Taxonomists have regarded the genus Glyceria as a "critical group". In this Thesis the three British species of Section Euglyceria have been studied in order to assess the occurrence of micro-evolutionary differentiation, both at the species level and within the species population. A biosystematics approach was adopted, and the taxonomy, cytology, breeding systems and population structure of the species were investigated. Ecologically each species occurs as a large number of small local populations in paludal places. These are often microgeographically isolated, and some were sampled for study. Before making a biometrical comparison of these samples, developmental studies were undertaken, with emphasis on leaf morphology, and the relation between leaf morphology and inflorescence initiation. The cyto-taxonomy of the species confirmed the existence of three taxa, which intergraded morphologically. Only one chromosome number was found in each of these three species, and the local populations are therefore not chromosomal races. A full study was made of the sterile tetraploid inter-specific hybrid, G, pedicellata. The results shed some light on the evolutionary relationships of the parent species. Biometrical comparison of local population samples showed that these were highly significantly different genetically, and analysis of the breeding systems and population structure of these species gave information about the possible ways in which micro-evolutionary differentiation had occurred. G. declinata (diploid) and G. plicata (tetraploid) are inbreeders, whilst G. fluitans (tetraploid) is and outbreeder, and much more variable, with a wider distribution. The first two species are probably very closely related. The factors influencing the genetic differentiation of the local populations have been analysed. The small inbreeding populations are reproductively isolated by their breeding system. In the outbreeder, gene exchange is limited due to micro-geographical isolation and the occurrence of genetic barriers to crossing between populations. In each species sub-population differentiation has taken place, but only the variable outbreeder G. fluitans appears to have the capacity for progressive evolutionary change.

As a necessary part of the measurement of the time course of Incorporation of a specific precursor of RNA, 14C-uridine, into the various species of cellular RNA a study of the role of the metabolic pool has been carried out. 2-14C-uridine was rapidly incorporated by suspension cultures of Acer pseudoplatanus L. over a wide range of concentrations. A substantial proportion was incorporated specifically into RNA without measurable delay whilst the remainder equil-ibrated with a large pool of phosphorylated compounds within the cell. In addition, as the external concentration of 2-14C-uridine was increased, an increasing proportion of the uridine entering the cells was rapidly degraded with release of 14Co2 from culture. The pattern of labelling of RNA throughout the growth cycle has indicated that a large proportion of the total cellular content of RNA is turned over daring one cell generation time in batch cultures and that there are separate pools for RNA precursor molecules and degradation products. Investigation of the time course of incorporation of 2-14C- uridine into the various species of total RNA, fractionated by sucrose density gradient sedimentation and by gel electrophoresis, in actively dividing cell cultures has revealed the presence of several distinct species of rapidly labelled high molecular weight nuclear RNA which have been tentatively identified as precursors to ribosomal RNA. One of these RNA components has an estimated molecular wight of 3.8 x 106 daltons which is much greater than any yet reported in higher plant tissues. A culture system in which cells can be maintained in synchronous cell division for several full cell cycles has been described. Measurement of the changes in the rates of incorporation of labelled thymidine, uridine and leucine through the cell cycle have shown that they correlate, in general, with changes in the rates of accumulation of total DNA, RNA and protein. An S phase of from 30-35 hours was indicated and there was evidence of peaks of RNA synthesis after mitosis and after cytokinesis.

The European species of Puccinellia Parl. (Gramineae) have been revised in the light of new information derived from, the study of lemma surfaces and a multivariate analysis of a number of quantitative and qualitative characters from selected taxa. These investigations enable conclusions to be made as to the relationships of several of the taxa studied. An account, which includes details of synonymy, descriptions, geographical distributions and chromosome numbers, has been prepared for eventual inclusion in Flora Europaea, 14 species are described and 14 new combinations are required. These new combinations will be validated in a forthcoming paper in the series "Notulae Systematicae ad Floram Europaeam spectantes". A sectional classification is included, where one new sectional name is proposed sect. Pseudosclerochloa Hughes (type: P. rupestris (With.) Fern, et Weath.) The genus is divided into 2 subgenera: subgenus Puccinellia and subgenus Pseudocolpodium (Tzvelev) Hughes. It is concluded that the genera Torreyochloa Church and Phippsia R. Br. should not be included within the limits of Puccinellia.

