<p>Auxin is an important plant growth regulator affecting many aspects of plant growth and development. Maturation is an epigenetic developmental process that affects several processes and traits of economic importance that are regulated by auxin. Among these are elongation growth, wood formation and adventitious rooting. We hypothesized that maturation blocks auxin-regulated pathways that carry out these processes. The objective of this study was to identify genes that are differentially expressed in mature and juvenile loblolly pine shoots after auxin treatment.We used microarray screening to identify auxin-induced genes from loblolly pine. From about 200 putatively differentially expressed ESTs, nine were chosen for further study. Induction levels from the microarray analysis were verified by northern blotting using the same RNA that was used in the microarray probe synthesis. We found good correspondence for seven of the nine clones. However, the induction levels of two clones were underestimated by the microarray. Sequence analysis of the nine clones indicated that they encode proteins that belong to different functional groups and pathways. Three clones encode methionine synthase (MS), serine hydroxymethyl transferase (SHM) and caffeoyl-CoA-methyltransferase (CCoAM). These three enzymes are involved in the pathway leading to production of lignin, supporting the role of auxin in the lignification process. Two other clones show homology to light-induced genes, indicating interaction between auxin and light signaling pathways. One of these clones (9228) is a repressor of plastid rRNA gene, suggesting that auxin may repress photomorphogenesis. Three clones encode proteins previously implicated in root-related developmental processes. One (6ca1) shows homology to a subtilisin protease (AIR3) and another (7cg8) to a proline rich protein (AIR1) from Arabidopsis thaliana. Both Arabidopsis genes are induced by auxin and are expressed during lateral root formation. The third clone (5ng4) showed homology to the MtN21 nodulin from Medicago truncatula that is induced after Rhizobium inoculation. Auxin may play an important role in nodule formation, possibly by triggering a lateral root developmental program that is later modified by the symbiont into nodule formation.We used northern blotting to characterize the expression patterns in mature and juvenile shoots treated with auxin. In addition to the nine clones identified in this study, we screened for differential expression five pine genes (LPEA1-5) that show homology to the Aux/IAA gene family. We found significant expression reduction in mature shoots only for 5ng4. This pine auxin induced nodulin1 (PAIN1) clone was further characterized. The full PAIN1 predicted protein sequence indicates that this is most likely a membrane protein with 10 transmembrane helices, the N- and C-termini in the cytoplasm and a pore-like region separating the two five- transmembrane domains with high sequence conservation in Arabidopsis, Oryza and Medicago. The high degree of conservation indicates essential function in plant growth and development. A large gene family of 38 members was identified in the Arabidopsis genome. The functional significance of this redundancy is most likely associated with functional divergence and/or the need for expression specificity of the different family members. PAIN1 induction is auxin-specific and is restricted to stems, hypocotyls and roots, all three competent to form lateral or adventitious roots. Differential expression of PAIN1 was also found in developing xylem forming top and bottom wood. The localization of a PAIN1:GFP fusion protein at the periphery of tobacco cells is consistent with the transmembrane sequence predictions. The occurrence of the fusion protein in punctate structures that could be part of the Golgi apparatus is consistent with the sequence predictions that the protein is targeted to the secretory pathway. Further determination of the biochemical and developmental function of the genes identified here should increase our understanding of auxin-dependent processes and how they are affected by maturation.<P>

<p><P> The primary focus of this thesis is the study of the regulation of NtPMT3 and NtQPT1 gene expression. Their spatial and temporal expression has been demonstrated to be regulated coordinately. In this work, a 2.05kb of 5' flanking region of NtPMT3 has been cloned from N. tabacum. Then a series of nested 5'-deletion of the NtPMT3 promoter driving a GUS reporter gene were generated. The chimeric reporter gene constructs, as well as a 35S CaMV promoter::GUS were introduced into tobacco N. tabacum cv. D-174 using Agrobacterium-mediated leaf disc transformation. Several independent transgenic lines for each construct were regenerated. Samples of leaf, stem and root were collected from different transformants and GUS activities were quantified by fluorometric assays. These deletion analyses showed that 320 base pairs from the site of transcription initiation is sufficient to direct its root-specific expression. A negative regulatory element that suppressed expression of the reporter gene in roots is located between -2.048kb to -1.632kb 5' of the transcription initiation site. In this 417bp region, six Auxin Response Elements (AuxREs) or AuxRE-like elements were found. Auxin is known to down regulate PMT transcription and this part of sequence might play an important role in its auxin response. Sequential deletion analyses also defined three positive regulatory elements, -1631bp to -1335bp, -601bp to -318bp and -317bp to -116bp. Deletion of these regions resulted in a decrease in their GUS expression. </P><P> Previous studies of the NtQPT1 have identified a Nic gene response element located between -731bp and -1056bp from the transcription initiation site. To isolate the trans-acting factors binding to this sequence, I screened a tobacco root lambda-gt11 cDNA expression library. I obtained 5 cDNA clones encoding three different proteins that bind to the target sequence. DNA sequence of one protein (~790bp) shows high homology to N. tabacum DNA-binding protein of HMG protein family gene (ID AF002226). DNA sequence of the second protein (550bp) is about 99% identical to an A. thaliana small nuclear ribonucleoprotein-like protein gene (ID AY042797). Two cDNAs encoding the third protein are about 2.3kb. 500bp of both 3' and 5' ends were sequenced and showed high homology to a putative jasmonate inducible gene of A. thaliana (ID AY035108). The homology of 3' untranslated region indicates that the N. tabacum root expression library was contaminated by Arabidopsis thaliana cDNA. </P><P>

