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PubMed1991

Persistent hepatitis B virus following interferon alfa therapy and liver transplantation.

A 44-year-old man with chronic hepatitis B virus infection and cirrhosis was treated with recombinant human interferon alfa for 67 days immediately before orthotopic liver transplantation and immunoprophylaxis with hyperimmune globulin to hepatitis B virus in the peritransplant period. Dot blots for hepatitis B virus DNA demonstrated marked reduction in viremia after 41 days of interferon alfa treatment. Southern analysis for hepatitis B virus in liver showed a pronounced decrement in actively replicating forms in the explant, although hepatic infection was still detectable. After liver transplantation, tests for serum hepatitis B virus DNA and hepatitis B surface antigen remained negative. The patient died 32 days after transplantation of causes unrelated to hepatitis B virus. DNA isolated from liver and other visceral organs at autopsy showed infection of the engrafted liver and the persistence of monomeric relaxed circular forms of hepatitis B virus DNA in pancreas, kidney, and spleen. Thus, graft reinfection occurred despite aggressive antiviral therapy and immunoprophylaxis combined with liver transplantation. Existing viral serological markers appear insufficiently sensitive to assess residual infectivity.

Adult↗

PubMed1987

Mechanisms of frameshift mutagenesis by aflatoxin B1-2,3-dichloride.

In order to characterize frameshift mutagenesis by aflatoxin B1-2,3-dichloride (AFB1Cl2), we have introduced a +1 (BK8) or a -1 (HS8) frameshift within the lacZ alpha gene segment contained in the phage M13mp8 to obtain lacZ alpha- derivatives. BK8 or HS8 replicative form DNA was modified with AFB1Cl2 in vitro, transfected into appropriate Escherichia coli hosts and lacZ alpha+ revertants scored and defined by DNA sequencing. The -1 frameshift (BK8) results suggest the following. (1) The E. coli recA gene is not absolutely required for AFB1Cl2-induced frameshift mutagenesis; however, in recA+ cells, ultraviolet light (SOS) induction enhances AFB1Cl2 mutagenesis, but such ultraviolet induction is not required. The plasmid pGW270 (mucAB+) significantly enhances the AFB1Cl2-induced frameshift mutagenesis. The uvrABC+ excision system plays a major role in the repair of AFB1Cl2-induced damage. (2) Sequence analysis reveals that AFB1Cl2 induces two classes of -1 frameshift mutations: the simple class in which the frameshift is due to the loss of one base-pair, and the complex class in which the loss of a base-pair is coupled to a vicinal base substitution. Both types of mutations occur predominantly at G.C runs, which are hotspots for AFB1Cl2 damage. The complex mutations appear to be concerted events targeted by a single AFB1Cl2 adduct. The frequency of these complex mutations is significantly enhanced by mucAB activity. In this system, recA activity is required for generation of significant levels of complex mutations. An analysis of the +1 frameshifts (HS8) reveals that AFB1Cl2 induces +1 frameshifts with an efficiency comparable to that for -1 frameshifts. Most +1 frameshifts occur by the addition of a base, and a third of the additions are complex mutations because they are accompanied by at least one base substitution. All simple additions occur at G.C runs; however, in a striking contrast to spontaneous insertions, a majority of the induced events introduce an A.T pair at these sites. Our data suggest a model for the generation of base substitution as well as simple and complex frameshift mutations induced by AFB1Cl2. To the extent determined, the frameshift specificity of aflatoxin B1 activated by metabolic enzymes is similar to that of AFB1Cl2.

Aflatoxin B1↗

PubMed1987

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes.

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.

Bacteriophages↗

PubMed1987

Cloning and expression of the filamentous bacteriophage Pf1 major coat protein gene in Escherichia coli. Membrane protein processing and virus assembly.

