Transformations and movement of P in the rhizosphere

Summary Wheat plants labelled with³³P were grown in thin layers of soil amended with³²P-labelled fertiliser. Roots were separated from the soil during plant growth by a porous membrane to overcome difficulties in measuring microbial P in rhizosphere soil. Over the 22 day growth period, net movement of³³P out of healthy growing roots varied from 0.9–4.9% of the total³³P translocated to the root. Over the same period the plants took up 12.0% and the microbial biomass 14.1% of the fertiliser³²P. On drying and rewetting of the soil after the plants were harvested, a large proportion of root P moved into soil fractions while³²P appeared to accumulate in the biomass and stable P forms.     

Lethal modifications of DNA via the transmutation of32P and33P incorporated in the genome of the S13 bacteriophage

Summary When circular single-stranded DNA of phage S13 is labelled with³²P or³³P, the transmutations very efficiently bring about a loss of phage infectiousness (efficiency = 1 for³²P and 0.73 for³³P). For both radionuclides, the lethal efficiencies as well as the lethal events are different. In the case of³²P, the lethal event is the loss of the circular integrity of the DNA molecule, occurring as a consequence of a systematic single strand-break caused by each³²P decay (100%). Conversely, in the case of³³P, the lethal events are either a single strand-break (40%) or a local stereochemical modification (33%). The same primary event, the substitution at each³³P decay of a phosphate by a sulfate molecule, leads to one of these lethal events in relation to the decay site. Moreover, neither the phage adsorption nor its genome injection into bacteria depends on the physical state of the genome, and thus lethality is revealed at only the genetic level.     

Contribution by two arbuscular mycorrhizal fungi to P uptake by cucumber (Cucumis sativus L.) from32P-labelled organic matter during mineralization in soil

An experiment was set up to investigate the role of arbuscular mycorrhiza (AM) in utilization of P from organic matter during mineralization in soil. Cucumber (Cucumis sativus L.) inoculated with one of two AM fungi or left uninoculated were grown for 30 days in cross-shaped PVC pots. One of two horizontal compartments contained 100 g soil (quartz sand: clay loam, 1:1) with 0.5 g ground clover leaves labelled with³²P. The labelled soil received microbial inoculum without AM fungi to ensure mineralization of the added organic matter. The labelling compartment was separated from a central root compartment by either 37 m or 700 m nylon mesh giving only hyphae or both roots and hyphae, respectively, access to the labelled soil. The recovery of³²P from the hyphal compartment was 5.5 and 8.6% for plants colonized withGlomus sp. andG. caledonium, respectively, but only 0.6 % for the non-mycorrhizal controls. Interfungal differences were not related to root colonization or hyphal length densities, which were lowest forG. caledonium. Both fungi depleted the labelled soil of NaHCO₃-extractable P and³²P compared to controls. A 15–25% recovery of³²P by roots was not enhanced in the presence of mycorrhizas, probably due to high root densities in the labelled soil. The experiment confirms that AM fungi differ in P uptake characteristics, and that mycorrhizal hyphae can intercept some P immobilization by other microorganisms and P-sorbing clay minerals.     

Primary Structural Relationship of p16 to m16 Ribosomal RNA

IMMATURE ribosomes contain RNAs which can be distinguished from the molecules present in mature ribosomes¹. The precursor of 16S rRNA (p16) can be identified in pulse labelled cells^2–4; the conversion of p16 to mature 16S rRNA (m16) occurs during the latter stages of ribosome assembly and demands concomitant protein synthesis¹. We have compared oligonucleotide fingerprints of E. coli p16 and m16 rRNA mixtures, labelled respectively with³²P and³H or³²P and³³P and now report that the precursor is of 10% greater molecular weight than the mature molecules. Identifiable sequences are cleaved from the precursor during the maturation process.     

Mutational analysis of the RNase-like domain in subunit 2 of fission yeast RNA polymerase II

Local sequence similarity exists between the subunit 2 of eukaryotic RNA polymerases II and the barnase-type bacterial RNases. The RNase-like domain from the Rpb2 ofSchizosaccharomyces pombe was expressed inEscherichia coli as a GST fusion protein and examined for its RNase activity. When the GST fusion protein was incubated in vitro with³²P-labeled RNA, the RNA degradation activity was less than 0.1%, if any, of the level of synthetic barnase. In order to check the in vivo function of this region, we constructed two mutantrpb2 alleles,rpb2 ^E357A andrpb2 ^H3a6L , each carrying a single amino acid substitution at the site correponding to one of the three essential amino acid residues forming the catalytic site in barnase (mutation of barnase at the corresponding sites results in complete loss of RNase activity) and five other mutantrpb2 alleles, each carrying a single mutation at various positions within the RNase-like domain but outside the putative catalytic site for RNase activity. When these mutantrpb2 alleles were expressed in anrpb2-disruptedS. pombe strain, all the mutants grew as well as the wild-type parent and did not show any clear defective phenotypes. These results suggest either that the RNase-like domain in Rpb2 does not function as an RNase in vivo or that the RNase activity of this domain, if present at all, is not essential for cell growth.     

