Tissue cultures of Acer pseudoplatanus L. and Atropa belladonna cultivar Lutea Doll have been used as experimental material for ultrastructural and growth studies. Cytoplasmic changes accompanying the growth of batch propagated Acer suspensions in synthetic medium are described, special attention being given to the structure of cell walls, mitochondria, plastids, and microbodies. The structural changes accompanying transfer of cells to liquid medium containing elevated sucrose concentrations, and/or enhanced levels of growth hormones are described. A study has been made of the general ultrastructural features of chlorophyllous Atropa root callus cells at the time of sub-culture, during division, and when senescent. By plating of suspensions of root callus, variant clones were isolated which differed in their morphology and pigmentation, and these differences were retained when the variants were propagated on semi-solidified medium and in suspension. Differences in chlorophyll and carotenoid biogenesis were apparent between the variants following exposure of the dark-grown callus cultures to light. The accumulation of pigments was correlated with chloroplast ontogeny. Investigations of the photosynthetic and autotrophic capabilities of green callus, together with the effect of auxins on growth and pigmentation of suspension cultures are reported; the latter experiments were aimed at enhancing chlorophyll production in cultured cells as a step towards establishing autotrophic cultures. The variant clones differed in their rate of cell division and the maximum cell density attained in suspension. The minimum inoculum density for sustained growth of these cultures was higher on agar than in liquid medium. The clones showed pH optima for growth at low densities. The differing responses of the cultures to "conditioned" medium suggested nutritional differences between the clones. Suspension cultures of green and ''hyaline" clones became aggregated on transfer to medium lacking auxin, or with a low auxin level. This aggregation was accompanied by expression of the totipotency of these cells by organogenesis, even though the clones had previously been propagated for a prolonged period in liquid medium. Cellular aggregates contained meristematic regions overlying highly vacuolated cells, and primordia arising from these meristematic regions differentiated into roots of normal structural organisation.

Little is known about the digestive processes in insects which feed on dung. They are of interest because the main components of the dung are cellulose and lignin and these substances are extremely difficult to digest. In this research the larvae and adults of Typhaeus typhoeus L. and the larvae of Oryctes boas Fabr. were studied. The composition of the food of the insects was investigated. The gut and mouthparts were examined to find how they are adapted for coprophagy. The micro-organisms in the gut were studied and compared in type and numbers with those in the food. The enzymes in the gut were investigated. Experiments were carried out to find what substances are removed from the food in the gut, and how long the passage of food takes. The food of Typhaeus typhoeus larvae contains a particularly high percentage of cellulose and lignin but the larvae cannot digest these substances. They feed by digesting micro-organisms which live in the dung. Bacteria are cultured in the hindgut sacs and provide an additional source of food. Some cellulose is digested in the gut by bacteria, and the digestion products are absorbed by the larvae. The food of Typhaeus typhoeus adults contains a relatively high percentage of substances other than cellulose and lignin and the insects produce enzymes which digest these. They can digest micro-organisms but are less dependent on them than the larvae. Oryctes boas larvae are found feeding on many different, substances. They utilise the easily digested substances in their food, if these are present, but they can also digest cellulose. It is uncertain whether cellulase is produced by the insect or by symbionts which have not been detected. The larvae digest bacteria which are cultured in the hindgut; these bacteria digest a little cellulose and hemicellulose and the larva absorbs the fatty acids produced.

