Panel of real-time PCRs for the multiplexed detection of two tomato-infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real-time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus-Israel (TYLCV-IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B.tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality-assurance purposes, two internal control assays were included in the assay panel for the co-amplification of solanaceous plant DNA or B.tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV-IL, 100 plasmid copies of ToLCV, 500fg B.tabaci MEAM1 and 300fg B.tabaci MED DNA. Evaluated methods for routine testing of field-collected whiteflies are presented, including protocols for processing B.tabaci captured on yellow sticky traps and for bulking of multiple B.tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality-assured diagnostic method for the identification and discrimination of tomato-infecting begomovirus and B.tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease-management programmes both in Australia and worldwide.

Studies on an enzyme reacting with isoniazid fromMycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis H37Rv possesses an enzyme (referred to as ‘Y enzyme’) which catalyses in the presence of INH and NAD, the formation of a product, which turns yellow on acidification. The requirements for the reaction, such as enzyme concentration, INH concentration, etc., have been standardized. The substrate specificity of the enzyme with respect to INH and NAD has been determined. The reaction is specific for the INH-sensitive strain and is totally absent in INH-resistant strains. Furthermore, the ‘Y enzyme’ shows some characteristic features of a peroxidase in its requirement for oxygen and sensitivity to inhibition by various reagents. The requirements of this enzyme which is involved in the action of isoniazid inM. tuberculosis H37Rv is described for the first time.

Effects of light conditions and plant density on growth and reproductive biology of Cascabela thevetia (L.) Lippold

Cascabela thevetia (L.) Lippold (Apocynaceae) is an invasive woody weed that has formed large infestations at several locations in northern Australia. Understanding the reproductive biology of C. thevetia is vital to its management. This paper reports results of a shade house experiment that determined the effects of light conditions (100% or 30% of natural light) and plant densities (one, two, four or eight plants per plot) on the growth, time to flowering and seed formation, and monthly pod production of two C. thevetia biotypes (peach and yellow). Shaded plants were significantly larger when they reached reproductive maturity than plants grown under natural light. However, plants grown under natural light flowered earlier (268 days compared with 369 days) and produced 488 more pods per pot (a 5-fold increase) over 3 years. The yellow biotype was slightly taller at reproductive maturity but significantly taller and with significantly greater aboveground biomass at the end of the study. Both biotypes flowered at a similar time under natural light and low plant densities but the yellow biotype was quicker to seed (478 versus 498 days), produced significantly more pods (364 versus 203 pods) and more shoot growth (577 g versus 550 g) than the peach biotype over 3 years. Higher densities of C. thevetia tended to significantly reduce the shoot and root growth by 981 g and 714 g per plant across all light conditions and biotypes over 3 years and increase the time taken to flower by 140 days and produce seeds by 184 days. For land managers trying to prevent establishment of C. thevetia or to control seedling regrowth once initial infestations have been treated, this study indicates that young plants have the potential to flower and produce seeds within 268 and 353 days, respectively. However, with plant growth and reproduction most likely to be slower under field conditions, annual surveillance and control activities should be sufficient to find and treat plants before they produce seeds and replenish soil seed banks. The most at-risk part of the landscape may be open areas that receive maximum sunlight, particularly within riparian habitats where plants would consistently have more favourable soil moisture conditions.

Portrait of Max Boehm ( 1871-1944 ).

The model is seated, facing the viewer and looking straight at him. He is wearing a business suit and holding a cigar. The pose is relaxed abd the color tonalities warm, with the yellow background dominant. Heavy impasto is used for face and hands. Neg. 37599 Signed upper right, also dated