In-situ determination of the P-relations around the primary root of maize with respect to inorganic and phytate-P

Autoradiographs of soil slices mapping the distribution of phytate-derived³³P around the primary root of 6-day-old maize seedlings were used to investigate the uptake of phytate by the root. Analysis of the autoradiographs with a laser densitometer and processing of the data with image analysing software resulted in a resolution of 40 μm. The effect of³³P-crossfire was corrected by analysis of the apparent³³P-gradient around a phosphate-impermeable teflon tube that was inserted into the labeled soil as a standard. In spite of the high resolution achieved, a significant depletion zone could not be detected when the soil was equilibrated with³³P-phytate. However, with³³P-inorganic phosphate, 2 concentric zones were obvious. Within the inner zone, P was accumulated by about 20%, while in the outer zone a corresponding depletion of P could be detected. The accumulation zone coincided with the extension of the root hair cylinder, whereas the depleted area was clearly beyond the range of the root hairs.     

The uptake,distribution, leakage,and incorporation of32P into organic compounds in alfalfa plants susceptible and resistant to the bacterial wilt and the effect ofCorynebacterium insidiosum upon these processes

Higher³²P uptake per plant was found in the healthy resistant (R) alfalfa (Medicago saliva L.) plants when compared with the healthy plants susceptible (S) to the bacterial wilt, following the exposure of the roots of intact plants to the radiophosphate solution. The bacterial infection markedly decreased³²P uptake and radioactivity levels per dry matter in most organs of the R-plants on the day 8 and 14 after inoculation withCorynebacterium insidiosum whereas in the S-plants a decrease in³²P uptake was only found on the day 8.³²P leakage rate from the infected R-plant roots to the nonradioactive nutrient solution was higher than from the healthy ones on the day 8. At the same time³²P content in the organic P fraction was somewhat increased due to the infection in the R-plant roots, whereas³²P content in DNA was decreased. After foliar application,³²P distribution pattern was similar in the tissues of both the S- and the R-plants and was not affected due to the infection in the course of the 3rd week after inoculation. However, the bacterial infection markedly increased³²P translocation from the primary leaf to the rest of the R-plant. An erratum to this article is available at .     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

Clinical performance of non-radioactive assays for HIV-1 DNA amplified by the polymerase chain reaction

A major factor preventing more widespread use of polymerase chain reaction in the clinical laboratory is the lack of convenient non-radioactive probe hybridization procedures which do not sacrifice sensitivity or specificity. In this report, we describe comparisons of probes labelled with biotin, digoxygenin, alkaline phosphatase, and³²P. We report the comparison of solution or liquid hybridization assay and Southern blotting with digoxygenin-labelled oligonucleotides on a total of 64 clinical specimens. Perfect diagnostic agreement between the³²P and digoxygenin probes was obtained. These data suggest that the non-radioactive assay as described is as sensitive and as specific as the assay with³²P-Iabelled probes.     

Changes in phosphorus metabolism in alfalfa plants induced by bacterial wilt

Radioactive phosphate was applied to the roots of intact alfalfa plants (Medicago sativa L.) on the 49th day after inoculation withCorynebacterium insidiosum (Me Culloch) Jensen and the³²P contents in different fractions of phosphoric compounds were determined. In inoculated plants, susceptible to bacterial wilt, the inorganio phosphate contents (³²Pinorg) was increased markedly and the³²P bound in organic compounds soluble in acids (³²Porg) decreased as compared with control. In roots of the same plants the³²P contents in phospholipid fraction and DNA were decreased. In tolerant inoculated plants the³²Pinorg increase and³²Porg decrease as compared with those changes in susceptible plants were less expressive. No expressive changes in determined³²P fractions have been proved in resistant plants without any visible disease symptoms.     

Changes in growth and in uptake,distribution and translocation of phosphorus in susceptible and resistant alfalfa plants induced byCorynebacterium insidiosum

The weight of alfalfa plants, especially roots of susceptible strain, decreased when inoculated withCorynebacterium insidiosum. At the 6th week after inoculation the³²P uptake per plant and its translocation into the above-ground organs were considerably decreased in susceptible plants. On the other hand, the³²P uptake was increased and the radiophosphorus was accumulated in above-ground organs in resistant plants.     

Une radiothérapie interne contre l’infection au VIH, la dernière pièce du puzzle ?

Although highly active antiretroviral therapy (HAART) has succeeded in many cases in suppressing ongoing viral replication, the HIV-infected cells are not eradicated by this therapy. In this article, the author proposes a new treatment protocol based on administration of radiophosphorus 32 (³²P) with HAART combination. The proposed new protocol consists of the following steps: HAART keeps viral replication below the level of detection. Then, ³²P is administered for the removal of the “residual disease”. By its beta emission, incorporated ³²P to viral RNA or proviral DNA may kill free virions and productively infected cells, reactivates latently infected cells and makes them vulnerable to immune competent cells and antiretroviral therapy. This new combination may exhibit synergistic interactions between virostatic effects of antiretroviral molecules and radiobiological effect of internal radiotherapy and provide a new tool to purge the viral reservoirs.     