Pollen of Burley tobacco gave rise to plantlets by anther culture. Not all anthers responded and in those that did the number of plants produced was different. Response varied with different batches of anthers, but this was not a result of the season. Chilling of anthers before culture was not beneficial. Androgenesis was influenced by the stage of pollen development tetrad stages failed completely, and the mitotic-early bicellular stage was optimal. Mature pollen gave rise to plants which were almost all haploid. A dimorphism existed in maturing pollen removed from the plant. At the time starch deposition occurred in normal grains, a small proportion of grains failed to lay down starch or continue increasing in diameter and lost their cytoplasmic staining reaction with aceto-carmine. The size of these S-grains was constant, but their number was different in different anthers, even from the same bud. During culture embryos arose only from S-grains. It is suggested that S-grain formation may be determined during meiosis. The anther wall contributes to the androgenic response. Although ethylene is produced by cultured anthers, neither this gas nor any other appears to be required at levels above those present in air, either in the culture vessels or within the closed anthers themselves. Using an extraction technique involving homogenisation of anthers, few plantlets were obtained from 4 day or 14 day cultured pollen, even using a nurse system involving high density embryogenic carrot cells. Damage to an anther resulted in rapid death of its pollen. The dehiscence lines of anthers could be artificially opened with no effect on androgenesis. Pollen removed by the dehiscence lines gave rise to plantlets on a simple medium with almost the same frequency as intact anthers, but only if a period of 14 days of intact culture was allowed before isolation.

Changes in extracellular peroxidase activity may be mediated by phytochrome as a means of regulating grown rate changes. This was tested in the first internode of light grown mustard seedlings. A correlation between an extracellular anionic peroxidase isoform (A4), extracted by infiltration/centrifugation, was found to decrease in activity by 50% when growth rate was enhanced by a low R:FR ratio. The preparation of protoplasts revealed A4 to be more or less exclusively extracellular. The phytochrome-regulation of A4, apparent in the small percentage of the enzyme that could be extracted by infiltration/centrifugation, was not repeated when the major pool of A4 was examined. A light regulated increase in peroxidase activity was however found in mustard hypocotyls. The light-regulation of cucumber hypocotyl growth was tested and changes in a ionically bound (IB) cationic peroxidase fraction examined. More specifically the use of a long hypocotyl, phytochrome B deficient cucumber mutant (lh), enabled further speculation as to the phytochrome species involved in these changes. When examined, the IB peroxidase activity increased in activity within two hours of the addition of supplementary FR light (a low R:FR ratio), correlating with a change in the rate of growth that could be detected using a mm scale ruler. Whether changes in extracellular peroxidase activity constitute a primary mechanism in the phytochrome-mediation of growth rate changes in light-grown cucumber hypocotyls remained indeterminable. Speculations and possible importance of the observed correlations are discussed. In etiolated cucumber seedlings phytochrome has been shown to control growth within minutes of exposure to light. The extracellular peroxidase activity however, remained unaltered until two days after the commencement of de-etiolation. Thus it was postulated that there appears to be two separate mechanisms in cucumber hypocotyls by which phytochrome-mediated growth rate changes and associated changes in cell wall extensibility are regulated.

The severe forms of listeriosis, have a very high mortality rate. In farm animals, especially sheep, these losses can be of considerable economic importance. The increase in cases of listeriosis in both man and animals over the last decade has stimulated research to develop an effective vaccine to protect against Listeria monocytogenes. However, attempts at protection using killed or chemically attenuated live vaccines have been disappointing. An alternative to these procedures is the development of strains with a defined mechanism of attenuation. Attempts were made to construct aromatic amino acid dependent mutant strains of L. monocytogenes and to investigate their efficacy as a vaccine. Two strategies were used for the transposon mutagenesis of L. monocytogenes. Suicide vectors carrying transposon Tn917 and unable to replicate in Listeria were constructed. To facilitate the transformation of these vectors into Listeria species an efficient electrotransformation system was developed. However, this procedure was unsuccessful in generating Tn917 insertion mutants. Insertional mutagenesis of L. monocytogenes EGD with Tn917 was achieved using a temperature sensitive plasmid. An aromatic amino acid requiring mutant deficient in chorismate mutase activity was isolated. The multiplication of this mutant was found to be unimpaired in both mouse tissues and cultured bone marrow derived macrophages. Organisms isolated from infected tissues were found to be prototrophic while still harbouring a Tn917 insertion. It was concluded that the original mutant carried a point mutation in the gene encoding chorismate mutase and that this had reverted on passage through the mouse. A transposon induced aromatic amino acid dependent mutant of L. monocytogenes found to be deficient in prephenate dehydratase activity was obtained for investigation. The virulence and multiplication of this mutant were reduced in the mouse. Vaccination with this mutant was found to stimulate a protective immune response in mice. The results indicate that aromatic amino acid dependent mutants of L monocytogenes protect against listeric infection and offer a new approach to the development of anti-listerial vaccines.