Australian bat lyssavirus infection in two horses

In May 2013, the first cases of Australian bat lyssavirus infections in domestic animals were identified in Australia. Two horses (filly-H1 and gelding-H2) were infected with the Yellow-bellied sheathtail bat (YBST) variant of Australian bat lyssavirus (ABLV). The horses presented with neurological signs, pyrexia and progressing ataxia. Intra-cytoplasmic inclusion bodies (Negri bodies) were detected in some Purkinje neurons in haematoxylin and eosin (H&E) stained sections from the brain of one of the two infected horses (H2) by histological examination. A morphological diagnosis of sub-acute moderate non-suppurative, predominantly angiocentric, meningo-encephalomyelitis of viral aetiology was made. The presumptive diagnosis of ABLV infection was confirmed by the positive testing of the affected brain tissue from (H2) in a range of laboratory tests including fluorescent antibody test (FAT) and real-time PCR targeting the nucleocapsid (N) gene. Retrospective testing of the oral swab from (H1) in the real-time PCR also returned a positive result. The FAT and immunohistochemistry (IHC) revealed an abundance of ABLV antigen throughout the examined brain sections. ABLV was isolated from the brain (H2) and oral swab/saliva (H1) in the neuroblastoma cell line (MNA). Alignment of the genome sequence revealed a 97.7% identity with the YBST ABLV strain.

Spatial variation of climate and the impact of disturbances on local climate and forest recovery in northern Finland

The structure and function of northern ecosystems are strongly influenced by climate change and variability and by human-induced disturbances. The projected global change is likely to have a pronounced effect on the distribution and productivity of different species, generating large changes in the equilibrium at the tree-line. In turn, movement of the tree-line and the redistribution of species produce feedback to both the local and the regional climate. This research was initiated with the objective of examining the influence of natural conditions on the small-scale spatial variation of climate in Finnish Lapland, and to study the interaction and feedback mechanisms in the climate-disturbances-vegetation system near the climatological border of boreal forest. The high (1 km) resolution spatial variation of climate parameters over northern Finland was determined by applying the Kriging interpolation method that takes into account the effect of external forcing variables, i.e., geographical coordinates, elevation, sea and lake coverage. Of all the natural factors shaping the climate, the geographical position, local topography and altitude proved to be the determining ones. Spatial analyses of temperature- and precipitation-derived parameters based on a 30-year dataset (1971-2000) provide a detailed description of the local climate. Maps of the mean, maximum and minimum temperatures, the frost-free period and the growing season indicate that the most favourable thermal conditions exist in the south-western part of Lapland, around large water bodies and in the Kemijoki basin, while the coldest regions are in highland and fell Lapland. The distribution of precipitation is predominantly longitudinally dependent but with the definite influence of local features. The impact of human-induced disturbances, i.e., forest fires, on local climate and its implication for forest recovery near the northern timberline was evaluated in the Tuntsa area of eastern Lapland, damaged by a widespread forest fire in 1960 and suffering repeatedly-failed vegetation recovery since that. Direct measurements of the local climate and simulated heat and water fluxes indicated the development of a more severe climate and physical conditions on the fire-disturbed site. Removal of the original, predominantly Norway spruce and downy birch vegetation and its substitution by tundra vegetation has generated increased wind velocity and reduced snow accumulation, associated with a large variation in soil temperature and moisture and deep soil frost. The changed structural parameters of the canopy have determined changes in energy fluxes by reducing the latter over the tundra vegetation. The altered surface and soil conditions, as well as the evolved severe local climate, have negatively affected seedling growth and survival, leading to more unfavourable conditions for the reproduction of boreal vegetation and thereby causing deviations in the regional position of the timberline. However it should be noted that other factors, such as an inadequate seed source or seedbed, the poor quality of the soil and the intensive logging of damaged trees could also exacerbate the poor tree regeneration. In spite of the failed forest recovery at Tunsta, the position and composition of the timberline and tree-line in Finnish Lapland may also benefit from present and future changes in climate. The already-observed and the projected increase in temperature, the prolonged growing season, as well as changes in the precipitation regime foster tree growth and new regeneration, resulting in an advance of the timberline and tree-line northward and upward. This shift in the distribution of vegetation might be decelerated or even halted by local topoclimatic conditions and by the expected increase in the frequency of disturbances.

