Bacteriophage phi X174 DNA synthesis in Escherichia coli HF4704S (dnaHts) cells.

The DNA synthesis of bacteriophage phiX174 in Escherichia coli HF470S, a mutant temperature sensitive in the initiation of DNA replication (dnaHts), has been examined. In HF4704S cells, phiX174 can grow normally at 27 degree C whereas the phage cannot grow after the cessation of DNA synthesis of the host cells at 42 degrees C. Upon infection, phiX174 DNA can be injected into the host cell and the parental replicative form can be formed, but the progency replicative form cannot be synthesized at 43 degrees C in the absence of host DNA synthesis. The progency replicative form cannot be synthesized at 27 degrees C in the presence of 30 mug chloramphenicol/ml in the host cell which has been incubated for 74 min at 43 degrees C followed by transfer to 27 degrees C in the presence of 30 mug chloramphenicol/ml. When 30 mug chloramphenicol/ml is added later than 5 min after the temperature shift-down to 27 degrees C, the progency replicative form synthesis is not inhibited. Thus, the host cell function, for which the gene dnaH is responsible, has been shown to be essential to the progency replicative form production.

Deoxyribonucleic acid synthesis induced with ultraviolet light in Brij 58-treated Bacillus subtilis spores germinated in the presence of chloramphenicol.

The direct measurement of ultraviolet light-stimulated DNA synthesis in the permeable Bacillus subtilis cells was performed. Bacillus subtilis spores germinated in the presence of chloramphenicol were treated with Brij 58 and irradiated with ultraviolet light, and (3H)dTTP was incorporated into these cells by the DNA polymerase assay system. Characteristics of the incorporation were distinct from those into spores germinated in the absence of chloramphenicol and treated with Brij 58, in the respect that the former incorporation did not require ATP and only partially depended on the presence of all four deoxyribonucleoside triphosphates. The incorporation of (3H)dTTP into DNA was confirmed by CsCl density gradient centrifugation. A DNA polymerase I-deficient strain, JBl 49(59) had no (3H)sTTP incorporating activity induced by ultraviolet light irradiation when the germinated spores were treated with Brij 58. Analysis of alkaline sucrose gradient centrifugation revealed that fragmented DNA caused by ultraviolet light irradiation was rejoined to the size of DNA of non-irradiated cells by incubating irradiated cells in the DNA polymerase assay mixture containing NAD+. The results also suggested that a machinery of DNA repair probably pre-existed in the spore.

Bacteriophage phiX174 growth in an Escherichia coli dnaIts mutant, KS810.

A bacteriophage phiX174-sensitive Escherichia coli dnaIts mutant, KS810, was constructed and growth of phiX174 in the cells was investigated. phiX174 and phiX174am3trD could grow normally at 43 degrees C as well as 27 degrees C, therefore we conclude that the growth of bacteriophage phiX174 is not dependent upon the host dnaI gene product.

Inactivation of infectivity of phiX174 DNA by menadione and reduced menadione.

Interaction of menadione and reduced menadione with phage phiX174 DNA was investigated. A concentration of 2-10(-4) M menadione inactivated 60% of the infectivity of phiX174 DNA to spheroplasts of Escherichia coli, while reduced menadione inactivated 97% of the infectivity of phiX174 DNA at the same concentration. Alkaline sucrose gradient centrifugation revealed 2-10(-5) M reduced menadione caused approximately 24% of phiX174 DNA to produce strand break under the condition of 80% lethanlity. DNA strand break was not observed even at 4 - 10(-4) M menadione. These results indicated that there were different mechanisms for inactivation of phiX174 DNA between menadione and reduced menadione.

Sequence-specific modification of DNA by 6-hydroxybenzo[a]pyrene.

6-Hydroxybenzo[a]pyrene cleaved phi X174 supercoiled DNA to open circular DNA in the presence of heavy metal ions. It induced an alkali-labile modification in DNA via an oxygen-radical-mediated reaction; the most frequent alkali-labile sites were on the 3' side of the pyrimidine residues of the pyrimidine cluster.