Investigation of contemporary pollen recruitment to lakes can help to elucidate the fossil record in lake sediments. In the English Lake District, pollen recruitment was studied at Blelham Tarn (area 10.2 hectares), into which two experimental tubes have been introduced. Each isolates a water column (45 m diameter X 11-12 m deep). Estimates were made of pollen deposited per unit area per unit time from the air (in traps on land and floating inside the tubes), and within the lake (at the mud surface and in traps submerged both inside and outside the tubes to catch seston). Samples from inflows were also analysed for pollen. Results indicated that: (i) Airborne pollen deposition varied in intensity, (a) seasonally, and (b) between years, with the magnitude of annual pollen production. (ii) Floating traps underestimated (by c. X 2) the actual intensity of pollen deposition on the water. (iii) Most pollen disseminated was deposited near the parent plants. Hence terrestrial surfaces form a reservoir from which pollen is recruited by surface runoff. (iv) Catches in the submerged traps varied seasonally with the relative recruitment of (a) fresh airborne pollen, and (b) pollen resuspended from the sediment surface by turbulence. (v) Delivery of streamborne pollen to the lake varied non-seasonally with rainfall/stream discharge. (vi) Ratios for pollen: seston trapped varied seasonally with plankton production, (vii) Pollen taxa were not dispersed with equal efficiency in air: poorly-dispersed taxa were recruited mainly to the lake by runoff. The effects are considered of recruitment processes and of limnological variables upon the following: (a) the spatial distribution of pollen over the mud surface; (b) the relationship between the pollen composition of lake sediment and vegetation composition around Blelham Tarn. Factors which apparently determine the recruitment of pollen to lake sediment today are discussed generally, and the possible effects of these factors upon the fossil record (pollen percentages and absolute pollen deposition rates) are considered.

Immature pollen of Nicotiana tabacum cv Wisconsin 38 and cv Burley, isolated at stages from the late uninucleate to early bicellular, was successfully cultured to give embryoids and green plantlets. This is the first report of successful culture of tobacco pollen isolated direct from the plant. The method represents a substantial advance on existing methods which relied on both prior treatment of the excised buds and a short period of anther culture, to induce the pollen into embryogenesis, The pollen was isolated and pretreated for about 7 days in an appropriate medium. For bicellular pollen, from which the largest number of embryoids was obtained, this medium was simply water; for uninucleate pollen, which required a higher osmotic pressure for survival, it was a solution of inorganic salts. After the pretreatment, nutrient medium with sucrose was added, upon which embryoids and plantlets formed. Not more than 6% of the pollen grains developed into microscopic embryoids. Still fewer survived to become plantlets. Attempts to induce more pollen grains into embryogenesis by means of growth substances and other empirical modifications of the pretreatment medium were unsuccessful. Plantlet yields were increased by increasing nutrient availability after the pretreatment. The low fraction of pollen grains induced into embryogenesis was consistent with findings of previous workers which suggested that embryogenesis is predetermined by events that precede culture. Isolation of the pollen into the complete nutrient medium instead of into water was deleterious to embryogenesis. This contrasts markedly with the situation in anther culture in which high yields of pollen plantlets can be obtained by supplying the complete medium at inoculation. The deleterious effect of sucrose and other organic compounds on embryogenesis in the case of isolated pollen suggests that in anther culture the anther tissues act as a screening agent preventing access of the deleterious compounds to the pollen during the initial induction period. Attempts to culture isolated pollen of N. sylvestris were less successful. A few microscopic embryoids were obtained, but none developed into plantlets. Isolated pollen of N. sylvestris may have more stringent nutritional and osmotic requirements than that of N. tabacum. Changes in the anatomy of N. sylvestris anthers during culture were investigated. They could not be correlated with the potential for embryogenesis. Details of anther and pollen development were, studied in Hevea brasiliensis as a preliminary to anther and pollen culture studies. Anthers or staminal columns bearing many anthers were cultured, but multicellular embryoids were not observed in any of the treatment tested. Time did not permit for the findings on the culture of isolated tobacco pollen to be tested in any detail in Hevea.

The uropathogenic Escherichia coli strain 20025 (04:K12:H-) elaborates at least four fimbrial antigens, Fy (F14), F12-rel, F13, F1C and secretes the toxin a-haemolysin. The work presented in this thesis demonstrates that the gene clusters encoding these five determinants are located within a 60-70kb region of the 20025 chromosome. The F14 fimbrial antigen produced by 20025 is a novel P-fimbriae serotype and exhibits a different binding specificity to the majority of P-fimbriae. To determine the basis of this apparent difference in cell surface receptor specificty a detailed molecular analysis of the F14 gene cluster is presented. The cloning and analysis of two other Escherichia coli surface antigens, the non-fimbrial adhesin NFA-1 and the K12 capsular polysaccharide is also described.

