Requirement for Phosphoglucose Isomerase of Xanthomonas campestris in Pathogenesis of Citrus Canker

A mutant (XT906) of Xanthomonas campestris pv. citri, the causal agent of citrus canker, was induced by insertion of the transposon Tn5tac1 and isolated. This mutant did not grow or elicit canker disease in citrus leaves but was still able to induce a hypersensitive response in a nonhost plant (the common bean). The mutant was also unable to grow on minimal medium containing fructose or glycerol as the sole carbon source. A 2.5-kb fragment of wild-type DNA that complemented the mutant phenotype of XT906 was isolated. Sequence analysis revealed that this DNA fragment encoded a protein of 562 amino acids that shows homology to phosphoglucose isomerase (PGI). Enzyme activity assay confirmed that the encoded protein possesses PGI activity. Analysis of the activity of the promoter of the pgi gene revealed that it was inhibited by growth in complex medium but induced by culture in plant extract. These results demonstrate that PGI is required for pathogenicity of X. campestris pv. citri.     

Cell fission and formation of mini cell bodies by high frequency alternating electric field.

We report the use of high frequency alternating electric fields (AC) to induce deformation of sea urchin eggs, leading to budding of membrane vesicles or fission of cells. Several mini cell bodies can be prepared from a single egg by carefully manipulating the frequency and amplitude of the AC field and the ratio between the interelectrode spacing and the cell diameter, alpha. alpha values between 2.2 and 3.5 have been found to be optimal for inducing fission of sea urchin eggs. In a typical experiment, a sea urchin egg (diameter = 75 microns), suspended in a low ionic medium (conductance < 2 mS/m), was located under the microscope between two platinum wire electrodes, separated by a distance of approximately 200 microns. A medium strength AC field (< 100 V/cm at 2 MHz) was applied to attract the egg to one of the two electrodes via dielectrophoresis. This process took place in a few seconds. The voltage was then slowly increased to approximately 1000 V/cm over approximately 30 s. The cell elongated and separated into two fragments, the larger one containing the nucleus. When the field was turned off, the mother cell and the daughter vesicle retracted to form spherical mini cell bodies that appear to be stable as assessed by the absence of swelling for the duration of the experiment (approximately 15 min). This indicates that membranes of these mini cell bodies were not leaky to ions and small molecules. This procedure could be repeated a few times to make several mini cell bodies from a single egg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Slow equilibration of a denatured protein: comparison of the cluster model with the proline isomerization model

The slow equilibration of the denatured state after rapid unfolding of a globular protein is examined by the cluster model of protein folding (Kanehisa &; Tsong, 1978). The detection of this process in ribonuclease A and its acid catalysis have been considered evidence for the proline isomerization model. Our calculation shows that similar kinetic behavior is also expected for the cluster model.     

Sex identification of owls (family Strigidae) using oligonucleotide microarrays

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.     

Increased binding of liposomes to cells by electric treatment.

The influence of electric field treatments on the interaction of large unilamellar vesicles (liposomes) with animal cells was monitored by the fluorescence assay based on the use of the liposomes loaded by a dye 1-hydroxypyrene-1,3,6-trisulfonic acid (HPTS). It was shown that application of a short electric pulse (100 microseconds of 3-4 kV/cm) to the suspension of cells in presence of vesicles resulted in significant (more than 2 times) increase of the fluorescence associated with cells. The pH-sensitivity of the excitation spectrum of the dye and its interaction with the quencher were used to determine the nature of the phenomenon as the increase of the liposome binding onto the cell surface but not the consequence of a promotion of liposome uptake into the cells by endocytosis. The higher affinity for the liposome caused by the electric field has a lifetime of several minutes. The possible relation of the effect described to the electroporation of cell membranes and to macroscopic changes in membrane structure is discussed.     

Kinetic evidence of microscopic states in protein folding.

Staphylococcal nuclease unfolds at acidic pHs and refolds at neutral pH. Previous kinetic analysis based on both the direct pH jump and the sequential pH jump, from a native condition (pH 7.0) to pHs beyond unfolding transition zones (pH 3.0 and pH 12), and vice versa, supports the mechanism, D3<-->D2<-->D1<-->N0, in which N0 is the native state and D's are the three substates of the denatured form [Chen, H.M., You, J.L., Markin, V.S., & Tsong, T.Y. (1990) J. Mol. Biol. 220, 771-778; Chen, H.M., Markin, V.S., & Tsong, T.Y. (1992) Biochemistry 31, 1483-1491]. Here we show that both the single- and the double-pH jump kinetics of folding and unfolding to the intermediate pHs (3.4-5.0, i.e., in the transition zone), in which both the native and the denatured states coexist, are not compatible with this simple sequential model. At 25 degrees C, log tau 1(-1) (for the D1<-->N0 step) and log tau 2(-1) (for the D2<-->D1 step) vs pH show a square root of-shaped dependence on the final pH, with minimal values (tau 1(-1) of 0.56 s-1 and tau 2(-1) of around pH 3.9. The third relaxation tau 3 (for the D3<-->D2 step, 35 s) was independent of pH in the range 3.4-8.5. The square root of-shaped dependence on pH of log tau 1(-1) and log tau 2(-1) cannot be reproduced by the above but can be accounted for if each of N0, D1, and D2 is composed of many microscopic states in rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential utilization of calcitonin gene regulatory DNA sequences in cultured lines of medullary thyroid carcinoma and small-cell lung carcinoma.

Regulation of expression of the human calcitonin gene was found to differ between two tumor lines of different tissue origin, medullary thyroid carcinoma (TT line) and small-cell lung carcinoma (DMS53 line). Distal 5' DNA elements between -750 and -2000 exhibited a stronger basal activity in DMS53 than in TT cells, whereas proximal DNA sequences between -132 and -252 mediated a dramatic cyclic AMP response in TT but not DMS53 cells.