A restriction fragment carrying the major coat protein gene (gene VIII) was excised from the replicative form (RF) DNA of the class II filamentous bacteriophage Pf1, which infects Pseudomonas aeruginosa. This fragment was cloned into the expression plasmid pKK223-3, where it came under the control of the tac promoter. In transformed Escherichia coli JM101 cells, in the presence of the inducer isopropyl-beta-D-thiogalactoside, the bacteriophage Pf1 gene was strongly expressed. The bacteriophage Pf1 coat protein displays the same pattern of negatively charged N-terminal region, hydrophobic middle region and positively charged C-terminal region as that of its counterpart in the class I bacteriophage fd, which infects E. coli, but otherwise the two proteins have no sequence homology. However, the Pf1 procoat protein was found to undergo processing and insertion into the E. coli cell inner membrane, like its fd counterpart, demonstrating that this part of the assembly process is the same for these different bacteriophages. The complete transcriptional unit, incorporating the tac promoter and rrnB transcription terminators flanking the Pf1 coat protein gene, was excised from the expression plasmid and cloned into the intergenic space of bacteriophage R252, an fd bacteriophage that carries an amber mutation in its own major coat protein gene. The Pf1 coat protein gene was again well expressed in infected E. coli cells but the chimeric bacteriophage had growth properties identical to those of the parent bacteriophage R252 on suppressor and non-suppressor strains of E. coli. The class I bacteriophage Pf1 coat protein evidently cannot be recognized by the class I bacteriophage assembly complex at or in the E. coli cell inner membrane, either at the point of initiation of assembly or during the elongation process.

Amino Acid Sequence↗

PubMed1990

Evidence for allosteric transitions in secondary structure induced by superhelical stress.

Previous studies suggest that the global secondary structures of native supercoiled and equilibrium linear DNAs may differ somewhat. Recent evidence also indicates that metastable secondary structure commonly persists following complete relaxation of the superhelical stress by intercalating dyes or by the action of topoisomerase I. In this work, the torsion constants (alpha) of pBR322, pUC8 and M13mp7 (replicative form) DNAs are determined by time-resolved fluorescence polarization anisotropy at various times subsequent to linearization. In all three cases, the torsion constants are relatively low immediately after linearization, and evolve for eight to ten weeks before reaching their apparent equilibrium values. It is shown in detail how the persistence of metastable secondary structure, subsequent to relaxation of superhelical stress, necessarily implies that one or more transitions in equilibrium secondary structure are induced as the superhelix density is varied from zero to native, or vice versa. Samples of pUC8 dimer (5434 base-pairs) with different superhelix densities are prepared by the action of topoisomerase I in the presence of various amounts of ethidium. Their median linking number differences are determined by standard band counting methods. The translational diffusion coefficient (Do) and the plateau diffusion coefficient (Dplat) characterizing internal motions over short distances (225 A) are determined by dynamic light-scattering. The torsion constant (alpha) between base-pairs and the circular dichroism spectrum are also measured for each sample. Curves of Dplat, Do, alpha and molar ellipticity ([theta]) (at the minimum near 250 nm) versus superhelix density (sigma) are constructed. The curve of Do versus sigma is very similar to that for sedimentation coefficient versus sigma for simian virus 40 (SV40) and polyoma DNAs. The curves of Dplat, Do, alpha and [theta] versus sigma show that, with increasing negative superhelix density, a structural transition occurs near sigma = -0.020 to an intermediate state with low torsion constant, and a second structural transition occurs near sigma = -0.035 to a state that exhibits more normal properties by sigma = -0.048. These data are consistent with the hypothesis that supercoiling induces two successive allosteric transitions to alternative global secondary structures. The data are much less consistent with the hypothesis that supercoiling induces some radical secondary structure at one or a few sites of small extent at sigma = -0.020, and at other sites at sigma = -0.035, or with hypotheses based on changes in tertiary structure alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Circular Dichroism↗

PubMed1991

Repair of helix-stabilizing anthramycin-N2 guanine DNA adducts by UVRA and UVRB proteins.

The transfectivity of anthramycin (Atm)-modified phi X174 replicative form (RF) DNA in Escherichia coli is lower in uvrA and uvrB mutant cells but much higher in uvrC mutant cells compared to wild-type cells. Pretreatment of the Atm-modified phage DNA with purified UVRA and UVRB significantly increases the transfectivity of the DNA in uvrA or uvrB mutant cells. This pretreatment greatly reduces the UVRABC nuclease-sensitive sites (UNSS) and Atm-induced absorbance at 343 nm in the Atm-modified DNA without producing apurinic sites. The reduction of UNSS is proportional to the concentrations of UVRA and UVRB and the enzyme-DNA incubation time and requires ATP. We conclude that there are two different mechanisms for repairing Atm-N2 guanine adducts by UVR proteins: (1) UVRA and UVRB bind to the Atm-N2 guanine double-stranded DNA region and consequently release the Atm from the adducted guanine; (2) UVRABC makes an incision at both sides of the Atm-DNA adduct. The latter mechanism produces potentially lethal double-strand DNA breaks in Atm-modified phi X174 RF DNA in vitro.

