Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Binding of penicillin to thiol-penicillin-binding protein 3 of Escherichia coli: identification of its active site

Summary In order to determine the active site of penicillin-binding protein 3 of Escherichia coli (PBP3), the serine residue at position 307 was replaced with alanine, threonine or cysteine by oligonucleotide-directed site-specific mutagenesis. Since a unique BanII site exists at the position corresponding to serine-307, BanII digestion of the plasmid DNA after mutagenesis resulted in significant enrichment of the mutant plasmids. For mutagenesis, the gene coding for PBP3 (ftsI) was inserted into the expression cloning vector pIN-IIB. The hybrid protein produced was able to bind penicillin while mutant PBP3 in which serine-307 was replaced with either alanine or threonine did not lead to any detectable binding. However, contrary to the report of Broome-Smith et al. (1985) thiol-penicillin-binding protein 3, in which serine-307 was replaced with cysteine, was still able to bind penicillin. Replacement of serine-445 with an alanine residue had no effect on penicillin binding to PBP3.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

The heterogeneity of human fibroblasts as determined from the effects of hydrocortisone on cell growth and specific dexamethasone binding

To elucidate the heterogeneity of human fibroblasts from lung and skin, the effects of hydrocortisone on cell proliferation and the specific dexamethasone binding to cells were studied. Hydrocortisone at physiological concentrations stimulated the proliferation in three strains of human fetal lung fibroblasts and inhibited it in two strains. There are two kinds of fibroblasts in the human fetal lung in addition to the human skin fibroblasts reported previously. Dexamethasone-binding experiments showed that human fibroblasts may be classified into two groups with respect to the dissociation constant (Kd) of the binding reaction. The heterogeneity of human fibroblasts shown by Kd could not be correlated to classification on the basis of the effects of hydrocortisone on cell proliferation. The differences in Kd for the binding reactions suggest differences in donor tissues from which human fibroblasts are derived.     

Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.     

A Study on Erythrocyte Membrane Plasmalogen in Myotonic Dystrophy

Phospholipid classes that included plasmalogens of erythrocyte membranes in seven myotonic dystrophy (MyD) patients and seven normal controls were analyzed by HPLC. No significant difference in phospholipid classes was found between patients with MyD and normal controls, but there was a visible difference in peak profiles of compounds of the phosphatidylethanolamine class. In the study of plasmalogens, we used two preparation methods: exposure to HCl and deacylation with mild alkaline. The area ratio of the plasmalogen form to the diacyl form in the phosphatidylethanolamine class of MyD erythrocyte membranes was significantly lower than that of normal controls. Fatty acid analyses showed that fatty acids of both phosphatidylethanolamine subclasses have less unsaturation in MyD.     

Effect of various catecholamine antagonists on prolactin secretion in conscious male rabbits

To clarify physiological roles of catecholaminergic systems in the control of rabbit prolactin (PRL) release, the effect of various catecholamine receptor antagonists on plasma PRL levels was examined in conscious, freely moving male rabbits. An intravenous (iv) injection of yohimbin (2.5 mg/kg body wt), an alpha 2-adrenoreceptor antagonist, but not prazosin (2 mg/kg body wt), an alpha 1-adrenergic receptor antagonist, resulted in a significant elevation of plasma PRL. Conversely, propranolol (2.5 mg/kg body wt, iv), a nonselective beta-adrenoreceptor antagonist, and metoprolol (2.6 mg/kg body wt, iv), a beta 1-adrenergic antagonist, slightly but significantly suppressed basal levels of plasma PRL. On the other hand, haloperidol (0.5 mg/kg body wt, iv), pimozide (0.3 mg/kg body wt, iv), sulpiride (5 mg/kg body wt, iv), chlorpromazine (3 mg/kg body wt, iv), and YM-09151-2 (0.2 mg/kg body wt, iv), all dopamine receptor antagonists caused a significant increase in plasma PRL. These results suggest that dopaminergic and alpha 2-adrenergic mechanisms exert a tonic inhibitory role and beta-adrenergic mechanisms, probably beta 1, a tonic stimulatory role in the regulation of PRL release in the rabbit.     

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

The morphology and anatomy ofHydrastis (Ranunculales): Systematic reevaluation of the genus

Evidence from morphology and anatomy (including embryology), as well as from palynology, chemistry and cytology, indicates thatHydrastis is quite divergent from Ranunculaceae (in which the genus has been most often included) as well as from both Glaucidiaceae and Berberidaceae. Distinctive features ofHydrastis, which demarcate it from Ranunculaceae but which are sometimes shared by Berberidaceae, are: the unique mode of origin of the vascular supply to stamens and carpels; the micropyle being formed by both integuments; the xylem not V-shaped in cross section; scalariform vessel perforations present; haploid chromosome number 13; pollen tectum consisting of a compound layer of striae; leaf mesophyll not differentiated; the unique course of stem medullary bundles; D-galactose present. Its distinctive higher haploid chromosome number, as well as its many less-specialized character states (in floral structure, leaf anatomy, and xylem and vessel morphology), suggest thatHydrastis is a relictual primitive group which diverged early from a common ancestral stock of Ranunculaceae, Berberidaceae and probably of Circaeasteraceae; at least some of the features shared byHydrastis and one or another of the families concerned seem to be a heritage from their common ancestor. We propose a reestablishment of a monotypic family, Hydrastidaceae.