Remote sensing of boreal land cover: estimation of forest attributes and extent

Remote sensing provides methods to infer land cover information over large geographical areas at a variety of spatial and temporal resolutions. Land cover is input data for a range of environmental models and information on land cover dynamics is required for monitoring the implications of global change. Such data are also essential in support of environmental management and policymaking. Boreal forests are a key component of the global climate and a major sink of carbon. The northern latitudes are expected to experience a disproportionate and rapid warming, which can have a major impact on vegetation at forest limits. This thesis examines the use of optical remote sensing for estimating aboveground biomass, leaf area index (LAI), tree cover and tree height in the boreal forests and tundra taiga transition zone in Finland. The continuous fields of forest attributes are required, for example, to improve the mapping of forest extent. The thesis focus on studying the feasibility of satellite data at multiple spatial resolutions, assessing the potential of multispectral, -angular and -temporal information, and provides regional evaluation for global land cover data. Preprocessed ASTER, MISR and MODIS products are the principal satellite data. The reference data consist of field measurements, forest inventory data and fine resolution land cover maps. Fine resolution studies demonstrate how statistical relationships between biomass and satellite data are relatively strong in single species and low biomass mountain birch forests in comparison to higher biomass coniferous stands. The combination of forest stand data and fine resolution ASTER images provides a method for biomass estimation using medium resolution MODIS data. The multiangular data improve the accuracy of land cover mapping in the sparsely forested tundra taiga transition zone, particularly in mires. Similarly, multitemporal data improve the accuracy of coarse resolution tree cover estimates in comparison to single date data. Furthermore, the peak of the growing season is not necessarily the optimal time for land cover mapping in the northern boreal regions. The evaluated coarse resolution land cover data sets have considerable shortcomings in northernmost Finland and should be used with caution in similar regions. The quantitative reference data and upscaling methods for integrating multiresolution data are required for calibration of statistical models and evaluation of land cover data sets. The preprocessed image products have potential for wider use as they can considerably reduce the time and effort used for data processing.

Population dynamics of Thaumastocoris peregrinus in Eucalyptus plantations of South Africa

Thaumastocoris peregrinus is a sap-sucking insect that infests non-native Eucalyptus plantations in Africa, New Zealand, South America and parts of Southern Europe, in addition to street trees in parts of its native range of Australia. In South Africa, pronounced fluctuations in the population densities have been observed. To characterise spatiotemporal variability in T. peregrinus abundance and the factors that might influence it, we monitored adult population densities at six sites in the main eucalypt growing regions of South Africa. At each site, twenty yellow sticky traps were monitored weekly for 30 months, together with climatic data. We also characterised the influence of temperature on growth and survival experimentally and used this to model how temperature may influence population dynamics. T. peregrinus was present throughout the year at all sites, with annual site-specific peaks in abundance. Peaks occurred during autumn (February–April) for the Pretoria site, summer (November–January) for the Zululand site and spring (August–October) for the Tzaneen, Sabie and Piet Retief monitoring sites. Temperature (both experimental and field-collected), humidity and rainfall were mostly weakly, or not at all, associated with population fluctuations. It is clear that a complex interaction of these and other factors (e.g. host quality) influence population fluctuations in an annual, site specific cycle. The results obtained not only provide insights into the biology of T. peregrinus, but will also be important for future planning of monitoring and control programs using semiochemicals, chemical insecticides or biological control agents.