Adenosine Triphosphatases↗

PubMed1994

Effects of DNA adduct structure and distribution on the mutagenicity and genotoxicity of two platinum anticancer drugs.

cis-Diamminedichloroplatinum(II) (cis-DDP) and cis,trans,cis-ammine(cyclohexylamine)-dibutyratodichloroplatinu m(IV) (ACDDP) are anticancer drugs that bind to DNA, forming replication blocking adducts. ACDDP, probably manifests its cytotoxicity through the metabolite cis-ammine(cyclohexylamine)dichloroplatinum(II) (ACDP). The biological effects of ACDP and cis-DDP were compared by studying polymerase inhibition in vitro and mutagenesis and genotoxicity in vivo in the duplex genome of bacteriophage M13mp18 replicated in Escherichia coli. cis-DDP and ACDP adducts were equally genotoxic within the statistical error limits of the analysis. Survival of genomes platinated by either drug, increased by threefold in cells pretreated with u.v. irradiation to induce the SOS functions of the host. In the M13mp18 lacZ' gene fragment, the mutagenicity of ACDP was lower than that of cis-DDP; the difference was approximately twofold at a dose of two adducts per 370 base-pair mutational target. Mutagenesis by both compounds was SOS-dependent. The structural basis for lower mutagenicity of ACDP is proposed to be its reduced reactivity at d(ApG) sites. This effect is attributed to an orientational isomerism that precludes the formation of one of two possible DNA adducts at d(ApG) residues. The types of mutations induced for both drugs were similar, but they occurred with different distributions. Both compounds induced primarily G-->T transversions at d(GpG) sites whereas G-->A transitions and A-->T transversions, many at d(ApG), d(GpNpG), and d(GpG) sites, were also well represented. The mapping of DNA adducts by DNA synthesis inhibition revealed excellent correlation between the location of DNA lesions and the sites of mutations. Analysis of the distribution of mutations and the distribution of potential platination sites revealed no sequence-dependent mutation hotspots; i.e. mutagenesis was random throughout the lacZ' region of the M13mp18 bacteriophage genome. These results offer insights into the molecular mechanism of mutagenicity of platinum anticancer drugs.

Antineoplastic Agents↗

PubMed1987

High-frequency spontaneous deletion of DNA sequences flanked by direct DNA repeats which also contain an internal palindrome.

The DNA sequences associated with a very high-frequency, spontaneous deletion event have been determined to be two 11-base direct repeats which also contain an internal 6-base palindrome. A parental M13 replicative form (RF) DNA harboring DNA fragments of the T4 denV gene contained these direct repeats and could only be maintained at 5% of the total RF DNA within an infected cell. The remaining RF DNA was deleted for all intervening sequences between the direct repeats (2.2-kb), but one copy of the direct repeat was retained after the deletion had occurred. This site-specific deletion was highly reproducible in that if parental-sized M13 RF DNA was gel purified and transformed back into cells, the deletion occurred at precisely the same sequence as before. Electron microscopic analyses of DNA extracted from cells transformed with parental-sized DNA revealed the presence of excised 2.2-kb double-stranded circular DNA molecules. This observation thus rules out a copy choice replication/deletion mechanism to account for this high-frequency deletion event.

Base Sequence↗

PubMed1988

Mutational spectrum and recombinogenic effects induced by aminofluorene adducts in bacteriophage M13.

Double-stranded replicative form (RFI) DNA of bacteriophage M13 strain M13mp10 which carries partial lacZ gene has been modified in vitro to various extents with N-hydroxy-2-amino-fluorene (N-OH-AF) and then transfected into E. coli cells. High-performance liquid chromatography (HPLC) analysis results demonstrate that the sole adduct (95%) formed in modified DNA is N-(deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF). Approximately 20 adducts per RFI molecule constitute 1 lethal event when plaque-forming ability is assayed on E. coli cells which have received no prior SOS induction. The mutagenicity of dG-C8-AF adducts was assayed by measuring loss of beta-galactosidase activity as a function of adducts per molecule. A dose-dependent increase in Lac- mutants was observed, with a 4-fold increase in mutants per survivor at 30 adducts/molecule. The mutations produced, characterized by DNA sequencing, occur predominantly at either G or C positions different from those observed in the spontaneous mutant spectrum. Restriction-mapping results show that in our assay system, dG-C8-AF adducts induce a previously unreported recombinogenic activity.

