Genetic and physiological studies of abortive infections of hydroxymethyluracil-containing bacteriophages in lysogens of temperate Bacillus subtilis bacteriophage SPO2.

Wild-type bacteriophage phie and IS (interference-sensitive) mutants of the related phage SP82G did not productively infect strains of Bacillus subtilis that were lysogenic for temperate phage SPO2. In these abortive infections, the sensitive phages adsorbed to and penetrated the nonpermissive host, phage-directed macromolecular syntheses were initiated, but both viral and bacterial nucleic acid production abruptly stopped about 15 min after addition of the phages. The cessation of RNA and DNA synthesis was preceded or coincident with a reduction in oxygen utilization by the infected cultures. Genetic studies of both phie and SP82G suggest sensitivity to SPO2-mediated abortive infection was controlled by a single gene. A mutant of SPO2, SPO2ehp4-, lysogens of which no longer interfere with the development of SP82GIs, was also isolated. The discovery of this ehp- variant suggests the normal SPO2 prophage synthesized a substance that alters cell physiology in some manner detrimental to SP82GIs development. Since SPO2ehp4- grew on and lysogenized bacteria sensitive to wild-type SPO2, the product of the eph gene was apparently not an essential function of this temperate phage.Overall, these observations exhibit remarkable similarities to the inhibition of T4rII mutants by the product of the rex gene of phage lambda.     

The role of temperate bacteriophage SP beta in prophage-mediated interference in Bacillus subtilis.

Virulent bacteriophage phi 1 grows on a variety of Bacillus subtilis strains, mutants of this virus which abortively infect the transformable bacillus. B. subtilis 168, while retaining the ability to productively infect related bacteria have been found. In the present study, we demonstrate that the inability of one such variant, phi 1m, to develop normally in strain 168 is mediated by cryptic prophage SP beta. The latter is a temperate bacteriophage which is carried by B. subtilis 168 and most strains derived from this bacterium. Phi 1 m infection of SP beta lysogens begins with apparently normal adsorption, penetration, and inititaion of virus-directed syntheses. At about the 20th min of the latent period, however, there is an abrupt cessation of nucleic acid synthesis and cellular respiration, accompanied by a change in cell permeability. This course of events can be altered to a permissive infection by mutation in the mpi gene of SP beta, by mutation in the spoOA gene of the host, or by growing SP beta lysogens at high temperature. In addition, we found a second class of phi 1 mutants which abortively infect B. subtilis 168 derivatives even in the absence of the SP beta prophage.     

Biosynthesis of chlorophyll P-680 of photosystem II in greening leaves of plants adapted to heat shock

In etiolated pea and maize leaves illuminated after incubation at 38 degreesC, a new dark reaction was shown manifested in the bathochromic shift of spectral bands and accompanied by esterification of the product of protochlorophyllide photochemical reduction--Chld 684/676: Chld 684/676 --> Chl 688/680. After completion of the reaction a rapid (20-30 sec) quenching of the fluorescence of the reaction product (Chl 688/680) was observed. The reaction Chld 684/676 --> Chl 688/680 is inhibited under anaerobic conditions and in the presence of cyanide; the reaction accompanied by Chl 688/680 fluorescence quenching is not observed in pea mutants with impaired function of photosystem II reaction centers. The spectral properties of the formed Chl form with the absorption maximum at 680 nm, fluorescence quenching, and simultaneous synthesis of pheophytin suggest that the reaction is connected with the chlorophyll of photosystem II reaction center--P-680.     

The antigenic composition of Neospora caninum

Neospora caninum is an apicomplexan parasite which causes neosporosis, namely stillbirth and abortion in cattle, and neuromuscular disease in dogs. Although N. caninum is phylogenetically and biologically closely related to Toxoplasma gondii, it is antigenically clearly distinct. In analogy to T. gondii, three stages have been identified. These are: (i) asexually proliferating tachyzoites; (ii) tissue cysts harbouring slowly dividing bradyzoites; and (iii) oocysts containing sporozoites. The sexually produced stage of this parasite has only recently been identified, and has been shown to be shed with the faeces from dogs orally infected with N. caninum tissue cysts. Thus dogs are definitive hosts of N. caninum. Tachyzoites can be cultivated in vitro using similar techniques as previously described for T. gondii. Methods for generating tissue cysts containing N. caninum bradyzoites in mice, and purification of these cysts, have been developed. A number of studies have been undertaken to identify and characterise at the molecular level specific antigenic components of N. caninum in order to improve serological diagnosis and to enhance the current view on the many open questions concerning the cell biology of this parasite and its interactions with the host on the immunological and cellular level. The aim of this paper is to provide an overview on the approaches used for detection of antigens in N. caninum. The studies discussed here have had a great impact in the elucidation of the immunological and pathogenetic events during infection, as well as the development of potential new immunotherapeutic tools for future vaccination against N. caninum infection.     

