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The genes psbB, psbH, petB and petD for the components in photosystem II and the cytochrome b6/f complex are clustered and co‐transcribed in liverwort Marchantia polymorpha chloroplasts. On the opposite DNA strand in the spacer region between the genes psbB and psbH, we deduced an open reading frame consisting of 43 sense codons, and designated it as the ORF43 gene. The ORF43 gene was actively transcribed in liverwort chloroplasts. The ORF43 transcripts were entirely complementary to a part of the primary transcripts of the psbB operon. Heterogeneous Northern hybridization showed that the mRNA transcripts for the ORF43 gene increased with greening in pea seedlings. This is the first demonstration of divergent overlapping transcription in chloroplasts.

Four isogenic strains of temperature-sensitive septationless mutants, whose mutations are located on different genes, were used to study the periods of action of the gene products required for the initiation of septum formation during the cell cycle of Bacillus subtilis. The shift-up experiments, in which portions of a synchronous culture of each mutant were transferred to the nonpermissive temperature, showed that the transition point, at which cells attained the ability to divide at the nonpermissive temperature in the cell cycle, was strain specific. Furthermore, the heat shock experiments, in which portions of a synchronous culture were subjected to the nonpermissive temperature before the transition point for a fixed period and shifted back to the permissive temperature, showed that the time interval between the shift-back and the subsequent cell division was specific to each strain but was independent of the age of heat shock. These results led us to the idea that the initiation of septum formation in B. subtilis requires the timed action of the four gene products, each of which functions at a specific stage in the cell cycle. In addition, the result with DNA elongation mutant MK-526, which is also septation defective, supported our previous findings that the initiation of septum formation requires the termination of DNA replication in the previous cell cycle.

Cell division of Escherichia coli K-12 strain PA3092 was inhibited by the addition of adenosine 3',5'-phosphate (cAMP), and the cellular morphology was changed from rods into filaments. Nucleoids in the filaments were regularly distributed and septum formation was perfectly inhibited. This inhibition of cell division by cAMP was reversed by the addition of guanosine 3',5'-monophosphate. To examine whether the inhibitory effect of cAMP on cell division in E. coli PA3092 was specific, its effect in several parental strains was investigated. Induction of cell filamentation by cAMP was observed in E. coli PA309 and P678, but not in E. coli W505, W1, Y10, or the wild-type strain. This result suggests that filamentation by cAMP in E. coli PA3092, PA309, and P678 was due to the mutagenesis by which E. coli P678 was derived from E. coli W595.

In mouse preadipocyte Ob1771 cells, transcription of the insulin-like growth factor-I (IGF-I) gene was stimulated by growth hormone (GH), and IGF-I protein combined with GH in medium was required for their differentiation to adipocytes. During induction of the differentiation, the intracellular expression of each class of IGF-I mRNA was analyzed by reverse transcriptase-polymerase chain reaction. When the cells were cultured in the presence of GH, the class 1del. IGF-I mRNA was a major molecular species among IGF-I mRNAs. In the presence of both GH and IGF-I, the splicing pattern of IGF-I mRNA changed from class 1del. to class 1. Moreover, as detected by Western blotting, the IGF-I protein was present in cells and in the medium only when the cells were cultured in the presence of both GH and IGF-I. We found that IGF-I secreted from Ob1771 cells could act in an autocrine/paracrine fashion and induce the differentiation of other Ob1771 cells. It was demonstrated that the translation efficiency of class 1 mRNA was higher than that of class 1del. mRNA in vitro. These results suggested that stimulation with exogenous IGF-I in the presence of GH was required for the production of class 1 IGF-I mRNA and that the production of the IGF-I protein was activated by increasing the translation efficiency through shifting the splicing pattern of IGF-I mRNA from class 1del. to class 1. Exogenous IGF-I triggered the differentiation by initiating the synthesis of endogenous IGF-I.

The cleavage by bleomycin-Fe(II) complex in the presence of dithiothreitol of 3‘-or 5‘-end-labeled DNA from the region of the bacteriophage G4 origin of complementary strand synthesis was investigated by using the DNA-sequencing technique. Bleomycin cleaved a single-stranded DNA substrate preferentially at inverted repeat sequences, which potentially form stem-and-loop structures, while it cleaved double-stranded DNA substrates with different specificity. The results support the formation of three adjoining stem-and-loop structures in the region of the phage G4 origin of complementary strand synthesis under the low-salt conditions used and suggest a difference in the form of the double helix between the stem and the double-stranded DNA fragment. Bleomycin appears to be a useful reagent for searching stem-and-loop structures. The results may also contribute to the understanding of the mode of action of bleomycin as an antitumor antibiotic.