Bacteriophages↗

PubMed1991

Genetic requirements for frameshift reversion induced by bulky DNA adducts in M13 DNA.

In order to analyze the genetic requirements and mechanisms of frameshift mutagenesis by activated aflatoxin B1 (AFB1), in vitro-modified phage M13 replicative form (RF) DNA was transfected into appropriate Escherichia coli cells and +1 or -1 frameshift revertants in the lacZ(alpha) gene were isolated. This analysis shows that both +1 and -1 frameshift mutagenesis by AFB1 is significantly reduced in a umuC- background. On the other hand, in the absence of RecA, +1 frameshift mutagenesis is partially reduced, but -1 frameshift mutagenesis is unaffected. DNA sequence analysis of +1 frameshifts induced by AFB1 in recA- cells suggests that the mutations occur at the same sites as in recA+ cells, but that there are significant differences in the specificity of the observed base changes. A model consistent with the observed effects in the absence of RecA suggests that an appreciable fraction of AFB1-adducted guanines can correctly template for a cytosine.

Animals↗

PubMed1993

Nonrandom distribution of O6-methylguanine in H-ras gene sequence from DNA modified with N-methyl-N-nitrosourea.

The distribution of O6-meG in the rat H-ras gene sequence was studied using PCR by transition of O6-meG to adenine during the reaction. In order to study the transition mutations the PCR product was cloned in a replicative form of phage M13mp18 and sequenced. The use of PCR for detection of O6-meG was validated by using oligonucleotides (61 bases) containing one O6-meG residue at a defined site. After treatment of rat liver DNA by N-methyl-N-nitrosourea in vitro, a striking nonrandom sequence distribution of O6-meG was observed. Sixty-eight per cent of O6-methylated Gs were found in the middle G of the sequences GGT and GGA in the H-ras gene whereas no methylation was found in the middle G of the sequences AGG, GGG, TGT, TGC, CGA and CGC. No O6-meG adduct was found in the 12th codon of H-ras (sequence GGA). The frequency of O6-meG formation as a function of two flanking nucleotides on each side of the target guanine was calculated as an approach to understanding more distant sequence effects. It was found that in the DNA sequence studied the formation of O6-meG was highest if the G was flanked by PyPu or PuPu on the 5' side (Py, pyrimidine and Pu, purine) whereas PuPu on the 3' side showed maximal inhibition of O6-meG formation.

Animals↗

PubMed1988

Capsid protein VP1 (p85) of Aleutian disease virus is a major DNA-binding protein.

The interaction between Aleutian disease virus (ADV) DNA and proteins isolated from ADV-infected cells or ADV virions, respectively, was examined. Proteins were separated on SDS-polyacrylamide gels, transferred to nitrocellulose, and probed with 32P-labeled restriction fragments of replicative form (RF) DNA or with single-stranded virion DNA. The ADV capsid protein VP1 was found to bind the 3'-terminal BamHI fragment of RF DNA extending from map units (m.u.) 0 to 15. No binding was observed with the fragment extending from m.u. 15 to 63 and only minor amounts of label were detected with the 5'-terminal EcoRV fragment extending from m.u. 63 to 100. With the latter fragment, small amounts of label were also detected bound to higher-molecular-weight proteins of about 100,000 Da. No binding of DNA to the ADV nonstructural protein NS1 or to the ADV capsid protein VP2 was detected. Additionally, single-stranded virion DNA was found to bind to VP1.

Aleutian Mink Disease Virus↗

PubMed1989

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo.

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3' proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (-) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermediates and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (-) RNA probes under denaturing conditions and (2) characterization of the 5' ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2'-methyltransferase.

Capsid↗

PubMed1986

Plasmid pU29, a vehicle for mutagenesis of the photosynthetic puf operon in Rhodopseudomonas capsulata.

