Methods for the isolation of pure, intact <i>Physarum</i> polysomal RNA have been employed to obtain polyadenylated RNA. <i>Physarum</i> polyadenylated RNA isolated by these methods has been demonstrated to be suitable for use as a template for the synthesis of <i>Physarum</i> single-stranded cDNA. These molecules ranged in size from 100--2000 nucleotides. The enzyme steps involved in the synthesis and cloning of double-stranded cDNA were investigated individually to determine the optimum reaction conditions. A globin mRNA template was employed to analyse conditions for single and double-stranded cDNA synthesis. It was demonstrated that synthesis of the second cDNA strand, S1 Nuclease treatment, and synthesis of homopolymer tracts are inefficient and variable features of the standard cDNA protocols employed at the time of the study. These difficulties have been reported by other workers, and it has been suggested as a result that alternative methods of cDNA synthesis and cloning should be employed. Several cDNA clones, obtained from a limited <i>Physarum</i> cDNA library, were analysed in detail. One, pPCF2, was demonstrated to hybridize to methylated, repetitive <i>Physarum</i> DNA by Southern hybridization. Therefore in this instance, methylation of CpG sequences, which has been implicated in the negative regulation of gene expression, did not inhibit transcription of DNA containing these sequences, as has been proposed. The DNA sequence which hybridized to pPCF2 contained internal, methylated sequences, and was not methylated at the flanking restriction endonuclease recognition sites. This is consistent with observations that methylation at the 5' promoter region of the gene, inhibits expression of that gene, while methylation of the structural region does not. Another <i>Physarum</i> cDNA clone, pPCF3, was demonstrated to hybridize exclusively to undermethylated, repetitive <i>Physarum</i> DNA sequences. A hypermethylated, highly repetitive, cloned <i>Physarum</i> DNA sequence, pPH29, was shown to hybridize to cytoplasmic and polysomal RNA. Recently, pPH29 has been identified as the middle sequence of a putative transposon-like element. Such elements are believed to transpose via an RNA intermediate, which is reverse-transcribed into cDNA. The demonstration that pPH29 hybridizes to <i>Physarum</i> RNA provides further evidence, in conjunction with structural information, that it may form part of a transposable element.

No description available.

This study has succeeded in identifying, cloning and sequencing three genes previously unknown in R. sphaeroides, che W, che R and che Y2. These genes are homologous to genes recently discovered in Rhizobium meliloti and also show a lesser homology to genes in other organisms including the well characterised enteric chemotaxis genes. A comparative analysis of the deduced proteins has been made to determine structural and functional similarities between the R. sphaeroides genes and their homologues in R. meliloti and in E. coli. Considerable conservation of functionally important amino acid residues was revealed. In vivo complementation was done using the R. sphaeroides Che W protein to complement Che W^- strain of E. coli. The R. sphaeroides protein complemented the E. coli strain empirically proving similarity of function of the protein between these widely divergent genera. Homologous recombination using transposon-interrupted genes and internal gene fragments was attempted and met with limited success because R. sphaeroides proved permissive to the suicide vectors used. The most significant result of this study has been not in the similarities revealed between the chemotaxis systems of R. sphaeroides and the enterics, but in the differences discovered. It is now known that R. sphaeroides possesses two Che Y proteins, Che Y and Che Y2, and that whereas the R. sphaeroides Che Y2 protein is closely related to the E. coli Che Y protein, the R. sphaeroides Che Y protein shows considerable evolutionary divergence from it's enteric homologue. The significance of a second copy of Che Y protein suggests that these two response regulators act independently at the motor/switch complex and that they represent the final elements of two functionally distinct chemotactic sensory transduction pathways. This dual pathway system is not present in the enteric bacteria (a member of the γ-group of proteobacteria) but, we propose, may be present in a large group of environmentally important bacteria, the α-group of proteobacteria. Caulobacter, Agrobacterium, Rhizobium and Azospirillum species (α-group proteobacteria) all show behavioural similarities, and there are genetic clues to suggest that these organisms possess a dual chemotactic sensory transduction similar to the one we have found in R. sphaeroides. Apart from being fundamentally important, the dual sensory pathway hypothesis explains the wild-type behaviour of R. sphaeroides, as well as the difficulty in obtaining behavioural mutant phenotypes using methods developed during the investigation of E. coli.

No description available.

No description available.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Thesis (M.S.)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Andreas M. Fisher. Includes bibliographical references (leaves 45-48).

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 6 uppsatser.

Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2002? / Bibliography: leaves 97-114.