P1 plasmid replication: measurement of initiator protein concentration in vivo.

To study the functions of the mini-P1 replication initiation protein RepA quantitatively, we have developed a method to measure RepA concentration by using immunoblotting. In vivo, there are about 20 RepA dimers per unit-copy plasmid DNA. RepA was deduced to be a dimer from gel filtration of the purified protein. Since there are 14 binding sites of the protein per replicon, the physiological concentration of the protein appears to be sufficiently low to be a rate-limiting factor for replication. Autoregulation is apparently responsible for the low protein level; at the physiological concentration of the protein, the repA promoter retains only 0.1% of its full activity as determined by gene fusions to lacZ. When the concentration is further decreased by a factor of 3 or increased by a factor of 40, replication is no longer detectable.     

Evaluation of the multitest medium for rapid presumptive identification of Vibrio cholerae from environmental sources

The multitest V. cholerae medium (VC medium) for rapid presumptive identification of Vibrio cholerae was evaluated. On the basis of reactions in the VC medium, 379 strains recovered during a yearlong ecological study in Calcutta were presumptively identified as V. cholerae. Further phenotypic characterization of these strains revealed that the reactions of 371 (97.9%) isolates were consistent with that of V. cholerae. False-positive reactions were exhibited by eight (2.1%) strains, three of which were identified as Vibrio fluvialis biotype 1. By slightly varying the basic formulation of the VC medium, we could eliminate some false-positive reactions. On the basis of the present evaluation, we recommend the routine use of the VC medium.     

A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins

A transposon-induced mutant (T8-1) of Bradyrhizobium japonicum (61A76) was unable to develop into the nitrogen-fixing endosymbiotic form, the bacteroid. Comparison between this mutant and T5-95, an ineffective (non-nitrogen fixing, Fix^-) mutant, confirmed that the process of bacteroid development is a distinct phase of differentiation of the endosymbiont and is independent of nitrogen fixation activity. The T8-1 mutant was able to induce normal-size nodules which differentiated two plant cell types and contained numerous infection threads. However, the infected cells were devoid of bacteroids. Electron microscopy revealed that the ends of the infection threads were broken down in a normal manner once the thread had penetrated the cells, but the mutant was not internalized by endocytosis. The lack of peribacteroid membrane (PBM) in nodules induced by this mutant was correlated with a reduced level of expression of plant genes coding for PBM nodulins. These genes were expressed in the T5-95 mutant, showing that the low expression in T8-1 was not due to the lack of nitrogen fixation. One of the PBM nodulins, nodulin-26, was found at normal levels in the nodules which lack PBM, suggesting that there are at least two developmental stages in PBM biosynthesis. These data suggest that a coordination of plant and Rhizobium gene expression is required for the release and internalization of bacteria into the PBM compartments of infected cells of nodules.author for correspondence     

Small-angle X-ray study on the structure of ribulose-1,5-bisphosphate carboxylase-oxygenase from Rhodospirillum rubrum

The quaternary structure of ribulose-1,5-bisphosphate carboxylase-oxygenase (rubisco) from Rhodospirillum rubrum, an enzyme consisting of two large subunits, L₂, was investigated by small-angle X-ray scattering. In the presence of HCO ₃ ^- and Mg²⁺, rubisco is in the active state and displays a radius of gyration of 2.96 nm, a maximum diameter of 9.5 nm and a volume of 170 nm³. A model is presented where the subunits are arranged back-to-back, rotated relative to each other by 90°, and shifted by 1.3 nm. Upon inactivation by removal of HCO ₃ ^- and Mg²⁺, the model swells slightly without any distinct changes in configuration. This contrasts with our previous observations with rubisco from Alcaligenes eutrophus, an enzyme composed of small (S) and large (L) subunits, L₈S₈, where inactivation gives rise to substantial changes in configuration.Abbreviations RuBP Ribulose-1,5-bisphosphate - 3-PGA 3-phosphoglyceric acid     

A study of sutural bones in different morphological forms of skulls

117 adult human skulls were classified for three morphological forms, i.e. dolichocephalic, mesocephalic and brachycephalic, and were examined for the presence of sutural bones in each form. Sutural bones occur in each forms of the skulls with no statistically significant differences. This finding is interpreted as indicating that sutural bones are not formed secondary to stress, otherwise the incidence of the sutural bones would have been differed in different morphological forms of skulls.     