In vitro conversion of S13 viral DNA in phage particles to the double-stranded DNA.

The conversion of single-stranded DNA in S13 intact phage particles to the double-stranded replicative form DNA was observed in cell extracts prepared from Escherichia coli H560 (S13s, polA, endA) cells lysed with lysozyme and the non-ionic detergent, Brij 58. The DNA product, which associated with a rapidly sedimenting component, was identified as RFII-DNA with a gap by sedimentation analysis. The conversion was inhibited by N-ethylmaleimide, but not by rifampicin, nicotinamide mononucleotide or polymyxin B. The dnaB gene product was involved in the replicative system. Similar extracts prepared from a S13-resistant E. coli strain K12W6 also catalyzed this synthesis.

Studies on pig serum lipoproteins. III. Affinity chromatography of native lipoproteins on concanavalin A-sepharose.

The comparison of the binding capacities of the three major classes of pig serum lipoproteins, very low-density, low-density and high-density lipoproteins, to concanavalin A, was demonstrated by affinity chromatography on concanavalin A-Sepharose. Very low-density lipoprotein was separated into two fractions (60 to 66% of total protein was adsorbed). Each fraction had different electrophoretic mobility in pore size gradient gel. The majority of the carbohydrate was found in the adsorbed fraction. The carbohydrate content of the unadsorbed fraction was 0.14% sialic acid. 0.47% hexosamine and 0.93% neutral sugars, and of the adsorbed fraction, 2.05, 3.21 and 4.20%, respectively. The adsorbed and unadsorbed fractions contained fucose, mannose and galactose in the molar ratio of 1.0 : 3.6 +/- 0.2 : 2.2 +/- 0.4 and 1.0 : 3.1 +/- 0.2 : 2.5 +/- 0.3, respectively. Based on these results, two different molecular species were proved to be present in very low-density lipoproteins. In high-density lipoproteins, 80 to 85% of the total protein was not adsorbed on concanavalin A-Sepharose in spite of the presence of mannose in the apoprotein. In contrast to these lipoproteins, low-density lipoprotein was completely adsorbed on concanavalin A-Sepharose. However, the separation of the subfractions of low-density lipoprotein as well as the subfractions of high-density lipoprotein could not be achieved by this affinity column. The carbohydrate content of eluted fractions of low-density and high-density lipoproteins was identical with the previously reported values obtained in native lipoproteins. This difference in affinities for concanavalin A was also evidenced by gel electrophoretic profiles in urea and in sodium dodecyl sulfate which showed different glycoprotein distribution in each class of lipoproteins.

Site-specific DNA damage caused by lipid peroxidation products.

DNA damage induced by autoxidized lipids was investigated using covalently closed circular (supercoiled) DNA and DNA fragments of defined sequence. DNA-strand-breaking substances accumulated during autoxidation of methyl linolenate, and strand breakage was measured with samples taken at different times. The DNA-strand-breaking activity reached its maximum a little after the peak value of peroxide and decreased upon further autoxidation. The peak of the DNA-strand-breaking activity did not always coincide with the peak of thiobarbituric acid reactants or of conjugated diene, either. The DNA-strand-breaking reaction was dependent on metal ions and was inhibited by potassium iodide and tiron and partially by catalase, suggesting the involvement of radical species and/or oxygen radicals. No direct cleavage of singly end-labeled 100-200 basepair DNA fragments by autoxidized methyl linolenate and cupric ion was detected under the conditions used. Cleavage occurred during subsequent heating in piperidine after the reaction. The alkali-labile damage was preferentially induced at pyrimidine residues, especially in dinucleotide sequences of pyrimidine-guanine (5'----3'), which was determined by sequencing.

Sequence-specific alkali-labile lesions in DNA caused by D-isoglucosamine.

