Sweet potato chlorotic stunt virus: Studies on viral synergism and suppression of RNA silencing

The studies presented in this thesis aimed to a better understanding of the molecular biology of Sweet potato chlorotic stunt virus (SPCSV, Crinivirus, Closteroviridae) and its role in the development of synergistic viral diseases. The emphasis was on the severe sweet potato virus disease (SPVD) that results from a synergistic interaction of SPCSV and Sweet potato feathery mottle virus (SPFMV, Potyvirus, Potyviridae). SPVD is the most important disease affecting sweetpotato. It is manifested as a significant increase in symptom severity and SPFMV titres. This is accompanied by a dramatic sweetpotato yield reduction. SPCSV titres remain little affected in the diseased plants. Viral synergistic interactions have been associated with the suppression of an adaptive general defence mechanism discovered in plants and known as RNA silencing. In the studies of this thesis two novel proteins (RNase3 and p22) identified in the genome of a Ugandan SPCSV isolate were shown to be involved in suppression of RNA silencing. RNase3 displayed a dsRNA-specific endonuclease activity that enhanced the RNA-silencing suppression activity of p22. Comparative analyses of criniviral genomes revealed variability in the gene content at the 3´end of the genomic RNA1. Molecular analyses of different isolates of SPCSV indicated a marked intraspecific heterogeneity in this region where the p22 and RNase3 genes are located. Isolates of the East African strain of SPCSV from Tanzania and Peru and an isolate from Israel were missing a 767-nt fragment that included the p22 gene. However, regardless of the absence of p22, all SPCSV isolates acted synergistically with SPFMV in co-infected sweetpotato, enhanced SPFMV titres and caused SPVD. These results showed that p22 is dispensable for development of SPVD. The role of RNase3 in SPVD was then studied by generating transgenic plants expressing the RNase3 protein. These plants had increased titres of SPFMV (ca. 600-fold higher in comparison with nontransgenic plants) 2-3 weeks after graft inoculation and displayed the characteristic SPVD symptoms. RNA silencing suppression (RSS) activity of RNase3 was detected in agroinfiltrated leaves of Nicotiana bethamiana. In vitro studies showed that RNase3 was able to cleave small interferring RNAs (siRNA) to products of ~14-nt. The data thus identified RNase3 as a suppressor of RNA silencing able to cleave siRNAs. RNase3 expression alone was sufficient for breaking down resistance to SPFMV in sweetpotato and for the development of SPVD. Similar RNase III-like genes exist in animal viruses which points out a novel and possibly more general mechanism of RSS by viruses. A reproducible method of sweetpotato transformation was used to target RNA silencing against the SPCSV polymerase region (RdRp) with an intron-spliced hairpin construct. Hence, engineered resistance to SPCSV was obtained. Ten out of 20 transgenic events challenged with SPCSV alone showed significantly reduced virus titres. This was however not sufficient to prevent SPVD upon coinfection with SPFMV. Immunity to SPCSV seems to be required to control SPVD and targeting of different SPCSV regions need to be assessed in further studies. Based on the identified key role of RNase3 in SPVD the possibility to design constructs that target this gene might prove more efficient in future studies.