Herpes simplex virus type 1 glycoprotein H is a minor viral constituent which has been implicated in viral mechanisms of cell entry and exit. Study of this molecule has been hindered by a relative lack of specific immunological reagents. This study reports the generation and characterisation of specific polyclonal antibodies. The purification of gH and the generation of polyclonal serum is described. Antigenic determinants reactive with polyclonal antibodies were determined using synthetic peptides derived from the primary amino acid sequence of the molecule and a similar aproach was applied using available monoclonal antibodies. A series of 833 hexapeptides, with five residue overlap, were synthesised representing all potential continuous epitopes comprised of six residues or less. Peptides, displaying structural homology with epitopes of the native molecule were identified by screening for antibody binding. To further validate the identity of epitopes, corresponding homologous peptides were conjugated to carrier molecules and used to generate anti-peptide antibodies. The biological and immunological properties of the resulting anti-peptide antibodies were determined. Three antipeptide antibodies appeared reactive with the glycoprotein either following western transfer or in immunoprecipitation confirming the identity of gH-1 epitopes.

No description available.

Phytochrome, a R/FR reversible photoreceptor that regulates plant photomorphogenesis, is encoded by a gene family of which there are at least five members. The best characterised member is phytochrome A which accumulates to high levels in etiolated tissue but is degraded in light, unlike other members of the family which are light-stable. A two-stage screen was designed for isolation of phyA mutant seedlings. The mutant phenotype was illustrated by deficiency in FR-HIR-mediated inhibition of hypocotyl elongation, which is exclusively mediated by phytochrome A while retaining R-HIR-mediated inhibition of hypocotyl elongation, which is mediated by one or more of the light-stable phytochrome species. Mutants isolated using this screen were divided into three complementation classes, fhy1, fhy2 and fhy3. Immunoblot and spectrophotometric analyses of these three mutant lines demonstrated that one of them, fhy2, was deficient in phytochrome A protein. Southern analysis of several alleles of the fhy2 mutation indicated that deficiency in phytochrome A resulted from structural alterations in the PHYA gene. Subsequently, the fhy2 mutants were designated as phyA mutants. The other two classes of long hypocotyl mutants isolated in this screen, fhy1 and fhy3, showed no structural alteration in the PHYA gene and had normal immunochemically and spectrophotometrically detectable levels of phytochrome A. Therefore, fhy1 and fhy3 are putative transduction chain mutants. Comparison of the photophysiology of phytochrome A mutants with that of wild-type seedlings allowed conclusions to be drawn about the roles of phytochrome A in selected, assayable photomorphogenetic responses. phyA mutant seeds exhibit wild-type germination responses to R but do not germinate in FR, suggesting that phytochrome A mediates a promotory response to FR. The importance of phytochrome A as the principal photoreceptor in FR is extended to de-etiolated plants where phytochrome A retains an inhibitory role in hypocotyl elongation, under supplementary FR or low R:FR ratio conditions. Phytochrome A also plays a role in flowering which becomes apparent under low fluence-rate incandescent day extensions, suggesting that in fluence-rate limiting conditions, phytochrome A is an important component of daylength perception. fhy1 and fhy3 mutants, selected for long hypocotyls in FR alongside phyA mutants, were also characterised and shown to have wild-type levels of phytochrome A protein with no apparent rearrangement of the PHYA gene. The mutation at FHY1 affects hypocotyl elongation but not germination, de-etiolation or flowering, suggesting that hypocotyl elongation is on a different branch of the transduction chain to these other responses and that FHY1 is a component of that branch. Hypocotyl elongation of fhy3 seedlings is affected in R, as well as in FR which may suggest that the transduction chain component encoded by FHY3 is shared by the phytochrome, or phytochromes, that control hypocotyl elongation in R. Characterisation of phyA and fhy mutants of Arabidopsis has led to elucidation of the roles of phytochrome A in different photomorphogenetic responses and some preliminary investigation of components of the transduction chain. This reseach has also further clarified the roles of other phytochromes and the extent of overlap of the spheres of action of distinct molecular species of phytochrome.

