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Abstract In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.

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.

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 lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

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 cryIVA gene encodes a component of the delta-endotoxin of Bacillus thuringiensis subsp. israelensis. By S1 nuclease mapping and primer extension analysis, we have identified the transcriptional initiation site of cryIVA. The transcriptional activity from the promoter was detected only for the sporulating cells more than 3 h after onset of the stationary phase. Upstream from the cryIVA transcriptional initiation site was found a nucleotide sequence partially homologous to the promoter consensus sequence for the E sigma E holoenzyme of Bacillus subtilis. Thus, it was strongly suggested that the identified cryIVA promoter, like some other crystal protein gene promoters, was under the control of sigma 35, the B. thuringiensis homolog of sigma E.

A 3.6-kb BglII-SmaI segment of the transfer region of IncI1 plasmid R64drd-11 was sequenced and characterized. Analysis of the DNA sequence indicated the presence of four genes, traA, traB, traC, and traD, in this region. The expression of the traB, traC, and traD genes was examined by maxicell experiments and that of the traA gene was examined by constructing the traA-lacZ fusion gene. The introduction of frameshift mutations into the four genes indicated that the traB and traC genes are essential for conjugal transfer in liquid medium and on a solid surface. Both were also required for the formation of the thin pilus, which is the receptor for phages I alpha and PR64FS. Upstream of the traA gene, a promoter sequence for sigma 70 of E. coli RNA polymerase was identified by S1 nuclease mapping and primer extension experiments.

Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease. We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues. The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M. xanthus lonV, E. coli lon, and Bacillus brevis lon genes, respectively. When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development. However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M. xanthus was found to be arrested at an early stage in these strains. The mutant strains were able to form neither fruiting bodies nor myxospores.

The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.

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.

P-glycoprotein modulators are respected to be multidrug resistance reversing agents in cancer chemotherapy. Some calcium channel blockers, calmodulin inhibitors or immunosuppressive agents have been used in clinical studies, although the dose of these drugs required to test in vitro experimental data might cause potent pharmacological effects which are not desirable in patients. By using LLC-GA5-COL150 cells that express P-glycoprotein specifically on the apical membranes, we examined the transport of anticancer drugs mediated by P-glycoprotein. Cepharanthin, a biscoclaurine alkaloid, potently inhibits the transport of vinblastine and daunorubicin, both commonly used anticancer agents. The 50% inhibitory concentration of cepharanthin on daunorubicin transport was 2.06 microM. Combined inhibitory effects on daunorubicin transport were observed when cepharanthin was used together with cyclosporin A, a potent immunosuppressive agent and P-glycoprotein modulator. Cepharanthin itself was transported by P-glycoprotein. Transcellular transport of cepharanthin across LLC-GA5-COL150 cell monolayers was saturable when its concentration was under 5 microM, and the transport was inhibited by P-glycoprotein modulators. These results indicate that cepharanthin can reverse multidrug resistance, and proper combination with other P-glycoprotein modulators could potentiate its inhibitory effect on expelling the anticancer drugs out of the cell via P-glycoprotein.

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage phi X174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB', but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB' was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB' protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the prepriming events--i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

Expression of the adenylate cyclase gene (cya) in synchronized Escherichia coli cells was investigated by using the cya-lacZ protein and operon fusion plasmids. The regulation of cya expression during the cell cycle is characterized as follows: cya is expressed during cell elongation; expression is repressed during cell division; regulation is exerted at the transcriptional level. To test cya expression during cell elongation, we constructed a plasmid (pLCR1) in which the lacUV5 promoter operator was fused to the structural gene of cya and investigated the effect of cya expression by isopropyl-beta-D-thiogalactopyranoside (IPTG) on the cell division of cells containing pLCR1. By the addition of IPTG, cell division was inhibited and filaments were formed. Such an inhibitory effect was antagonized by adding cyclic GMP to the culture medium and was not observed in the crp mutant.