Detection of surface-associated and intracellular glycoconjugates and glycoproteins in Neospora caninum tachyzoites

The surface-associated molecules of the invasive stages of apicomplexan parasites such as Neospora caninum and Toxoplasma gondii are most likely crucially involved in mediating the interaction between the parasite and its host cell. In N. caninum, several antigens have recently been identified which could participate in host cell adhesion and/or invasion. These are antigens which are either constitutively expressed on the outer plasma membrane, or antigens which are only transiently localised on the surface as they are expulsed from the secretory vesicles either prior, or after host cell invasion. Some of these proteins have been characterised at the molecular level, and it has been shown that they are, with respect to protein sequences, closely related to homologous counterparts in T. gondii. Nevertheless, there is only a low degree of cross-antigenicity between the two species. In microbial interactions it has been shown that carbohydrates could also play a crucial role in host cell recognition and immunological host parasite interactions. In this study we present data which strongly suggest that the surface of N. caninum tachyzoites is glycosylated. In SDS-PAGE, glycoproteins comigrated largely with glycosylphosphatidylinositol-anchored proteins which were identified using in vivo [3H]ethanolamine labelling followed by autoradiography. The lectin Con A reacted strongly with the surface of these parasites, binding of which is indicative for the presence of N-glycans. Additional surface binding was observed, although only in a subpopulation of all tachyzoites, for wheat germ agglutinin and Jacalin. Intracellular binding sites for Con A were mainly associated with the parasite dense granules. By lectin labelling of Western blots of N. caninum protein extracts, glycoproteins were identified which reacted specifically with the lectins Con A, wheat germ agglutinin, Jacalin and soy bean agglutinin.     

Phosphate Regulation of Nitrate Assimilation in Soybean

It is known that phosphorus deficiency results in alterationsin the assimilation of nitrogen. An experiment was conductedto investigate mechanisms involved in altered ¹⁵NO^–₃ uptake,endogenous ¹⁵N translocation, and amino acid accumulation insoybean (Glycine max L. Merrill, cv. Ransom) plants deprivedof an external phosphorus supply for 20 d in solution culture.Phosphorus deprivation led to decreased rates of ¹⁵NO^–₃uptake and increased accumulation of absorbed ¹⁵N in the root.Both effects became more pronounced with time. Asparagine, theprimary transport amino acid in soybean, accumulated in largeexcess in roots and stems. In roots of phosphorus-deprived plants,concentrations of ATP and inorganic phosphate declined rapidly,but dry weight accumulation was similar to or above that ofthe control even after 20 d of treatment. Arginine accumulationin leaves was greatly enhanced, even though ¹⁵N partitioninginto the insoluble reduced-N fraction of leaves was unaffected.The results suggest that decreases in NO^–₃ uptake in lowphosphorus plants could be caused by feedback control factorsand by limited ATP availability. The decline in endogenous Ntransport from the root to the shoot may be associated withchanges in membrane properties, which also result in paralleleffects on hydraulic conductance and the upward flow of waterthrough the plant. Key words: Phosphorus stress, nitrate uptake, nitrate translocation, arginine     

PCR-based assays of mendelian polymorphisms from anonymous single-copy nuclear DNA: techniques and applications for population genetics

This paper outlines a PCR-based approach for population genetics that offers several advantages over conventional Southern blotting methods for revealing restriction-fragment-length polymorphisms (RFLPs) in nuclear DNA. Primers are constructed from clones isolated from a nuclear DNA library, and these primers subsequently are employed in in vitro syntheses of homologous regions. Amplified products are then screened directly for RFLPs by using gel-staining procedures. Population applications for this PCR-based approach, including potential strengths and weaknesses, are exemplified by two RFLP data sets generated to estimate (a) male-mediated gene flow in the green turtle (Chelonia mydas) and (b) geographic population genetic structure in the American oyster (Crassostrea virginica). Restriction assays of amplified products from 14 or 15 independent primer pairs in each species revealed polymorphisms at several loci that proved highly informative in the population genetic analyses. In general, the Mendelian polymorphisms produced by this PCR-based approach will provide useful genetic markers for population studies, particularly in situations where simpler and less expensive allozyme methods have failed, for whatever reason, to provide adequate information.      

The Repetitive Microtubule-Associated Proteins MARP-1 and MARP-2 of Trypanosoma brucei

The microtubular membrane skeleton of Trypanosoma brucei contains two closely related, repetitive, high-molecular-weight microtubule-associated proteins, MARP-1 and MARP-2 (MARP for icrotubule- ssociated epetitive roteins). Their structure is unusual in that they consist of tandemly arranged, strongly conserved 38-amino-acid repeat units over almost their entire length of about 320 kDa. Their nonrepetitive N and C ends are comparatively short. The predicted amino acid sequences reveal a gradient of similarity between MARP-1 and MARP-2 which increases from the N-terminus (no significant similarity) through the repeat domain (50% similarity) to the C-terminus (94.5% similarity). Transfection of mammalian cell lines with recombinant fragments of MARP-2 demonstrate that the nonrepetitive C-terminus of MARP-2 binds specifically to microtubules. This C-terminus does not show sequence similarity with any other microtubule-associated proteins and thus appears to represent a novel type of microtubule-binding domain.