Meningococcal meningitis is one of the most serious infectious diseases, with high mortality rates in spite of antibiotic therapy. Although polysaccharide-based vaccines have been developed to Neisseria meningitidis (meningococcus) groups A, C, W135 and Y, there is, as yet, no effective vaccine against serogroup B meningococcus. The capsular polysaccharides of Neisseria meningitidis group B and Escherichia coli K1 are identical and the pathways of biosynthesis similar. Molecular genetic analysis, performed prior to any published data, has revealed the organisation of the genes involved in biosynthesis and polymerisation of alpha2,8- linked N-acetylneuraminic acid in N. meningitidis group B. The biosynthetic region contains four genes, three of which appear to be organised in an operon. Northern blot analysis revealed a 2.85kb transcript which correlates well with the size of the predicted transcript for the operon. The proteins encoded by the four genes are similar to the products of the neuA, neuB, neuC and neuS genes of the E. coli K1 capsular polysaccharide gene cluster. In contrast to previous studies (Echarti et al., 1983), Southern hybridisation analysis demonstrated similarity between the cloned meningococcal group B capsule genes and the cloned E. coli K1 capsular polysaccharide genes. The DNA and predicted protein similarities substantiate the theory of the common evolutionary origin of encapsulation in these two unrelated bacteria. Additional novel findings are the demonstration of a similar biosynthetic pathway of capsule production in all sialic acid-encapsulated meningococcal serogroups, and a common mechanism of transport. Studies on the molecular organisation and control of capsule gene expression are fundamental to the successful development of a meningococcus group B vaccine, or to the progression of alternative therapeutic approaches for treatment of infections by sialic acid-encapsulated organisms.

The importance of salicylic acid (SA) in both local resistance to pathogens and the subsequent establishment of systemic acquired resistance has been investigated in tobacco. In order to assess the role of SA in the plant defense response, it was decided to try to stop the accumulation of SA by expression of an enzyme able to degrade it. A gene encoding salicylate hydroxylase (SH-L) was cloned from Pseudomonas putida and shown to be functional in E. coli. This gene was fused to various plant promoters which should allow temporal and spatial alteration of salicylate accumulation in pathogen-challenged tobacco. The promoters chosen included the CaMV35S promoter which allowed constitutive expression of SH-L and thus total inhibition of SA accumulation. An AoPR1 promoter was used to inhibit the early accumulation of SA around developing lesions. The tobacco acidic PR1a gene promoter was used to drive SH-L expression in response to endogenous SA accumulation both locally and systemically following localised pathogen attack. Two pathogen systems, one viral, tobacco mosaic virus (TMV) and one bacterial. Pseudomonas syringae, were used to analyse how their interactions with tobacco were altered in various SH-L backgrounds by examining lesion phenotypes, defence gene expression and SA levels. It was found that local PR protein (at least PR1a) induction is dependent on salicylate-mediated signalling and that SA is absolutely required in the development of HR to limit virus spread and kill bacteria. It was also confirmed that SA is required for the establishment of systemic acquired resistance (SAR). A recent hypothesis on the mechanism of action of SA is that SA may function in plants by inhibiting catalase thus allowing the accumulation of hydrogen peroxide (H2O2) which can then act as a second messenger to switch on defence gene expression and activate SAR. The theory was tested using transgenic plants unable to accumulate salicylate. It was concluded that SA does not function downstream of hydrogen peroxide in the induction of PR proteins.