Chum Wing Yan Winnie. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 190-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

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Bibliography: leaves 363-381.

"May 2002" Bibliography: leaves 157-176. Through the use of a variety of biochemical and genetic techniques, the importance of the nuclear localisation of PBL was examined, as well as the function of its RadECl and BRCT domains. The RadECl/BRCT domains were found to be required in the cytoplasm for cytokinesis, extending the range of function attributed to these domains. PBL was also shown to shuttle between the nucleus and the cytoplasm, providing an explanation for the observed ability of nuclear PBL to influence cytoplasmic structure.

Mesorhizobium loti possesses a symbiosis island (ICEMlSym[R7A]) that confers upon the bacterium the ability to form a symbiotic association with legumes of the genus Lotus. Nodulation (nod, nol and noe) genes located on the ICEMlSym[R7A] encode enzymes that are responsible for the production of a species-specific signaling molecule, named Nod factor. Perception of Nod factors by plant receptors triggers several plant responses and facilitates bacterial invasion, leading to the formation of root nodules. The studies in this thesis aimed to examine the impact of various structural components of the M. loti Nod factor on host specificity and recognition within Lotus species. The minimal gene requirement for eliciting nodule development on Lotus plants was also determined. The M. loti strain R7A Nod factor has a backbone of five N-acetyl-D-glucosamine (GlcNAc) residues. The non-reducing terminal GlcNAc residue carries an acyl chain of either a vaccenic acid (C[18:1]) or palmitic acid (C[16:0]), a carbamoyl group and a methyl group, while an acetylfucose is present at the reducing terminus. Analysis of loss-of-function [Delta]nodZ and [Delta]nolL mutants showed that the acetylfucose at the reducing terminus was required for efficient nodulation of Lotus species, especially during the initiation of infection threads and for induction of symbiotic gene, NIN. Upon inoculation with R7A[Delta]nodZ, nodulation of Lotus corniculatus and L. filicaulis was significantly delayed and reduced, while only a delay in the onset of nodulation was observed with L. japonicus. Interestingly, nodulation of L. burttii induced by R7A[Delta]nodZ was as efficient as that induced by R7A. Hence, the absolute requirement for the acetylfucose during nodulation was host-dependent. In planta complementation and domain swap experiments using transgenic L. japonicus nfr1 and nfr5 mutants were employed to investigate the role of the reducing terminal acetylfucose in the perception of Nod factor. Nodulation of complemented L. japonicus nfr1 and nfr5 mutants inoculated with R7A[Delta]nodZ was poor, whereas similar plants inoculated with R7A nodulated well. This suggests that the in planta complementation was inefficient and as a result accentuated the effect of the acetylfucose on the Nod factor recognition. The responses of recombinant inbred lines (RILs) derived from a cross between L. filicaulis and L. japonicus to inoculation with strain R7A[Delta]nodZ suggested that at least two genetic loci on chromosome 4, in addition to the Nfr1 and Nfr5 genes, contribute to Nod factor perception and in particular the host-specific recognition of the acetylfucose, This suggests the involvement of multiple receptors or a receptor with multiple components in the perception of Nod factors. A gain-of-function study demonstrated that the presence of nodulation genes alone in nonsymbiotic mesorhizobia was sufficient to induce nodulation and bacteroid formation on Lotus plants, indicating that no other ICEMlSym[R7A] genes were required for infection thread formation or bacterial release. Nodulation assays of four Lotus species indicated host-specific requirements for nodulation genes. The presence of the nodA, nodC, nodD1, nodD2, nodZ, noeL and nolK genes was sufficient to permit nodulation of L. burttii, but was insufficient to induce nodulation of L. japonicus, L. corniculatus and L. filicaulis. The importance of the carbamoyl and methyl groups, and the influence of Nod factor concentration during nodulation were also implicated in this study. A model for the Nod factor perception in Lotus was proposed.