Zeaxanthin biofortification of sweet-corn and factors affecting zeaxanthin accumulation and colour change

Zeaxanthin, along with its isomer lutein, are the major carotenoids contributing to the characteristic colour of yellow sweet-corn. From a human health perspective, these two carotenoids are also specifically accumulated in the human macula, and are thought to protect the photoreceptor cells of the eye from blue light oxidative damage and to improve visual acuity. As humans cannot synthesise these compounds, they must be accumulated from dietary components containing zeaxanthin and lutein. In comparison to most dietary sources, yellow sweet-corn (Zea mays var. rugosa) is a particularly good source of zeaxanthin, although the concentration of zeaxanthin is still fairly low in comparison to what is considered a supplementary dose to improve macular pigment concentration (2 mg/person/day). In our present project, we have increased zeaxanthin concentration in sweet-corn kernels from 0.2 to 0.3 mg/100 g FW to greater than 2.0 mg/100 g FW at sweet-corn eating-stage, substantially reducing the amount of corn required to provide the same dosage of zeaxanthin. This was achieved by altering the carotenoid synthesis pathway to more than double total carotenoid synthesis and to redirect carotenoid synthesis towards the beta-arm of the pathway where zeaxanthin is synthesised. This resulted in a proportional increase of zeaxanthin from 22% to 70% of the total carotenoid present. As kernels increase in physiological maturity, carotenoid concentration also significantly increases, mainly due to increased synthesis but also due to a decline in moisture content of the kernels. When fully mature, dried kernels can reach zeaxanthin and carotene concentrations of 8.7 mg/100 g and 2.6 mg/100 g, respectively. Although kernels continue to increase in zeaxanthin when harvested past their normal harvest maturity stage, the texture of these 'over-mature' kernels is tough, making them less appealing for fresh consumption. Increase in zeaxanthin concentration and other orange carotenoids such as p-carotene also results in a decline in kernel hue angle of fresh sweet-corn from approximately 90 (yellow) to as low as 75 (orange-yellow). This enables high-zeaxanthin sweet-corn to be visually-distinguishable from standard yellow sweet-corn, which is predominantly pigmented by lutein.

Condensed phase reactions in solid propellants

The paper investigates the cause for the difference between differential scanning calorimetric results and mass spectrometric studies on polystyrene (PS) ammonium perchlorate (AP) propellants as related to the method of preparation of the propellant and the difference in experimental conditions by the use of mass spectrometry. Sufficient time is given for the product sublimates to interact with each other and attain equilibrium. It is shown that the propellant decomposition is a nonadditive phenomenon and that even a physical mixture of AP and PS does not yield additive decomposition products of its components. Results on the identification of a yellow compound containing chlorine in the bulk of the propellant suggest a condensed phase reaction. The occurrence of the reaction in the porous condensed phase of the propellant may explain the larger exothermicity of the propellant compared to the additive heats of decomposition of its components.

Time to rest : Signals in shoot apex developmental transitions underlying dormancy