We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected by cAMP. The inhibition of cya expression by cAMP was also observed in the cya-lac fused operon system, although it was necessary to increase the amount of cAMP or CRP in the cells to detect the effect. The results indicate that cAMP-CRP is a negative regulator of cya expression at the level of transcription.

A 126-kilobase (kb) physical and genetic map of the Inc-I alpha plasmid R64 was constructed by using the restriction enzymes, BamHI, SalI, XhoI, HindIII, and EcoRI. The replication (Rep) and incompatability (Inc) functions of this plasmid were located in a 1.75-kb segment of an EcoRI fragment, E10 (3.3 kb). In addition, the genes determining growth inhibition of phage BF23 (Ibf), suppression of dnaG ( Sog ), resistance to tetracycline (Tetr), and resistance to streptomycin ( Strr ) were located on the 5.5-kb HindIII-XhoI fragment, the 8.1-kb EcoRI fragment (E5), the 4.6-kb HindIII fragment (H8), and the 4.1-kb HindIII fragment (H10), respectively. The map of R64 was compared with that of ColIb, which belongs to the Inc-I alpha group.

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.

The nucleotide sequences of fic-1 involved in the cell filamentation induced by cyclic AMP in Escherichia coli and its normal counterpart fic were analyzed. The open reading frame of both fic-1 and fic coded for 200 amino acids. The Gly at position 55 in the Fic protein was changed to Arg in the Fic-1 protein. The promoter activity of fic was confirmed by fusing fic and lacZ. The gene downstream from fic was found to be pabA (p-aminobenzoate). There is an open reading frame (ORF190) coding for 190 amino acids upstream from the fic gene. Computer-assisted analysis showed that Fic has sequence similarity with part of CDC28 of Saccharomyces cerevisiae, CDC2 of Schizosaccharomyces pombe, and FtsA of E. coli. In addition, ORF190 has sequence similarity with the cyclosporin A-binding protein cyclophilin.

PA3092 is an Escherichia coli mutant that forms filaments at 43 degrees C in the presence of cyclic AMP (cAMP). The mutation responsible for this phenotype is called fic-1. We cloned fic-1 from PA3092 by selection for the neighboring argD gene. The fic-1 gene product had a relative molecular mass of 21 kilodaltons by the maxicell method. A strain with the fic gene completely deleted was constructed by replacing fic with a kanamycin resistance gene. In one of the fic-deleted strains derived from PA3092, cAMP did not induce cell filamentation at 43 degrees C, but it did in the same strain harboring a plasmid containing the fic-1 gene. These results indicate that the fic-1 gene product is necessary for the induction of cell filamentation by cAMP but is dispensable to the cell. We also found that high levels of NaCl suppressed the cell filamentation induced by cAMP.

Myxococcus xanthus was pulse-labeled with [3H]thymidine immediately after germination of dimethyl sulfoxide-induced spores. The restriction enzyme digests of the total chromosomal DNA from the pulse-labeled cells were analyzed by one-dimensional as well as two-dimensional agarose gel electrophoresis. Four PstI fragments preferentially labeled at a very early stage of germination were cloned into the unique PstI site of pBR322. By using these clones as probes, a restriction enzyme map was established covering approximately 6% of the total M. xanthus genome (330 X 10(3) base pairs). The distribution of the specific activities of the restriction fragments pulse-labeled after germination suggests a bidirectional mode of DNA replication from a fixed origin.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.

The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP- N- acetylglucosamine:alpha-3-d-mannoside beta-1, 2- N- acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N- glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c-Myc epitope tag at the C-terminus elucidates that the N - terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.