Influx and efflux of p in roots of intact maize plants : double-labeling with p and p

Rates of P influx and efflux were determined in whole plants at ambient P concentrations comparable to those found in soil solutions. Maize (Zea mays L. var NC+59) seedlings were trimmed (endosperm and adventitious roots removed) and grown in a greenhouse in solution cultures at P concentrations of approximately 0.4 and 1.8 micromolar. Roots of intact plants previously exposed to ³²P-labeled solutions at 0.2 and 2.0 micromolar P for 48 hours were rinsed 10 minutes in P-free solution and exposed to ³³P solutions at 0.2 and 2.0 micromolar for 10 minutes. Net depletion of ³³P from and appearance of ³²P in the ambient solution were used to measure influx and efflux. The ration of ³²P efflux to ³³P influx was about 0.68 at 0.2 micromolar and 0.08 at 2.0 micromolar. When plants were allowed to deplete P from solutions, the P concentration in the medium dropped to about 0.15 micromolar within 24 hours and 0.05 micromolar within 60 hours. Results indicate that P efflux is a substantial component of net P accumulation at P concentrations normally found in soil solutions.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

Veal heart ribonuclease P has an essential RNA component

The activity of RNase P (EC 3.1.26.5) from veal heart can be abolished by pretreatment of the enzyme preparation with micrococcal nuclease, pancreatic RNase A, or RNase T₁. This indicates that veal heart RNase P contains an RNA component essential for function of the enzyme as has also been shown for $\text{E. coli}$ RNase P (1–3). Additionally, veal heart RNase P has a buoyant density in Cs₂SO₄ of 1.33 g/cm³, which is intermediate between that of protein and nucleic acid.     

Nutrient acquisition from different soil depths by pedunculate oak

Eight oak trees (Quercus robur L.) received ³²P at a soil depth of 50 cm and ³³P at a soil depth of 15 cm at the end of June 2002 through plastic tubes inserted into the mineral soil. The phosphorus uptake from different soil depths was estimated by analysing the concentration of ³²P and ³³P in the foliage of oak growing in a mixed stand in southern Sweden. ³²P and ³³P were recovered in the leaves/needles after 21 and 39 days. The recovery of labelled P in oak was higher from 15 cm soil depth than from 50 cm, however, more than 4% of the total amount of labelled P was taken up from 50 cm. This indicates that oak can utilize deep soil layers for nutrient uptake. A study on the uptake of Cs (as an analogue to K) and ¹⁵N into the leaves was performed on the same trees and detectable amounts of ¹⁵N and Cs were recovered in leaves and buds. This indicates that ¹⁵N and Cs can be used to study nutrient uptake of mature trees from the mineral soil.     

Chemical and biological consequences ofβ-decay

Summary In Part 1 of this article physical and chemical effects of-decay in labelled molecules were reviewed and their potential importance for breaking predetermined and specific bonds were pointed out. After incorporation of labelled biomolecules in living systems, such as viruses, phages or cells, the radioactive decay of the label alters the biological behaviour of the system, in the extreme case causing loss of the ability to reproduce, the extent of these consequences depending strongly on the type of radioisotope.Now Part 2 includes a brief discussion of biological effects associated with-decay, emphasizing the relative importance of local transmutation and internal radiation effects from the decay of³H,¹⁴C,³²P,³³P,³⁵S and¹²⁵I. Attempt is also made, whenever possible at the present stage of understanding, to correlate biological effects with chemical processes on a molecular level.     

Modular construction for function of a ribonucleoprotein enzyme: the catalytic domain of Bacillus subtilis RNase P complexed with B. subtilis RNase P protein

The bacterial RNase P holoenzyme catalyzes the formation of the mature 5′-end of tRNAs and is composed of an RNA and a protein subunit. Among the two folding domains of the RNase P RNA, the catalytic domain (C-domain) contains the active site of this ribozyme. We investigated specific binding of the Bacillus subtilis C-domain with the B.subtilis RNase P protein and examined the catalytic activity of this C-domain–P protein complex. The C-domain forms a specific complex with the P protein with a binding constant of ~0.1 µM. The C-domain–P protein complex and the holoenzyme are equally efficient in cleaving single-stranded RNA (~0.9 min^–1 at pH 7.8) and substrates with a hairpin–loop 3′ to the cleavage site (~40 min^–1). The holoenzyme reaction is much more efficient with a pre-tRNA substrate, binding at least 100-fold better and cleaving 10–500 times more efficiently. These results demonstrate that the RNase P holoenzyme is functionally constructed in three parts. The catalytic domain alone contains the active site, but has little specificity and affinity for most substrates. The specificity and affinity for the substrate is generated by either the specificity domain of RNase P RNA binding to a T stem–loop-like hairpin or RNase P protein binding to a single-stranded RNA. This modular construction may be exploited to obtain RNase P-based ribonucleoprotein complexes with altered substrate specificity.