Tobacco lines with high copy number of replicating recombinant geminivirus vectors after biolistic DNA delivery

The feasibility of obtaining clonal lines with replicating, multicopy geminivirus vectors by direct DNA trans-formation of cultured tobacco cells was studied. The replicating vectors pTGA32 and pST31 are based on the tomato golden mosaic virus (TGMV) A genome and encode the neomycin phosphotransferase type II (NPT-II) enzyme that confers kanamycin resistance to plant cells. Following introduction into plant cells, unit-length viral genomes were released from the tandem repeats and replicated. In protoplasts, replication of unit-length pTGA32 and pST31 was about as efficient as replication of unit-length DNA A from plasmid pTGA26, which contains 1.5 copies of wild-type DNA A. Tobacco suspension culture cells were bombarded with the recombinant DNA A constructs and selected for kanamycin resistance. The number of kanamycin-resistant clones per bombardment was about the same when the TGMV DNA A vectors or a non-replicating plasmid (pLC14) which also encodes NPT-II was used. Replicating, unit-length DNA A in up to approximately 1000 copies per cell was found in about 10% of the kanamycin-resistant clones selected following bombardment of cells with TGMV vectors. The results suggest that geminiviruses may serve as useful multicopy vectors in cultured cells.     

The effects of slip velocity at a membrane surface on blood flow in the microcirculation

Closed-form solutions are presented for blood flow in the microcirculation by taking into account the influence of slip velocity at the membrane surface. In this study, the convective inertia force is neglected in comparison with that of blood viscosity on the basis of the smallness of the Reynolds number of the flow in microcirculation. The permeability property of the blood vessel is based on the well known Starling's hypothesis [11]. The effects of slip coefficient on the velocity and pressure fields are clearly depicted.     

Barley yield under saline water cultivation

Summary In a microplot experiment conducted during the winter seasons of 1979–80 and 1980–81 on a sandy loam soil in the semi-desert tract, the accumulation of salts was found to be highest in March after harvest of the barley crop grown with saline water of EC values ranging from 2.2 to 24 mmhos/cm. The average EC of saturation extract of the surface soil layer (0–15 cm) was 0.79 times that of the applied irrigation water at the time of crop harvest, however, accumulated salts of the winter season were leached by the following monsoon rains. The average SAR of saturation extract of soil was 1.5 times that of the irrigation water in March but quite low in November. Highly significant correlations, (+0.90 to 0.99) at the post irrigated period between ECse of soils and EC of waters and SARse of soils and SAR of waters have been observed. Barley could be grown economically with irrigation water upto EC 16 mmhos/cm; however an average reduction in grain yield or not more than 43.5% compared to the yield under irrigation with tube well water of EC 2.2 mmhos/cm, was obtained. The starch, N and P contents decreased and that of K and Na increased in the grain with the use of saline waters. The performance of DL-85 variety was best and its K/Na ratio was also higher than that of other tested varieties.     

Structural and immunochemical studies on Pterospermum suberifolium gum

The water-soluble polysaccharide from Pterospermum suberifolium gum is composed of l-rhamnose (24.0%), d-glucose (5.6%), d-galacturonic acid (32.4%), and d-glucuronic acid (19.7%), and it precipitated 77% of the antibody nitrogen from anti-Pneumococcal Type XXIII serum. From the results of methylation, periodate oxidation, and partial hydrolysis studies on the gum and its carboxyl-reduced product, a structure was assigned to its repeating unit. Inhibition of the cross-precipitation using the monosaccharides and the oligosaccharides obtained from the polysaccharide indicated that l-rhamnose and d-glucose were immunospecific, the former to the greater extent.     

Structural mapping of Kelch13 mutations associated with artemisinin resistance in malaria

Mutations in Plasmodium falciparum gene kelch13 (pfkelch13) are strongly and causally associated with resistance to anti-malarial drug artemisinin, but their effects on PfKelch13 structure and function remain unclear. Utilizing the publicly available three-dimensional structure of PfKech13 (PDB ID: 4yy8), we find that most of the mutations in its propeller domain occur in two spatial clusters. Of these, one cluster is enriched in surface exposed residues which may drive PfKelch13-centered protein interactions, and the second cluster mostly contains residues which are buried and whose mutations may destabilize PfKelch13 structure. The most prevalent resistant mutations C580Y and Y493H are distal from the above two clusters. The C580Y mutation creates sterically unfavourable contacts while Y493H possibly alters the hydrophobic core of the propeller domain. These analyses will facilitate further experimental studies aimed at understanding how mutations in pfkelch13 lead to artemisinin resistance.