When R64 DNA was digested with EcoRI, two DNA fragments not equimolar to the plasmid DNA were produced. A DNA region including these fragments was cloned (pKK009), and the pKK009 DNA sample was found to be a mixture of six or more DNA species with EcoRI, PstI, and AvaI cleavage sites at different positions, suggesting a complex rearrangement of DNA. When a part of the pKK009 DNA was removed by HindIII digestion, 33 different types of plasmids (pKK010-series plasmids) were obtained out of 58 clones tested, but no DNA rearrangement could be observed. On the basis of a comparison of the detailed restriction maps of these pKK010-series plasmids, we propose a model in which four DNA segments invert independently or in groups within the 1.95-kilobase region of R64, so that the arrangements of these four segments change randomly. The fixed pKK010-series plasmid DNA was again rearranged in the presence of R64, indicating that trans-acting gene function may be present to mediate the DNA rearrangement. The gene (tentatively designated as rci) was located on a 4.5-kilobase E9' fragment of R64.

Cyclic AMP (cAMP) inhibited septum formation in Escherichia coli PA3092 and induced cell filamentation at elevated temperatures. This phenomenon was first observed in E. coli PA3092 and is due to a temperature-sensitive mutation. We tentatively named this mutation fic (filamentation induced by cAMP). The fic gene was located near rpsL (formerly strA) on the E. coli K-12 map. the inhibitory effect of cAMP on cell division and filamentation in a fic mutant was not observed in a crp mutant. When cAMP was removed from the culture medium, filaments were divided into rods as the intracellular cAMP level decreased. These results suggest that the cAMP-cAMP receptor protein complex causes filamentation in the fic mutant, E. coli PA3092.

Post-translational modification of proteins was examined during the life cycle of Myxococcus xanthus. A specific pattern of protein phosphorylation was observed in vegetative cells. When spore formation was induced by glycerol, significant changes in the pattern of protein phosphorylation were observed, including the phosphorylation of two membrane proteins. In in vitro experiments, the same membrane proteins were phosphorylated by ATP when the membrane preparation from cells treated with glycerol was used. Changes in the pattern of protein methylation were also observed during spore formation induced by glycerol or fruiting body formation. These results suggest that post-translational protein modification may be required for spore formation or fruiting body formation.

The relationship between protein synthesis and processes of cell division was studied by using synchronized cells of Bacillus subtilis 168. The addition of chloramphenicol at the beginning of synchronous growth prevented septum formation and cell division, suggesting the requirement of protein synthesis for the processes of cell division. Experiments in which the drug was added to the cells at different cell ages showed that the protein synthesis required for the initiation of septum formation was completed at about 15 min and that the protein synthesis required for cell division was completed at about 45 min. By interpreting the result from the concept of the transition point for protein synthesis, it was suggested that the processes of cell division in B. subtilis require at least two kinds of protein molecules which are synthesized at distinct stages in the cell cycle. This was supported by the result of an experiment in which starvation and the readdition of a required amino acid to exponentially growing cells induced two steps of synchronous cell division. Further, the two transition points are in agreement with the estimations obtained by residual division after the inhibition of protein synthesis in asynchronous cells. The relationship of the timing between the completion of chromosome replication and the two transition points was also studied.

Oligomerization of Pd(OAc)2(L)2 (L = pyridine derivatives), a catalyst for the air oxidation of alcohols, is studied with MALDI TOF mass, using dithranol as the matrix. The degree of the Pd oligomerization is influenced by the pyridine ligands, and this ligand effect is similar to one observed for Pd black formation in the catalysis.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.

A shufflon or clustered inversion is a novel type of DNA rearrangement originally discovered in the IncI1 plasmid R64 (T. Komano, A. Kubo, and T. Nisioka, Nucleic Acids Res. 15:1165-1172, 1987). In a 1.95-kilobase region of R64 DNA, four DNA segments inverted independently or in groups, resulting in a complex DNA rearrangement. We found similar types of shufflon in other IncI1 plasmids, including delta, pIP111, pIP565, pIP112, pIP186, R144, R163, R483, and R621a. A variant type of shufflon occurs in the IncI1 plasmid ColIb.