The site-specific induction of DNA damage by 1-amino-1-deoxy-D-fructose (D-isoglucosamine) was investigated. When 32P-end-labeled DNA restriction fragments of known sequence were reacted with D-isoglucosamine in the presence of Cu2+, and the DNA products were analyzed on high-resolution denaturing polyacrylamide gels after treatment with aqueous piperidine (1 M) at 90 degrees C for 30 min, the DNA strands were cleaved at pyrimidine residues at a statistically significant frequency, and 80.5% of the extensively damaged sites were induced at pyrimidine residues in dinucleotide sequences of pyrimidine-purine (5'----3'). These cleavages were scarcely observed without piperidine/heat treatment. The damaged DNA sites increased in proportion to the reaction time and concentration of D-isoglucosamine. Metal-chelating agents (EDTA, diethylenetriaminepentaacetic acid) and some oxygen radical scavengers inhibited the induction of alkali-labile lesions. These results indicate that some oxygen radicals are involved in the induction of alkali-labile lesions.

Specific drug binding by purified lipid-reconstituted P-glycoprotein: dependence on the lipid composition.

We fused P-glycoprotein with beta-galactosidase at the C-terminus aiming to study the mechanism of drug binding of P-glycoprotein in reconstitution experiments. Expression of the fusion protein in NIH 3T3 cells conferred a multidrug-resistant phenotype, suggesting that beta-galactosidase fusion at the C-terminus does not affect the functions of P-glycoprotein. The fusion protein was partially purified by simple immunoprecipitation with anti-beta-galactosidase polyclonal antibody, and its [3H]azidopine binding property was investigated in the presence of various compositions of liposomes. The purified P-glycoprotein, after reconstitution into liposomes, was capable of binding [3H]azidopine. When the cholesterol content of liposomes was increased to a weight ratio of 20%, the specific binding activity of the partially purified fusion protein was stimulated, and when the cholesterol content was increased higher, the binding activity decreased. The binding was specifically decreased by competition with vinblastine. Stigmasterol was less effective, and ergosterol was the least effective in stimulating the specific binding.

DNA damage induced by ascorbate in the presence of Cu2+.

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.

Regulation of glpD and glpE gene expression by a cyclic AMP-cAMP receptor protein (cAMP-CRP) complex in Escherichia coli.

The glpE gene of E. coli was found to be transcribed divergently with respect to glpD, which is adjacent to glpE head-to-head on the E. coli chromosome. We constructed glpD- and/or glpE-lacZ fusion plasmids, which provided glpD and lacZ as reporter genes. The expression of glpD and glpE, under the control of the cAMP-CRP complex, was examined by measuring the activities in E. coli cells of beta-galactosidase encoded by lacZ and glycerol-3-phosphate dehydrogenase encoded by glpD. In the double-reporter-gene system, the expression of glpD and glpE was found to be positively regulated by cAMP-CRP. We also confirmed that intracellular levels of the translation products and the transcripts from glpD and glpE were positively regulated by cAMP-CRP. The cAMP-mediated induction of gene expression of glpD and glpE was significantly affected by structural alterations of the single CRP-binding site between glpD and glpE. These results indicate that the single CRP-binding site is a cis-acting element involved in the positive regulation of the expression of both glpD and glpE at the transcriptional level.

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein.

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.

Identification of a vegetative promoter in Myxococcus xanthus. A protein that has homology to histones.

A physical map of 330 x 10(3) base-pairs near the replication origin of Myxococcus xanthus chromosome has been established already. Using DNA fragments from this region, Northern blot hybridization analysis was carried out in order to identify the genes expressed during vegetative growth. One of the genes, tentatively designated as vegA, was cloned and its entire DNA sequence was determined. The amino acid sequence of the gene product deduced from the DNA sequence reveals that the VegA protein is a very basic protein with a molecular weight of 18,700. The gene was expressed in Escherichia coli using an expression vector, and its gene product was identified using SDS/polyacrylamide gel electrophoresis. From the results of S1 nuclease mapping, the vegA promoter was found to contain the sequence TAGACA at the -35 region and the sequence AAGGGT at the -10 region. These two regions are separated by 18 nucleotides. Genetic analysis suggests that the vegA gene may be essential for the growth of M. xanthus. From a computer-aided search for homologies to know protein structures, it was found that the VegA protein has homologies to histone H4 of Tetrahymena thermophila and histone H2B of sea urchin.