The dairy yeast Kluyveromyces lactis contains two cytoplasmic linear plasmids pGKL1 (K1) and pGKL2 (K2) which are associated with a killer and immunity phenotype. DNA sequence analyses have revealed that both K1 and K2 contain ORFs (ORFs 1 and 2, respectively) with the potential to encode Class B DNA polymerases (Dpols). This is consistent with a growing body of evidence which indicates that many 'linear plasmids' are dependent for their replication upon self-encoded DNA and RNA polymerases. Attempts to over-express the native Dpol genes of both K1 and K2, in E. coli, were largely unsuccessful. Fragments, ranging from 390 bp to the entire 3 kb gene, were cloned into a variety of expression vectors, but no protein products were observed. The lack of expression arose due to problems at the transcriptional, translational and post-translational level, reflecting the difficulty E. coli probably experiences expressing DNA of such a high A+T content. Experiments using gene fusions revealed a dramatic reduction in the level of product yield when the native K-plasmid DNA was coupled to the highly expressed amino terminal stabilising moiety. In vitro transcription data also revealed that transcription of the native genes appeared to be prematurely terminating, probably due to the occurrence of fortuitous transcriptional terminator sequences in the A+U rich mRNA. A 168 bp fragment of the extreme 5' end of the putative K2 Dpol was chemically resynthesised, incorporating an optimal codon bias for high level expression in E. coli. The gene fragment was designed such that a second section of the gene could easily be added at a later date, doubling the size of the potential product. This fragment was cloned into a fusion vector which directed the expression of heterologous genes as C-terminal fusions with the 27.5 kDa enzyme glutathione S-transferase (GST). Upon induction, strains bearing this plasmid expressed the GST-Dpol fusion protein to over 10% of total cellular protein. The addition of the extra 54 amino acids to the GST was, however, sufficient to render most of the fusion product insoluble. In the absence of a stabilising conjugant peptide, the small resynthesised gene was transcribed, but a protein product failed to accumulate. Peptides corresponding to two potentially antigenic sites, within the N-terminus of the K2 Dpol, were chemically synthesised. The two 8-residue peptides were coupled to tetravalent Multiple Antigen Peptide cores, and were used to immunise chickens. The peptides, in this form, however, failed to elicit an immune response from the recipients. The presence of covalently attached terminal proteins and the cytoplasmic location of these plasmids has severely impaired the ability to manipulate these plasmids. However, homologous recombination in vivo has been developed and used to incorporate selectable markers, to disrupt plasmid-borne genes and to re-introduce modified versions of endogenous genes. An affinity tag, consisting of six adjacent histidine residues was incorporated into the 3' end of the putative K1 Dpol gene. The modified gene was then coupled to a selectable marker and successfully re-introduced into K1. A K. lactis nuclear vector was constructed which expressed an anti sense RNA complementary to the extreme 5' end of the putative K1 Dpol gene. Despite constitutive high level expression of this RNA, the replication of plasmid K1 appeared to be unaffected.

In order to determine the aetiology and epidemiology of gastrointestinal infections in Saudi Arabia; viral, bacterial and parasitic causes of diarrhoea at a major referral centre were examined. Bacterial enteropathogens were found in 7.7% of patients; Salmonella species (51.7%) were found to be the most frequent pathogens followed by Campylobacter jejuni (28%) and Shigella species (14.9%). Clostridium difficile was also found in 9.5% of patients examined but no correlation could be found with presence of faecal leukocytes or pH. Susceptibility patterns of 15,467 isolates of Enterobacteriaceae to 14 antibiotics over 6 years showed that resistance increased in all the Enterobacteriaceae. Imipenem and ciprofloxacin were the only agents to remain active. Protozoan or metazoan parasites were detected in 27.8% of patients examined, the most common being Giardia lamblia and Hymenolopsis nana. Of the patients tested for viruses in stools, 14% had rotavirus, 8.5% adenovirus, 1.5% SRSVs and 0.3% coronavirus. Adenoviruses in stools were detected and serotyped for the first time in Saudi Arabia. Data were correlated with clinical history and serology which showed that immunosuppression was a major factor for onset of gastroenteritis. Type 40/41 were most prevalent followed by types 1,2,3, and 5. Most infections were in children under five years. Astroviruses were detected by PCR and gave an incidence of 1.5%. Rotavirus electropherotypes were determined by polyacrylamide gel electrophoresis and exhibited mostly the long electropherotype characteristic of group A subtype II. Environmental surfaces on a hospital ward were examined over a six month period in which rotavirus was found in 7% of sites tested and equated with areas involving most human activity and occurrence of rotavirus infections in patients. Diagnostic methods such as biotinylated DNA probe and latex agglutination (LA) were evaluated for rapid detection of enteric infections. LA was found to be suitable as a screening method but the DNA probe showed very low sensitivity and specificity for diagnostic use.

