Involvement of DnaK protein in mini-F plasmid replication: temperature-sensitive seg mutations are located in the dnaK gene

Summary The seg mutants (seg-1 and seg-2) of Escherichia coli cannot support the replication of the F factor and mini-F plasmids at 42°C. We cloned the wild-type E. coli chromosomal DNA fragment complementing the seg-1 and seg-2 mutations and found that both mutations were complemented by the wild-type dnaK gene coding for a heat shock protein. Transduction with phage P1 indicated that the seg-2 mutation is located at about 0.3 min in the region containing the dnaK gene in the order trpR-thrA-seg-2-leuB, consistent with the locus of the dnaK gene. Cloning and sequencing of the dnaK gene of the seg mutants showed that there was one base substitution within the dnaK gene in each mutant causing an amino acid substitution. These results indicate that the seg gene in which the seg-1 and seg-2 mutations occurred is identical to the dnaK gene. The mini-F plasmid pXX325 did not transform a dnaK null mutant to ampicillin resistance at 30°C in contrast to plasmids pBR322, pACYC184 and pSC101, which did. The active dnaK (seg) gene product is therefore essential for replication of the mini-F plasmid at both 30° and 42°C.     

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO₃-insensitive ATPase. The ATPase has high specificities both for substrate and Mg²⁺ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl⁻. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.     

The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli.

An Escherichia coli temperature sensitive mutant which produces spontaneously normal size anucleate cells at low temperature was isolated. The mutant is defective in a previously undescribed gene, named mukB, located at 21 min on the chromosome. The mukB gene codes for a large protein (approximately 180 kd). A 1534 amino acid protein (176,826 daltons) was deduced from the nucleotide sequence of the mukB gene. Computer analysis revealed that the predicted MukB protein has distinct domains: an amino-terminal globular domain containing a nucleotide binding sequence, a central region containing two alpha-helical coiled-coil domains and one globular domain, and a carboxyl-terminal globular domain which is rich in Cys, Arg and Lys. A 180 kd protein detected in wild-type cell extracts by electrophoresis is absent in mukB null mutants. Although the null mutants are not lethal at low temperature, the absence of MukB leads to aberrant chromosome partitioning. At high temperature the mukB null mutants cannot form colonies and many nucleoids are distributed irregularly along elongated cells. We conclude that the MukB protein is required for chromosome partitioning in E. coli.     

Further study on selective transmission of mitochondrial DNA in heteroplasmic lines of Drosophila melanogaster.

The temperature-dependent transmission of mitochondrial DNA (mtDNA) was investigated in heteroplasmic lines of Drosophila melanogaster established by germ-plasm transplantation. Using D. melanogaster, D. simulans and D. mauritiana as germ-plasm donors, five recipient-donor combinations of heteroplasmy, differing from those previously examined (Matsuura et al., 1991), were constructed. For intraspecific reciprocal combinations, donor mtDNA in one combination was retained at 25 degrees C but was almost lost by the tenth generation at 19 degrees C. In the reciprocal, the proportion of the same type of recipient mtDNA decreased more quickly at 19 degrees C than 25 degrees C. Decreasing rates at 19 degrees C in the reciprocals differed from each other. For interspecific combinations, two species were used as germ-plasm donors. Donor mtDNA derived from D. simulans was lost at both temperatures and the rate of decrease was greater at 19 degrees C than 25 degrees C. The proportion of donor mtDNA derived from D. mauritiana increased at a greater rate at 25 degrees C than 19 degrees C when using two different strains of D. melanogaster as recipients. These results suggest that both the nuclear and two types of mitochondrial genomes are involved in the selective transmission of mtDNA.     

Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant.

The partition-proficient mini-F plasmid pXX325 was stably maintained in the mukB null mutant, which is defective in chromosome partitioning into the two daughter cells. In the null mutant, the plasmid was partitioned into both nucleate and anucleate daughter cells, independently of host chromosomes.     

Chromosome partitioning in Escherichia coli: novel mutants producing anucleate cells.

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named mukA1, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mukA1 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin E1 protein, in addition to anucleate cell formation. Cloning of the mukA gene indicates that the mukA1 mutation is recessive and that the mukA gene is identical to the tolC gene coding for an outer membrane protein.     

Investigation of Brassica oleracea and Nasturtium officinale seeds for the presence of epithiospecifier protein

Cabbage seeds contain 5 glucosinolates and on autolysis produce, in particular, 1-cyano-2-3-epithiopropane. Watercress seeds contain 4 glucosinolates, but none capable of forming a cyanoepithioalkane. Cabbage exhibited behaviour commensurate with possession of an epithiospecifier protein (ESP) whilst watercress did not show any such activity.     

Reduced stroke volume during exercise in postural tachycardia syndrome.

Postural tachycardia syndrome (POTS) is characterized by excessive tachycardia without hypotension during orthostasis. Most POTS patients also report exercise intolerance. To assess cardiovascular regulation during exercise in POTS, patients (n = 13) and healthy controls (n = 10) performed graded cycle exercise at 25, 50, and 75 W in both supine and upright positions while arterial pressure (arterial catheter), heart rate (HR; measured by ECG), and cardiac output (open-circuit acetylene breathing) were measured. In both positions, mean arterial pressure, cardiac output, and total peripheral resistance at rest and during exercise were similar in patients and controls (P > 0.05). However, supine stroke volume (SV) tended to be lower in the patients than controls at rest (99 +/- 5 vs. 110 +/- 9 ml) and during 75-W exercise (97 +/- 5 vs. 111 +/- 7 ml) (P = 0.07), and HR was higher in the patients than controls at rest (76 +/- 3 vs. 62 +/- 4 beats/min) and during 75-W exercise (127 +/- 3 vs. 114 +/- 5 beats/min) (both P < 0.01). Upright SV was significantly lower in the patients than controls at rest (57 +/- 3 vs. 81 +/- 6 ml) and during 75-W exercise (70 +/- 4 vs. 94 +/- 6 ml) (both P < 0.01), and HR was much higher in the patients than controls at rest (103 +/- 3 vs. 81 +/- 4 beats/min) and during 75-W exercise (164 +/- 3 vs. 131 +/- 7 beats/min) (both P < 0.001). The change (upright - supine) in SV was inversely correlated with the change in HR for all participants at rest (R(2) = 0.32), at 25 W (R(2) = 0.49), 50 W (R(2) = 0.60), and 75 W (R(2) = 0.32) (P < 0.01). These results suggest that greater elevation in HR in POTS patients during exercise, especially while upright, was secondary to reduced SV and associated with exercise intolerance.