ESR studies of phase transitions in double propionates

Detailed ESR investigations of Mn2+ substituting for Ca2+ in Ca2Sr(C2H5COO)6 (DSP), Ca2Pb(C2H5COO)6 (DLP) and Ca2Ba(C2H5COO)6 (DBP), in single crystals and powders, over the temperature range from 200°C to -180°C have been carried out to study the successive phase transitions in these compounds. (DSP: [Tetragonal] ← 8.5°C → [tetragonal, ferroelectric] [tetragonal] ← -169°C → [monoclinic, ferroelectric]; DLP : [tetragonal] ← 60°C → [tetragonal, ferroelectric] ← -71.5°C → [monoclinic, ferroelectric]; [Cubic] ← -6°C → [orthorhombic] ← -75°C → [?]). Spectra have been analysed in terms of axial spin Hamiltonians and the temperature dependences of the parameters studied. In DSP and DLP across the I ↔ II transition, new physically and chemically inequivalent sites appear indicating the disappearance of the diad axes on which the propionate groups are located, bringing out the connection between the motional states of the propionate groups and the occurence of ferroelectricity. The II ↔ III transition also causes chemically inequivalent sites to develop, indicating that the transitions may not be isomorphous as believed previously. In DBP, the -6°C transition leads to (i) a doubling of both physically and chemically inequivalent sites (ii) a small (150 G at -6°C to 170 G at -8°C), but abrupt change in the magnitude of the zero-field splitting tensor D, and (iii) displacements of the orientations of the D tensors. Results are interpreted in terms of alternate rotations of the oxygen octahedra, showing participation of the carboxyl oxygens in the transition. No drastic changes in the parameters occur across the -75°C transition consistent with its second order nature. Similarities and dissimilarities of the ESR spectra of the three compounds in relation to the phase transitions, are discussed.

Synthetic studies towards bioactive frondosins: rapid framework access and diversity creation

A very concise and diversity-oriented approach to rapidly access frondosin-related frameworks from commercially available building blocks is outlined.

Mechanistic studies on the action of ammonium phosphate on polymer fire retardancy

The mechanism of fire retardant action of mono- and diammonium phosphates on polystyrene has been investigated. Ignition delay and mass burning rate studies reveal that the phosphates bring down both parameters considerably though to different extents. This has been adequately explained on the basis of the existing combustion models and physicochemical behavior of the material. Similar to their action on cellulosic materials, phosphates bring about fire retardancy in polystyrene via char formation. This is suggested to occur through a series of processes consisting of initial peroxide formation, decomposition to alcohols and aldehydes, formation of alkyl-phosphate esters, dehydration and subsequent char formation. Infrared and mass spectral studies support this mechanism.

Studies on diglycine sulphate

Both diglycine sulphate (DGS) and diglycine sulphate monohydrate (DGS.H2O) are reported to crystallize from solution with pH < 1(1,2). DGS.H2O (point group 2/m; Z = 4) shows a dielectric anomaly at 72°C suggestive of antiferroelectric transition(1). The crystals obtained by us from solution with pH < 0.5 at 20-25°C were always DGS (point group mmm; Z = 8) as confirmed by X-ray studies. The measurement of its dielectric constant along [100], [010] and [001] did not indicate any phase transition in the range 5-400°K. Thus DGS is a normal dielectric unlike TGS. The polarized Raman spectra and the infrared spectra were recorded to examine the configuration of glycine in DGS(3). The vibration spectra reveals that both the glycines in DGS exist as NH3+CH2COOH, thus precluding the hydrogen bond of the type N+-H…O- which exists between two glycine units in TGS. This seems to be a good reason for the difference in the dielectric behaviour of these two glycine sulphates.

Studies on Thermal Polymerization of Acrylamide

Thermal polymerization of acrylamide has been followed by the DSC technique, and the activation energy (E) values at different stages of the fraction polymerized (a) have been determined from the exotherm of the thermograms obtained. The trend of variation of E with agr shows that E remains constant up to agr = 0.5 and decreases with a further increase in agr. A close look at the composite nature of the exotherms, agr-t, and agr-T curves shows that the polymerization of acrylamide involves two processes. The first process is the formation of linear polyacrylamide and the second is the simultaneous cross-linking of the linear chains together with the formation of linear polyacrylamide. Experiments such as NH3 detection by differential thermal analysis techniques and annealing studies have been made to shed further light on the polymerization process.