Restriction enzyme, molecular cloning and DNA annealing techniques have been used to study mRNA and DNA coding for the embryonic feather keratins of the chicken and the DNA genomes of coliphages 186 and P2. The coliphage DNAs were used to develop the techniques for application to the keratin system which awaited the availability of appropriate bio - hazard containment facilities before being undertaken. The following results were obtained. 1. Restriction endonuclease cleavage of chick DNA with BamHI, BgïII, EcoRI, or HindIII, fractionation on agarose gels, immobilization on nitrocellulose filters and annealing to DNA complementary to purified 12S mRNA isolated from the developing embryonic feather and coding for embryonic feather keratins, yielded a complex pattern of major and minor bands. These patterns consisted of 4 - 6 major bands and many minor bands. No simple repeat length could be deduced from these patterns, suggesting that keratin - coding DNA is heterogeneous in coding sequences, non - coding sequences or both. 2. Keratin gene expression was shown to be independent of DNA rearrangement, as the complex pattern of restriction fragments was identical in DNA isolated from germ - line tissue ( sperm ) the differentiated feather tissue and somatic tissue not synthesizing keratins ( erythrocytes ). Keratin gene expression must therefore involve the activation of pre - existing control regions in the DNA. 3. The purified 12S mRNA coding for feather keratin was transcribed into double - stranded DNA and individual species isolated by molecular cloning in E. coli. Sequence variation between species was confirmed by restriction enzyme analysis. 4. Preliminary analysis of the cloned species revealed the existence of two distinct groups of species comprising 12S mRNA : Group I ( the more abundant group ) and Group II ( the less abundant ). The fact that filter - bound DNA of individual Group I species bound more 12s cDNA than equal amounts of Group II species DNA and that pure Group I species and total 12S mRNA sequences ( coding for keratins in cell - free translation systems ) annealed to exactly the same complex set of EcoRI, HindIII, or BgïII restricted chick DNA fragments, compels the conclusion that Group I species represent true keratin coding sequences. Group II species annealed to restricted chick DNA fragments which were totally different to those annealing, to either Group I species or total 12S mRNA sequences. Different Group II species appeared to anneal to certain common fragments, suggesting that this less abundant group was comprised of a family of sequence related species and were not simply contaminating mRNA species coding for ' housekeeping ' functions. Their exact nature is at present, however, uncertain. 5. Group I species, the presumptive keratin - coding species, are members of a family of homologous species present in the chick genome. This is demonstrated by the fact that the two Group I species which have been examined so far, shown to be non - identical by restriction analysis, and total 12S mRNA sequences from which they were derived, annealed to the same set of between 20 and 30 BglII, HindIII or EcoRI restricted chick DNA fragments under annealing and washing conditions of low stringency, ( high salt ). Under stringent ( low salt ) washing conditions, however, all except between 1 and 3 of the duplexes formed by these fragments and the Group I species were differentially lost from the filter, indicating that the majority of duplexes were mis - matched and therefore that these multiple copies were homologous and not identical. In addition the two non - identical Group I species annealed to EcoRI generated chick DNA fragments of different sizes under the stringent ( low salt ) washing conditions, demonstrating that differences must exist in the sequence of adjacent non - coding and / or intervening sequences ( should they exist ) for these two species. 6. Although the two Group I species discussed above annealed to different EcoRI generated chick DNA fragments under the stringent ( low salt ) washing conditions, they both annealed under these conditions to a HindIII generated chick DNA fragment of size 3.0 kb. Assuming that this is a single fragment and not two fragments co - electrophoresing by chance, sequences identical to or with very close homology to both of these species lie on the same fragment and are therefore linked in the genome. The exact nature of this linkage and of the extent of gene clustering, should it exist, was not determined. 7. Restriction cleavage maps of coliphages 186 and P2 were determined for the enzymes BamHI, BglII, EcoRI, HindIII, PstI, SaïI, XbaI, and XhoI. These maps were used to analyse four insertion or deletion mutants affecting the major control region of 186. 186ins2 and 186ins3 were shown to be insertions of an IS3 element in the cI. gene and int gene respectively. 186dell and 186del2 were shown to carry the same deletion affecting the cI gene, but 186del2 carried a cryptic insert in the repressor binding site ( operator ). / Thesis (Ph.D.)--Department of Biochemistry, 1979.