A membrane protein with a molecular weight of 40,000 (40K protein) was induced concurrently with cell filamentation by cyclic AMP (cAMP) in a fic mutant. In the crp mutant and the wild-type strain, cell filamentation by cAMP was not observed, and the 40K protein was not induced. Induction of the 40K protein is regulated by the cAMP-cAMP receptor protein complex and is closely related to cell filamentation by cAMP in the fic mutant.

phi X174am3trD, a temperature-resistant mutant of bacteriophage phi X174am3, exhibited a reduced ability to grow in a dnaP mutant, Escherichia coli KM107, at the restrictive temperature (43 degrees C). Under conditions at which the dnaP gene function was inactivated, the amount and the rate of phi X174am3trD DNA synthesis were reduced. The efficiency of phage attachment to E. coli KM107 at 43 degrees C was the same as to the parental strain, E. coli KD4301, but phage eclipse and phage DNA penetration were inhibited in E. coli KM107 at 43 degrees C. It is suggested that the dnaP gene product, which is necessary for the initiation of host DNA replication, participates in the conversion of attached phages to eclipsed particles and in phage DNA penetration in vivo in normal infection.

The mechanism of a renal tubular digoxin-cyclosporin A interaction was elucidated using a kidney epithelial cell line and the isolated perfused rat kidney. The cells expressed an excess amount of human P-glycoprotein on the apical membranes by transfection with MDR1 cDNA. Cyclosporin A inhibited the transepithelial transport of digoxin mediated by human P-glycoprotein; net basal-to-apical transport across the cell monolayer was 22.8, 21.2, 6.61 and 0.91 pmol/mg of protein/3 hr in the presence of 0, 1, 5 and 10 microM cyclosporin A, respectively. Cyclosporin A also reduced the renal tubular secretion of digoxin by the kidney. The ratio of fractional excretion/filtration fraction for digoxin was 2.88 +/- 0.71 (mean +/- S.D.) in the control, and this was decreased to 1.21 +/- 0.09 and 1.05 +/- 0.13 in the presence of 1 and 5 microM cyclosporin A, respectively. Because no signs of acute nephrotoxicity were observed, a direct effect of cyclosporin A accounted for the reduced secretion. On the other hand, digoxin did not affect cyclosporin A transport by P-glycoprotein. These findings indicate that serum concentrations of digoxin in patients should be carefully monitored when administered concurrently with cyclosporin A. The present transepithelial transport system using the transfectant cells is a simple and useful screening system for predicting drug interactions that can occur in a clinical situation.

This article represents the first evidence that the renal secretion of the commonly used drug, digoxin, is mediated by P-glycoprotein. In this study, it was demonstrated that digoxin is a substrate of P-glycoprotein, and the mechanism of a clinically important drug interaction, such as digoxin-quinidine, was elucidated. Human P-glycoprotein was expressed on the apical membrane of the porcine kidney epithelial cell line, LLC-PK1 by transfecting with human MDR1 cDNA. The expression and function of P-glycoprotein were confirmed by Southern and Western blotting, RNase protection assay, immunostaining and transporting activity for vinblastine. The transepithelial transport of [3H]digoxin was measured across the cell monolayers grown on microporous polycarbonate membrane filters. The transfectant cells exhibited markedly greater basal-to-apical transport and less apical-to-basal transport than the host cells, and the former was 8-fold greater than the latter. The augmented transepithelial transport resulted from the increased efflux from cells to apical side. This oriented transport was inhibited by the presence of 20 microM vinblastine, quinidine or verapamil. The rate of efflux to the apical side was 2-fold greater than that to the basal side. Quinidine inhibited the efflux to the apical side but did not affect transport into the basal side. These findings demonstrate that digoxin is transported by human P-glycoprotein, which is a previously undiscovered drug transport system in the kidney other than organic cation and anion transport systems, and suggest a molecular mechanism for the renal tubular secretion of digoxin as well as clinically important digoxin-quinidine interaction via P-glycoprotein.

The role of cyclic AMP (cAMP) in the cell cycle of Escherichia coli K-12 was studied in three mutant strains. One was KI1812, in which the cya promoter is replaced by the lacUV5 promoter. In KI1812, isopropyl-beta-D-thiogalactopyranoside induced the synthesis of cya mRNA, and at the same time cell division was inhibited and short filaments containing multiple nuclei were formed. The other strains were constructed as double mutants (NC6707 cya sulB [ftsZ(Ts)] and TR3318 crp sulB [ftsZ(Ts)]). In both double mutants, filamentation was repressed at 42 degrees C, but it was induced again by addition of cAMP in strain NC6707 and introduction of pHA7 containing wild-type crp in TR3318. These results indicate that lateral wall synthesis in the E. coli cell cycle is triggered by the cAMP-cAMP receptor protein complex.