Vuodenajat rytmittävät monivuotisten kasvien elämää pohjoisella pallonpuoliskolla, jolla varmin merkki lähestyvästä talvikaudesta on asteittain lyhenevä päivänpituus. Kun päivänpituus on lyhentynyt tiettyyn raja-arvoon saakka, kasvu hiipuu ja kasvin kehityksessä tapahtuu suuria muutoksia. Väitöskirjatyössäni tutkittiin mekanismeja, jotka liittyvät pituuskasvun päättymiseen, silmujen lepotilan kehittymiseen ja kärkisilmun muodostumiseen hybridihaavan ja koivuntaimilla lyhyen päivänpituuden seurauksena kasvihuoneolosuhteissa. Vain lepotilaiset silmut selviytyvät luonnossa ankaran talvikauden yli, joten etenkin lepotilan kehittymisen tutkiminen on keskeistä pyrittäessä selvittämään monivuotisille kasveille tyypillisen kasvutavan mekanismeja. Jo pitkään on tiedetty, että täysikasvuiset lehdet vastaanottavat tiedon päivänpituudesta ja lähettävät signaaleja varren johtojänteissä ylöspäin kohti kasvin kärkiosaa. Sen sijaan varren kärjen ja kärkikasvupisteen roolia lepotilan kehittymisessä on selvitetty vain vähän. Kuitenkin juuri kärkikasvupisteen selviytyminen vuodesta toiseen on tärkeää, koska sen jakautumiskykyiset solukot tuottavat kasvin maanpäälliset osat. Tässä työssä tehdyissä varttamiskokeissa osoitettiin, että varren kärki ei ainoastaan vastaanota signaaleja lehdistä ja ajoita toimintaansa niiden mukaan, vaan myös kärjellä itsellään on aktiivinen rooli lepotilan kehittymisessä. Erityisesti kiinnitettiin huomiota kärkikasvupisteen eri alueiden, ns. apikaalimeristeemin ja rib-meristeemin erilaisiin tehtäviin ja pääteltiin, että molemmat vaikuttavat lepotilan kehittymiseen. Kokeissa käytettiin normaalien hybridihaapojen lisäksi siirtogeenisiä hybridihaapoja, jotka eivät lopeta kasvuaan lyhyt päivä –olosuhteissa. Siirtogeeniset hybridihaavat ilmensivät voimakkaasti fytokromi A -nimistä valon vastaanottajamolekyyliä rib-meristeemin alueella, mikä saattoi osaltaan vaikuttaa poikkeavaan pituuskasvukäyttäytymiseen. Myös useiden lepotilan kehittymiseen liittyvien geenien ilmenemisessä havaittiin poikkeavuuksia verrattuna ei-siirtogeenisiin kontrolleihin, joiden silmuissa kehittyi lepotila lyhyt päivä –altistuksen seurauksena. Väitöskirjatyössäni havaittiin, että myös kaasumainen kasvihormoni etyleeni toimii viestinvälittäjänä silmujen lepotilan kehittymisessä ja vaikuttaa etenkin lepotilan oikeaan ajoittumiseen. Etyleenillä huomattiin olevan määräävä rooli päätesilmun muodostumisessa: siirtogeeniset koivut, jotka eivät aisti etyleeniä, eivät muodostaneet päätesilmua. Silti siirtogeeniset koivut vaipuivat lepotilaan, joskin myöhemmin kuin ei-siirtogeeniset kontrollit. Tämän perusteella todettiin, että lepotilan ja päätesilmun kehittyminen ovat erillisiä kehitystapahtumia, vaikka ne saattavatkin ajoittua osaksi päällekkäin.

Ultrastructural features of the early secretory pathway in Trichoderma reesei

We have systematically analysed the ultra structure of the early secretory pathway in the Trichoderma reesei hyphae in the wild-type QM6a, cellulase overexpressing Rut-C30 strain and a Rut-C30 transformant BV47 overexpressing a recombinant BiP1-VenusYFP fusion protein with an endoplasmic reticulum (ER) retention signal. The hyphae were studied after 24h of growth using transmission electron microscopy, confocal microscopy and quantitative stereological techniques. All three strains exhibited different spatial organisation of the ER at 24h in both a cellulase-inducing medium and a minimal medium containing glycerol as a carbon source (non-cellulase-inducing medium). The wild-type displayed a number of ER subdomains including parallel tubular/cisternal ER, ER whorls, ER-isolation membrane complexes with abundant autophagy vacuoles and dense bodies. Rut-C30 and its transformant BV47 overexpressing the BiP1-VenusYFP fusion protein also contained parallel tubular/cisternal ER, but no ER whorls; also, there were very few autophagy vacuoles and an increasing amount of punctate bodies where particularly the recombinant BiP1-VenusYFPfusion protein was localised. The early presence of distinct strain-specific features such as the dominance of ER whorls in the wild type and tub/cis ER in Rut-C30 suggests that these are inherent traits and not solely a result of cellular response mechanisms by the high secreting mutant to protein overload.

