode Caenorhabdits elegans is an important model organism. Genetics studies of a gene in nematodes help us to better understand the functioning of hornologs in more complex organisms. These studies investigate the nematode homolog of ABF-1, CeABF-1, and its potential role in the development of C. elegans. The BLAST Database (http://www.ncbi.nlm.nih.gov) predicted a bHLH protein in C. elegans, located on cosmid ZK682.4, of 170 amino acids with an overall 51% similarity to human ABF-1. Importantly, it had a 72% similarity to human ABF-1 within the bHLH domain. Genomic and eDNA clones of CeABF-1 were isolated using whole nematodes and PCR methods. Clones were constructed that would allow us to use green fluorescent protein (GFP) to localize CeABF-1 expression. We also used RNA interference to determine the function of CeABF-1. No obvious phenotype was observed in nematodes unable to produce the CeABF-1 protein. Preliminary studies suggest that CeABF-1 is not an essential gene for development in C. elegans. Constructs were also made for protein induction and antibody studies. Further studies are necessary to determine how CeABF-1 is involved in nematode development and its interaction with other proteins.

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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

The structure and expression of a novel Euglena gracilis chloroplast ribosomal protein operon was studied by gene mapping, molecular cloning, nucleotide sequencing primer extension and Northern analyses. The nucleotide sequence (12,240 bp) was determined for 100% of both strands encoding the 12 genes, rpl23 - rpl2 - rps19 - rpl22 - rps3-(2.8 kb region)- rpl16 - rpl14 - rpl5 - rps8 - rpl36 - trnI - rps14. The gene organization resembles the S10 and spc ribosomal protein operons of E. coli. The rpl5 gene was a new chloroplast gene not previously reported for any chloroplast genome nor described as a nuclear gene. The presence of numerous introns and an unusual 2.8 kb rps3-rpl16 intercistronic region were additional features that were unparalleled in other chloroplast DNAs. At least 15 introns were identified in the genes. Evidence is presented from primer extension analysis of chloroplast RNA for the correct in vivo splicing of six of the introns. Two introns within rps8 flanked an 8 bp exon, the smallest exon yet characterized in a chloroplast genome. Four introns shared structural properties with group II organelle introns. The remaining 11 introns were defined as new category of organelle intron, now designated "group III." The presence of additional introns in several intercistronic regions is proposed. Conserved regions in the predicted polypeptides were identified from the alignments with related proteins from other chloroplasts and bacteria. Evidence from Northern hybridization experiments with gene-specific probes supported the interpretation that 11 ribosomal protein genes, the 2.8 kb rps3-rpl16 intercistronic region and trnI were co-transcribed and encoded in a single operon. The co-transcription of genes coding for proteins and a tRNA is a novel finding for a chloroplast operon. Several stable polycistronic transcripts were identified, including a common 8.3 kb pre-mRNA. Stepwise processing pathways proposed for the mRNAs are described. Most mRNAs appeared to be fully spliced. The 5$\sp\prime$ ends of mRNAs for the first gene in the operon, rpl23, were mapped by primer extension. Plastid mRNAs from dark and light grown Euglena were analyzed on Northern blots.

Induction of vertebrate heart development requires inhibition of canonical/<i>β</i>-catenin dependent Wnt signalling, activation of non-canonical/<i>β</i>-catenin independent Wnt signalling and transcription factor activation. Wnt/<i>β</i>-catenin signalling may also have a later regulatory role in cardiogenesis. The recent discovery of Wnt6 expression next to and within the developing heart during the relevant stages of cardiomyogenesis, combined with knockdown and over-expression data suggests that Wnt6 may have a role in the regulation of this process. Inhibition of canonical signalling leads to increased expression of cardiac associated transcription factors such as members of the Nkx2 and GATA family. These families are expressed in overlapping regions which specify the early heart field prior to the expression of the later cardiomyocyte-specific genes. This study demonstrates the ability of <i>β</i>-catenin to inhibit cardiogenesis during later developmental stages, before the cardiac mesoderm begins to differentiate into myocardium (heart muscle) and that the newly discovered Wnt6 exerts inhibition of cardiogenesis in a <i>β-</i>catenin<i> </i>dependent manner. This inhibition of cardiogenesis by <i>β-</i>catenin can occur in a cell-autonomous manner and is a result of direct inhibition of cardiac transcription factors of the GATA family. Over-expression of these pro-cardiogenic transcription factors GATA4 and GATA6 can restore the cardiomyogenic differentiation programme in embryos where it has previously been inhibited by <i>β</i>-catenin. In conclusion GATA factors are the relevant targets of Wnt/<i>β-</i>catenin signalling in the inhibition of normal cardiac development. The subsequent loss of cardiac gene expression observed is therefore a result of insufficient GATA expression and function.

The Nrd1-Nab3-Sen1 (NNS) complex carries out the RNA polymerase II (Pol II) transcription termination of non-coding RNAs (ncRNAs) in yeast, although the detailed interactions among its subunits remain obscure. In this dissertation, we have identified three sequence motifs in Sen1 that mediate direct interactions with the Pol II CTD interaction domain (CID) of Nrd1, determined the crystal structures of these Nrd1 interaction motifs (NIMs) bound to the CID, which elucidated the molecular basis for their recognition by Nrd1 CID, and characterized the interactions in vitro and in yeast. Although the Sen1 NIMs are not essential for supporting viability from the in vivo studies, termination defects were observed from NIM deletions in a reporter assay. In addition, the conservation of Sen1 NIMs suggests these interactions are very likely to promote NNS function. This dissertation also describes the structural studies of the flowering time control protein FPA in plants, which regulates the alternative 3’-end processing of the FLOWERING LOCUS C (FLC) antisense RNA. FPA belongs to the split ends (SPEN) family of proteins, which contain N-terminal RNA recognition motifs (RRMs) and a SPEN paralog and ortholog C-terminal (SPOC) domain. The SPOC domain is highly conserved among FPA homologs in plants, but the conservation with the domain in other SPEN proteins is much lower. We have determined the crystal structure of Arabidopsis thaliana FPA SPOC domain at 2.7 Å resolution. Structural and sequence analyses identify a surface patch that is conserved among plant FPA homologs. Mutations of two residues in this surface patch did not disrupt FPA functions, suggesting that either the SPOC domain is not required for the role of FPA in regulating RNA 3’-end formation or the functions of the FPA SPOC domain cannot be disrupted by this combination of mutations.