Thesis (MSc. (Faculty of Health Science))--University of Pretoria, 2002 / Summary in English and Afrikaans. Includes bibliographical references.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--University OF MICHIGAN.

Thesis (Ph. D.)--Middle East Technical University, 2004. / Keywords: sto, Arabidopsis thaliana, Nicotiana tabacum, Transgenic plants, Salt stress, MDA.

Uterine leiomyomata (UL), commonly known as fibroids, are a major public health problem given their extreme prevalence (>70%), severity of associated symptoms, and indication for hysterectomies in women of reproductive age. Familial aggregation and twin studies have provided evidence for a genetic component to predisposition to develop UL. To date, a small number of genes involved in UL biology, including HMGA2, have been discovered through cytogenetic studies of the tumors. HMGA2 is involved in recurrent translocations in UL and a TC repeat polymorphism in the gene is associated with UL diagnosis. In this thesis, I investigate the possible role of the TC repeat in HMGA2 expression. In 293T cells, the TC repeat number did not affect promoter activation, however, in the more relevant UL and myometrial cells, HMGA2 promoter activation was severely impaired and a definitive conclusion could not be made. Genome-wide linkage and association studies provide a promising, unbiased approach for revealing additional regions of the genome associated with UL. In this thesis, I describe results from the first genome-wide linkage and association studies performed in white women affected with UL. A genome-wide linkage study of affected white sister pairs revealed two significant linkage peaks in 10p11 (LOD=4.15) and 3p21 (LOD=3.73) with five suggestive peaks (LOD>2.00) in 2q37, 5p13, 11p15, 12q14, and 17q25. A meta-analysis of genome-wide association results in two independent cohorts of white women revealed a single nucleotide polymorphism (SNP) with genome-wide significance that is associated with UL diagnosis (rs4247357, P=3.05E-08, odds ratio (OR) =1.299). The candidate SNP is located under the UL linkage peak at 17q25 and is in a large block of linkage disequilibrium (LD) which spans three genes: fatty acid synthase (FASN), coiled-coil domain containing 57 (CCDC57) and solute carrier family 16, member 3 (SLC16A3). FAS transcripts and/or protein levels are up-regulated in various neoplasms and have been implicated in tumor cell survival. By tissue microarray immunohistochemistry, we found FAS protein expression elevated three-fold in UL when compared to matched myometrial tissue implicating FASN as the first UL risk allele identified in white women by a genome-wide, unbiased approach.

To understand how the nervous system processes information, a map of the connections among neurons is essential. Viral transsynaptic transmission has gained popularity as a method for labeling neural circuits. In particular, the development of retrograde monosynaptic tracing vectors has enabled visualization of the pre-synaptic inputs onto defined sets of postsynaptic neurons. This system utilized the rabies virus (RABV), in which the glycoprotein gene in the virus was deleted, and re-supplied in trans. In order to build alternative, more flexible tracers, we made recombinant VSV genomes, first developing the use of vesicular stomatitis virus (VSV) for tracing neuronal connections. Viruses encoding several different fluorescent proteins were made, giving brilliantly labeled neurons, bright enough for live imaging and characterization of the detailed morphologies of cells. Expression was very rapid, facilitating identification of neurons both in vivo and in ex vivo applications. In addition, the use of an avian glycoprotein (ASLV-A) allowed specific targeting to cells expressing an avian glycoprotein receptor (TVA). This allowed monosynaptic tracing from defined starter cells. In order to alter the direction and cell type specificity of transmission, we then fitted VSV with a glycoprotein from one of multiple other viruses. Glycoproteins such as the rabies virus glycoprotein (RABV-G) endowed VSV with the ability to spread in a retrograde transsynaptic pattern, while the glycoproteins from viruses such as the lymphocytic choriomeningitis virus (LCMV) gave an anterograde pattern of transsynaptic spread. This anterograde or retrograde spread was observed in all species tested, and even for other non-VSV viruses, such as lentiviruses. We also developed transsynaptic tracing viruses which direct viral spread between defined cell types, instead of from a defined cell type to any upstream of downstream cell. In all, we developed an extensive transsynaptic tracing repertoire for tracing neuronal connections.