Photobiological studies of Baltic Sea phytoplankton

Phytoplankton ecology and productivity is one of the main branches of contemporary oceanographic research. Research groups in this branch have increasingly started to utilise bio-optical applications. My main research objective was to critically investigate the advantages and deficiencies of the fast repetition rate (FRR) fluorometry for studies of productivity of phytoplankton, and the responses of phytoplankton towards varying environmental stress. Second, I aimed to clarify the applicability of the FRR system to the optical environment of the Baltic Sea. The FRR system offers a highly dynamic tool for studies of phytoplankton photophysiology and productivity both in the field and in a controlled environment. The FRR metrics obtain high-frequency in situ determinations of the light-acclimative and photosynthetic parameters of intact phytoplankton communities. The measurement protocol is relatively easy to use without phases requiring analytical determinations. The most notable application of the FRR system lies in its potential for making primary productivity (PP) estimations. However, the realisation of this scheme is not straightforward. The FRR-PP, based on the photosynthetic electron flow (PEF) rate, are linearly related to the photosynthetic gas exchange (fixation of 14C) PP only in environments where the photosynthesis is light-limited. If the light limitation is not present, as is usually the case in the near-surface layers of the water column, the two PP approaches will deviate. The prompt response of the PEF rate to the short-term variability in the natural light field makes the field comparisons between the PEF-PP and the 14C-PP difficult to interpret, because this variability is averaged out in the 14C-incubations. Furthermore, the FRR based PP models are tuned to closely follow the vertical pattern of the underwater irradiance. Due to the photoacclimational plasticity of phytoplankton, this easily leads to overestimates of water column PP, if precautionary measures are not taken. Natural phytoplankton is subject to broad-waveband light. Active non-spectral bio-optical instruments, like the FRR fluorometer, emit light in a relatively narrow waveband, which by its nature does not represent the in situ light field. Thus, the spectrally-dependent parameters provided by the FRR system need to be spectrally scaled to the natural light field of the Baltic Sea. In general, the requirement of spectral scaling in the water bodies under terrestrial impact concerns all light-adaptive parameters provided by any active non-spectral bio-optical technique. The FRR system can be adopted to studies of all phytoplankton that possess efficient light harvesting in the waveband matching the bluish FRR excitation. Although these taxa cover the large bulk of all the phytoplankton taxa, one exception with a pronounced ecological significance is found in the Baltic Sea. The FRR system cannot be used to monitor the photophysiology of the cyanobacterial taxa harvesting light in the yellow-red waveband. These taxa include the ecologically-significant bloom-forming cyanobacterial taxa in the Baltic Sea.

Plasmids and aromatic degradation in Sphingomonas for bioremediation : Aromatic ring cleavage genes in soil and rhizosphere