DNA rearrangement of the shufflon determines recipient specificity in liquid mating of IncI1 plasmid R64.

The shufflon is a unique DNA rearrangement found in plasmid R64. R64 shufflon consists of four DNA segments, which are flanked and separated by seven 19-bp repeat sequences. Site-specific recombination between any inverted repeats results in a complex DNA rearrangement where four DNA segments invert independently or in groups. The shufflon is a biological switch to select one of the seven C-terminal segments of the pilV gene. To examine the biological significance of the shufflon, R64 derivatives, where the pilV gene was fixed in seven C-terminal segments, were constructed and used as donor cells for liquid mating. The transfer frequencies depended markedly on the combinations of recipient bacterial strains and C-terminal segments of the pilV gene in donor cells, indicating that the shufflon determines the recipient specificity in liquid mating of R64. The products of the pilV genes were found to be a component of thin pili produced by R64.

Structure and organization of Marchantia polymorpha chloroplast genome. IV. Inverted repeat and small single copy regions.

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

Two single-strand DNA initiation signals located in the oriV region of plasmid RSF1010.

Two single-strand initiation signals (ssi) are found in the oriV region of broad-host-range plasmid RSF1010, using a plaque assay system with a mutant M13 phage which lacks the greater part of the complementary DNA strand origin (oric). These two signals, designated ssiA and ssiB, have RSF1010-specific properties, because they require one or more RSF1010-specific factors provided in trans. The functional activity of ssiA is higher than that of ssiB. The two signals are located on separate DNA strands, so that the DNA chain elongations initiated from them in the opposite directions may pass each other. It is conceivable that these signals, ssiA and ssiB, direct DNA priming functions at the initiation stage in vegetative DNA replication of RSF1010.

Replication origin (oric) on the complementary DNA strand of Escherichia coli phage G4: biological properties of mutants.

Phage G4 origin of complementary DNA strand synthesis (oric) consists of three stable secondary loop structures. In a cloned 274-bp DNA fragment that is active as an ori in the filamentous phage cloning vector R199, insertion mutants have been constructed by introducing EcoRI and HindIII linkers at the base of loop III. The in vivo activity of these oric mutants (conversion of single-strand form to replicative form in the presence of rifampicin) was significantly reduced (50-70%) but not completely abolished. Nucleotide sequences and/or potential secondary structure of loop III centered at the AvaII site are therefore an important functional part of oric.

Plasmid pACYC184 contains an ssi signal for initiation of single-strand phage DNA replication.

Using the plaque assay system for screening the single-strand (ss) initiation determinant (ssi) sequences, we have found that 119-bp region in pACYC184, a derivative of the plasmid P15A of Escherichia coli, can direct such ss DNA initiation. This region is located downstream from the P15A origin of replication and conserves consensus sequences of the ssi signals found in the other plasmids. Signals for ss DNA initiation are defined as nucleotide sequences present on ss DNA templates and required for priming DNA synthesis. The direction of chain elongation in DNA synthesis is opposite to that of the leading strand. In this region, we found a potential stem-and-loop structure. The 119-bp DNA segment of plasmid pACYC184 cloned in f1R199 filamentous phage could direct rifampicin-resistant conversion of the ss DNA to the double-stranded replicative form.

Identification of a promoter for the crystal protein-encoding gene cryIVB from Bacillus thuringiensis subsp. israelensis.

The cryIVB gene of Bacillus thuringiensis subsp. israelensis (Bti) codes for a 135-kDa insecticidal crystal protein, which is specifically toxic to dipteran larvae. We have identified a transcription start point (tsp) of cryIVB by a primer extension experiment. The promoter sequence alignment, together with the chronology of appearance of the transcript, suggested that cryIVB is transcribed by RNA polymerase containing sigma 35 (E sigma 35). This was confirmed by investigation of cryIVB transcription in several Bacillus subtilis sporulation mutants. Unlike the lepidopteran-specific crystal protein-encoding genes [cryIA(a) and cryIB], transcription of which is regulated by both sigma 35 and sigma 28, cryIVB transcription was controlled only by the sigma 35-dependent promoter at the midsporulation stage.