Photo-Electron Spectroscopic Studies Of The Adsorption Of Organic-Molecules With Lone Pair Orbitals On Transition-Metal Surfaces

Ultraviolet and x-ray photoelectron spectroscopy have been employed to investigate the adsorption of methanol, ethanol, diethylether, acetaldehyde, acetone, methyl acetate and methylamine on surfaces of Fe, Ni and Cu. All these molecules adsorb molecularly at low temperatures (≤100 K). Lone pair orbitals of these molecules are stabilized on these metal surfaces (by 0·4–1·0eV) due to molecular chemisorption. The molecules generally undergo transformations as the temperature is raised to 120 K or above. The new species produced seems to depend on the metal surface. Some of the product species identified are methoxy species, formaldehyde and carbon monoxide in the case of methanol and methyl acetate, ethoxy species in the case of ethanol and 2-propanol in the case of acetone.

Interfacial structure and crystallographic studies of transformations in betat' and beta Cu---Zn alloys-II. martensitic formation of alpha1' plates in beta'

A TEM study of the interphase boundary structure of 9R orthorhombic alpha1' martensite formed in beta' Cu---Zn alloys shows that it consists of a single array of dislocations with Burgers vector parallel to left angle bracket110right-pointing angle beta and spaced about 3.5 nm apart. This Burgers vector lies out of the interface plane; hence the interface dislocations are glissile. Unexpectedly, though, the Burgers vectors of these dislocations are not parallel when referenced to the matrix and the martensite lattices. This finding is rationalized on published hard sphere models as a consequence of relaxation of a resultant of the Bain strain and lattice invariant shear displacements within the matrix phase.

Mechanistic studies on polymer flame retardancy Effect of Tribromoaniline

Abstract is not available.

Electronic Structure of some Substituted azolidines.CNDO/2, NMR and vibratational spectoroscopic studies

The 1H and 13C chemical shifts, characteristic vibrational frequencies and force constants for some substituted azolidines are correlated with the results of the CNDO/2 calculations. The influence of the exo and endo heteroatoms on the electronic structure of the heterocyclic ring are discussed.

Studies on follicular atresia: role of tropic hormone and steroids in regulating cathepsin-D activity of preantral follicles of the immature rat

The ovary of the immature female rat is comprised of primary and medium-sized preantral follicles. Upon stimulation with FSH or PMSG, the cathepsin-D activity, a representative lysosomal enzyme of granulosa cells, is reduced by 50% (P < 0.01). 17β-Estradiol at the doses tried was unable to mimic this effect. Blockade of steroidogenesis with cyanoketone also had no effect on the cathepsin-D activity of isolated granulosa cells. Dihydrotestosterone (DHT), however, at a dose of 1 mg/rat was able to inhibit PMSG's tropic action. It brought about an increase in cathepsin-D activity and reduction in steroidogenic activity of isolated granulosa cells. The atretogenic activity of DHT could be relieved by supplementation with exogenous FSH. DHT was observed to significantly reduce (P < 0.01) endogenous FSH and LH levels within 12–18 h of its injection suggesting that its atretic effect was due to its action at the pituitary rather than the gonad. In addition to the above the ability of 15 IU of PMSG to reduce cathepsin-D activity of granulosa cells was also significantly reduced (P < 0.01) if endogenous FSH was neutralized by a specific FSH antiserum. The present study suggests that as far as small and medium-sized primary and preantral follicles are concerned, FSH lack is the essential signal for onset of atresia.

Studies on the effects of in vitro methyiation on aminoacylation of transfer RNA

In vitro methyiation of Escherichia coli transfer ribonucleic acid by cell free extracts of Mycobacterium smegmatis leads exclusively to the formation of 1-methyl adenine [Vani, B. R., Ramakrishnan, T., Taya, Y., Noguchi, S., Yamaiuzumi, Z. and Nishimura, S.(1978) J. Bact., 137,1085]. We have studied the effect of this modification on aminoacylation of Escherichia coli tRNA by mycobacterial enzymes. Aminoacylation with total algal protein hydrolysate as well as several individual aminoacids like methionine, valine, tyrosine, aspartic acid and lysine were monitored. In all the cases methyiation had a positive effect on the extent of aminoacylation by mycobacterial enzymes. Decreased aminoacylation in vitro was observed when hypomethylated transfer RNA from ethionine treated cells was used as the substrate for aminoacylation.