Microbial degradation pathways play a key role in the detoxification and the mineralization of polyaromatic hydrocarbons (PAHs), which are widespread pollutants in soil and constituents of petroleum hydrocarbons. In microbiology the aromatic degradation pathways are traditionally studied from single bacterial strains with capacity to degrade certain pollutant. In soil the degradation of aromatics is performed by a diverse community of micro-organisms. The aim of this thesis was to study biodegradation on different levels starting from a versatile aromatic degrader Sphingobium sp. HV3 and its megaplasmid, extending to revelation of diversity of key catabolic enzymes in the environment and finally studying birch rhizoremediation in PAH-polluted soil. To understand biodegradation of aromatics on bacterial species level, the aromatic degradation capacity of Sphingobium sp. HV3 and the role of the plasmid pSKY4, was studied. Toluene, m-xylene, biphenyl, fluorene, phenanthrene were detected as carbon and energy sources of the HV3 strain. Tn5 transposon mutagenesis linked the degradation capacity of toluene, m-xylene, biphenyl and naphthalene to the pSKY4 plasmid and qPCR expression analysis showed that plasmid extradiol dioxygenases genes (bphC and xylE) are inducted by phenanthrene, m-xylene and biphenyl whereas the 2,4-dichlorophenoxyacetic acid herbicide induced the chlorocatechol 1,2-dioxygenase gene (tfdC) from the ortho-pathway. A method to study upper meta-pathway extradiol dioxygenase gene diversity in soil was developed. The extradiol dioxygenases catalyse cleavage of the aromatic ring between a hydroxylated carbon and an adjacent non-hydroxylated carbon (meta-cleavage). A high diversity of extradiol dioxygenases were detected from polluted soils. The detected extradiol dioxygenases showed sequence similarity to known catabolic genes of Alpha-, Beta-, and Gammaproteobacteria. Five groups of extradiol dioxygenases contained sequences with no close homologues in the database, representing novel genes. In rhizoremediation experiment with birch (Betula pendula) treatment specific changes of extradiol dioxygenase communities were shown. PAH pollution changed the bulk soil extradiol dioxygenase community structure and birch rhizosphere contained a more diverse extradiol dioxygenase community than the bulk soil showing a rhizosphere effect. The degradation of pyrene in soil was enhanced with birch seedlings compared to soil without birch. The complete 280,923 kb nucleotide sequence of pSKY4 plasmid was determined. The open reading frames of pSKY4 were divided into putative conjugative transfer, aromatic degradation, replication/maintaining and transposition/integration function-encoding proteins. Aromatic degradation orfs shared high similarity to corresponding genes in pNL1, a plasmid from the deep subsurface strain Novosphingobium aromaticivorans F199. The plasmid backbones were considerably more divergent with lower similarity, which suggests that the aromatic pathway has functioned as a plasmid independent mobile genetic element. The functional diversity of microbial communities in soil is still largely unknown. Several novel clusters of extradiol dioxygenases representing catabolic bacteria, whose function, biodegradation pathways and phylogenetic position is not known were amplified with single primer pair from polluted soils. These extradiol dioxygenase communities were shown to change upon PAH pollution, which indicates that their hosts function in PAH biodegradation in soil. Although the degradation pathways of specific bacterial species are substantially better depicted than pathways in situ, the evolution of degradation pathways for the xenobiotic compounds is largely unknown. The pSKY4 plasmid contains aromatic degradation genes in putative mobile genetic element causing flexibility/instability to the pathway. The localisation of the aromatic biodegradation pathway in mobile genetic elements suggests that gene transfer and rearrangements are a competetive advantage for Sphingomonas bacteria in the environment.

Variations in the Levels of Chloroplast tRNAs and Aminoacyl-tRNA Synthetases in Senescing Leaves of Phaseolus vulgaris

The relative amounts of chloroplast tRNAs(Leu), tRNA(Glu), tRNA(Phe), tRNAs(Thr), and tRNA(Tyr) and of chloroplastic and cytoplasmic aminoacyl-tRNA synthetases were compared in green leaves, yellowing senescing leaves, and N(6)-benzyladenine-treated senescing leaves from bean (Phaseolus vulgaris, var Contender). Aminoacylation of the tRNAs using Escherichia coli aminoacyl-tRNA synthetases indicated that in senescing leaves the relative amount of chloroplast tRNA(Phe) was significantly lower than in green leaves. Senescing leaves treated with N(6)-benzyladenine contained higher levels of this tRNA than untreated senescing leaves. No significant change in the relative amounts of chloroplast tRNAs(Leu), tRNAs(Thr), and tRNA(Tyr) was detected in green, yellow senescing, or N(6)-benzyladine-treated senescing leaves. Relative levels of chloroplast tRNAs were also estimated by hybridization of tRNAs to DNA blots of gene specific probes. These experiments confirmed the results obtained by aminoacylation and revealed in addition that the relative level of chloroplast tRNA(Glu) is higher in senescing leaves than in green leaves. Transcription run-on assays indicated that these changes in tRNA levels are likely to be due to a differential rate of degradation rather than to a differential rate of transcription of the tRNA genes. Chloroplastic and cytoplasmic leucyl-, phenylalanyl-, and tyrosyl-tRNA synthetase activities were greatly reduced in senescing leaves as compared to green leaves, whereas N(6)-benzyladenine-treated senescing leaves contained higher enzyme activities than untreated senescing leaves. These results suggest that during senescence, as well as during senescence-retardation by cytokinins, changes in enzyme activities, such as aminoacyl-tRNA synthetases, rather than reduced levels of tRNAs, affect the translational capacity of chloroplasts.