Plasmid pU21, which carries the reaction center and light-harvesting genes (puf operon) of Rhodopseudomonas capsulata, has been redesigned by site-specific mutagenesis. Five restriction sites have been removed and three unique restriction sites have been introduced into this 11,589-bp pBR322 derivative. The modifications divide the puf structural genes into four regions separated by five unique and nonmutagenic restriction sites. These four fragments have been subcloned into the M13-mp series of vectors to facilitate oligonucleotide-mediated site-specific mutagenesis experiments on the photosynthetic apparatus structural genes. The inserts can then be returned from the M13 replicative form to the redesigned pU21 derivative. The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts. Additionally, tandem homologous sequences (the reaction center L and M subunits) within the puf operon are now separated on different phage vectors, eliminating problems encountered in the targeting of mutagenic oligonucleotides to only one of the two homologous sites.

Chromosome Mapping↗

PubMed1988

A runaway-replication plasmid pSY343 contains two ssi signals.

Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively. We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199. A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A. Van der Ende, R. Teerstra, H. Van der Avoort, and P.J. Weisbeek, 1983, Nucleic Acids Res. 11, 4957-4975) was found, three copies in 170P and one in 93F. These two ssi signals contain possible stem and loop structures. The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region. Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection. The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection.

Base Sequence↗

PubMed1995

Minute virus of mice (MVM) nucleic acid production in susceptible and resistant strains of mice and F1 hybrids.

Susceptibility of neonatal mice to the lethal effects of the virulent allotropic variant of minute virus of mice is controlled by host genotype. We quantitated MVM-DNA production in resistant C57BL/6 (B6), susceptible C3H/He (H), and (B6 x H) F1 (F1) hybrid mice. Total DNA extracted from six organs of infected mice on post inoculation days (PID) 3, 5, 7, and 9 was analyzed by dot blot and Southern blot assays. 7 to 100-fold more viral DNA was produced in H mice than in B6 mice on PID 7 depending on the organ and F1 mice phenotypically resembled B6 mice. Southern blot analysis of DNA extracted from infected mice on PID 7 showed a prominent single-stranded band (5 kb, virion DNA) and a faint replicative form (5 kb, double-stranded) in all strains of mice. Total RNA extracted from the organs of B6, H, and F1 mice was analyzed by Northern blot. Viral mRNA production was generally below detection limits. In kidneys, hearts, and intestines of some H mice on PID 7, however, a 1.9 kb transcript was detected, smaller than transcripts for R1, R2 or R3. This study confirms that susceptibility to lethal MVMi infection correlates with upregulation of MVM DNA production, a recessive trait, and perhaps with the production of a novel 1.9 kb transcript.

Animals↗

PubMed1986

A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-lpr/lpr mouse: specificity and idiotype in serum immunoglobulins.

Anti-double stranded (ds) DNA antibodies in the sera of lupus-prone MRL/Mp-lpr/lpr (MRL/l) mice were determined by an enzyme-linked immunosorbent assay in parallel with anti-single stranded (ss) DNA, anti-left-handed Z-DNA and anti-poly(ADP-ribose) antibodies. The serum levels of these antibodies in these mice increased with age, and in particular anti-dsDNA antibodies appeared in mice more than 14 weeks old, along with progressive lymphadenopathy. We therefore established a hybridoma producing monoclonal anti-dsDNA antibody (2C10) from an 8-month-old MRL/l mouse. Monoclonal antibody 2C10 did not react with either poly(dT) or poly(I), which are major cross-reactants with previously reported monoclonal MRL mouse autoantibodies. Antibody 2C10 showed preference for phi X-174 replicative form DNA and calf thymus dsDNA over ssDNA. 2C10 idiotype (Id) was present in the sera of MRL/l mice, but only occasionally at high levels even in the aged mice tested. This result suggested that many Ids with anti-dsDNA antibody activity may contribute to lupus pathogenesis in this strain of mouse.

Aging↗

PubMed1986

Detection of double-stranded RNA by immunoblot electrophoresis.

A procedure is described which permits the serological detection of double-stranded RNA (dsRNA) which has been electrophoretically transferred from agarose gels to Zeta-Probe blotting membranes. Cucumber mosaic virus replicative forms of RNAs 1-3 and cassava (Manihot esculenta) clone 'Secundina' dsRNAs can be routinely detected from 1 g of leaf tissue. This procedure allows for the detection and size estimation of dsRNAs in crude nucleic acid preparations.

Electrophoresis, Agar Gel↗