Selected Potamogeton taxa were studied to establish the levels of gene flow within species, and to test the identity of putative hybrids. 390 individuals from twelve British populations of the anemophilous aquatic Potamogeton coloratus were analysed using starch gel electrophoresis of isozymes. Low levels of variability were found, with evidence of considerable inbreeding and/or clonal spread. Only two of the populations sampled are polymorphic; both inhabit sites with a long post-glacial history as wetlands. Populations of recent origin, as well as some of older vintage, contain only a single multi-locus isozyme genotype. Evidence for a duplicated IDH locus is presented. A further 647 individuals of Potamogeton coloratus from 60 ditches in the Gordano Valley, Somerset were analysed for variation at two polymorphic PGM loci. High levels of partitioning of genetic variation between ditches was observed with FST=0.575. A population of pondweeds from the River Stour in Dorset intermediate in morphology between Potamogeton natans L. and P. nodosus Poir. is shown by means of isozyme evidence to be the hybrid P. x schreberi G. Fisch. It is represented by a single multi-enzyme phenotype which, together with its sterility, suggests it reproduces vegetatively and may well constitute a single clone. It is genetically distinct from the morphologically similar hybrid between P. lucens L. and P. natans (P. x fluitans Roth). Genetic variation in Potamogeton pectinatus and P. filiformis was studied using isozymes. The overall levels of variability are similar to some other well-studied hydrophilous species, as well as to the average for terrestrial clonal species. Variation is shown to be distributed more between than within populations. Isozyme evidence supports the hypothesis that plants morphologically intermediate between Potamogeton pectinatus and P. filiformis are hybrids of these two species. Variation in enzyme banding patterns suggests that this hybrid has arisen on at least eleven occasions. In a review of molecular population genetic studies of aquatic plants, no significant difference was found between the levels of genetic diversity in aquatic plants compared to that found in clonal terrestrial plants. Many populations of aquatic plants do however show high levels of subdivision between populations.

A historical account of the lichen genus Alectoria Ach. from pre-Linnean to modern authors is given. The anatomy and morphology of the genus as a whole are described and in this the scanning electron microscope gave valuable assistance. The lichen acids of the genus have been investigated, and the taxonomic treatment of chemical races in lichens in general is reviewed and a working hypothesis suggested. A cluster-analysis numerical taxonomic study of 47 taxa of Alectoria and 8 species from allied genera, using both chemical and morphological characters, proved useful in the construction of a supraspecific classification of the genus. Four subgenera, Alectoria, Bryopogon (Link)Massal., Sulcata (Du Rietz)D.Hawksw., and Tortuosa D.Hawksw. are recognised, of which Bryopogon is subdivided into five sections. The relationships of the genus to other genera of lichens are discussed. A detailed account of the nomenclature, variability, ecology, and distribution of the British species shows that the following taxa occur in Britain:- A. bicolor (Ehrh.)Nyl. (incl. f. melaneira (Ach.)Nyl.), A. capillaris (Ach.)Cromb. (incl. f. fuscidula (Arnold)D.Hawksw.), A. chalybeiformis (L.)Gray (incl. f. prostrat-eosteola (Gyeln.)D.Hawksw.), A. fuscescens Gyeln. (incl. f. pallida (Savicz)D.Hawksw., var. canescens Bystrek, var. positiva (Gyeln.)D. Hawksw.), A. lanestris (Ach.)Gyeln., A. nigricans (Ach.)Nyl. (incl. f. subchalybeiformis Ras.), A. nitidula (Th.Pr.)Vain., A. ochroleuca (Hoffm.)Massal. (incl. var. citrina (Ras.)D. Hawksw.), A. pseudofuscescens Gyeln., A. pubescens (L.)Howe (incl. f. subciliata (Nyl.)D.Hawksw.), A. sarmentosa (Ach.) Ach. (incl. subsp. sarmentosa and subsp. vexillifera (Nyl.)D. Hawksw.), A. smithii Du Rietz, and A. subcana (Nyl. ex Stizenb.) Gyeln. A revised check-1ist and key to the British species is presented. Maps of the distribution of the British species within Britain and in the Northern Hemisphere are given, full details of all the British records being included in an appendix. Notes on the typification, characteristics, and idenities of non-British taxa studied in the course of my work are given separately. Reprints of some of my papers which are relevant to this thesis are also enclosed.