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Expression of the Autographa californica nuclear polyhedrosis virus p10 gene: effect of polyhedrin gene expression.

Two major late proteins, polyhedrin and p10, are synthesized in large quantities in baculovirus infected insect cells. This and the fact that both proteins are dispensable for virus replication, form the basis for the use of these viruses as vector for foreign gene expression. To address the question whether the Autographa californica nuclear polyhedrosis virus p10 promotor-driven expression is influenced by the concurrent expression of the polyhedrin gene, several recombinants were constructed with various deletions in the polyhedrin gene. The Escherichia coli lacZ gene was used as a marker to allow direct comparison between p10 and polyhedrin-driven expression. None of the deletions in the polyhedrin gene did result in higher expression of the p10 promoter-controlled gene. This suggested that the transcriptional and/or translational activity of the p10 and polyhedrin gene are independently regulated. To compare the level of polyhedrin and p10 promoter driven expression, recombinants with the lacZ gene cloned behind either promoter were studied. No significant difference in level of expression was observed. In cells infected with a recombinant with the lacZ gene present behind both promoters a reduced level of expression was observed, whereas a considerable increase was expected. This may be due to instability of the viral genome, as two copies of the lacZ gene were present.

Animals↗

Comparative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates.

The genome of citrus tristeza closterovirus (CTV) consists of a 20 kb single-stranded RNA encapsidated in a 2000 nm long, flexuous particle. Double-stranded (replicative form) RNAs purified from CTV-infected tissue were used to prepare complementary DNA libraries that involved initial first-strand cDNA synthesis followed by selective amplification of the coat protein gene. CTV-specific antisera were used to select clones expressing the coat protein. The coat protein genes of seven Florida and four exotic isolates that differ in their biological properties were cloned and sequenced. The gene is 669 base pairs long and encodes a 223 amino acid protein. There was a greater than 80% homology at both nucleotide and amino acid levels among all the isolates examined. However, comparisons showed that each isolate was found to have several unique amino acid residues. Several blocks of amino acid residues were conserved among all the isolates. A cluster dendrogram showed greater similarities among groups of mild and severe Florida isolates that differed significantly from those of the geographically distinct, exotic CTV isolates.

Amino Acid Sequence↗

Detection of a phage genome carrying a zonula occludens like toxin gene (zot) in clinical isolates of Stenotrophomonas maltophilia.

During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called phiSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage phiLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on phiSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.

Amino Acid Sequence↗

Construction of a food-grade multiple-copy integration system for Lactococcus lactis.

A food-grade vector system was developed that allows stable integration of multiple plasmid copies in the chromosome of Lactococcus lactis. The vector consists of the plus origin of replication (Ori+) of the lactococcal plasmid pWV01, the sucrose genes of the lactic acid bacterium Pediococcus pentosaceus PPE1.0 as selectable marker, a multiple-cloning site, and a lactococcal DNA fragment of a well-characterized chromosomal region. The system includes two L. lactis strains, LL108 and LL302, which produce the pWV01 RepA protein essential for replication of the Ori+ vectors. These helper strains allow the construction and isolation of the replicating form of the integration plasmids from a homologous background. Single-crossover integration of the plasmids in L. lactis MG1363 resulted in amplifications to a level of approximately 20 copies/chromosome after selection of the transformants on medium containing sucrose as the only fermentable sugar. The amplifications were stable under selective growth conditions. In glucose-containing medium a limited loss of integrated plasmid copies was detected at a rate of (7.5-15) x 10(-2) copies per generation. One strain, MG124, was isolated that had retained 11 integrated copies after a period of 120 generations of non-selective growth. These results show that the single-cross-over integration system described here represents a simple procedure for the engineering of stable food-grade strains carrying multiple copies of a gene of interest.

Blotting, Southern↗

Genetic characterization of a putative Densovirus from the mealybug Planococcus citri.

Total genomic DNA preparations from the citrus mealybug, Planococcus citri, contained a DNA band corresponding to 5.5 kilobases. This DNA was a linear molecule and was cloned into pUC18. Nucleotide sequence determination indicated that it was the replicative form of a densovirus, most closely related to the virus from Periplaneta fuliginosa (smokybrown cockroach).

Animals↗

Structure of the telomeric ends of mt DNA, transcriptional analysis and complex I assembly in the dum24 mitochondrial mutant of Chlamydomonas reinhardtii.

The dum24 mutant of Chlamydomonas reinhardtii contains four types of altered mitochondrial linear genomes: two types of deleted monomers and two types of dimers resulting from fusions between some monomers via their deleted ends. All molecules lack at least cob, nd4 and the 3' end of nd5, three adjacent genes located in the left part of the genome. We present evidence showing that in dum24, as in other deletion mutants, the deletions extend to the left telomeric end, and propose that the only replicative forms in the mutants are the dimeric DNA molecules that possess intact telomeric structures at both ends. Two abnormally large transcripts produced from chimeric genes are detected in dum24, which throws some light on the location of potential promoter sequences and processing signals in the mitochondrial genome. Using BN-PAGE analysis and immunological methods to detect complex I, we further show that dum24 mitochondria do not possess the normal multimeric complex I (850 kDa), but produce a smaller, partially assembled, complex (650 kDa), demonstrating a role for ND4 and/or ND5 subunits(s) in complex I assembly.

Animals↗

The ultrastructural morphology of native hepatitis B virus.

Cell lines (2.2.15 cells) capable of supporting the replication of hepatitis B virus (HBV) DNA and intact viral particles have been established by HBV DNA transfection into HepG2 cells. The purpose of this study was to determine the ultrastructural morphology of native HBV particles without purification in the culture supernatants and in sera from patients. Electron microscopy (EM) and immunogold EM of the samples were carried out using polyclonal and monoclonal anti-hepatitis B surface antigen antibodies. HBV particles in the purified samples from the culture supernatants by density-gradient centrifugation were examined to compare the morphology with that of unpurified samples. EM and immunogold EM studies demonstrated the presence of Dane particles (41.8 nm in diameter), cobra-shaped (head diameter, 42.4 nm), and horn-shaped (head diameter, 43.5 nm) particles in the culture supernatants and in the sera from two patients. The tail of the cobra-like particles had a diameter of 21.0 nm and a length of 214 nm. The hornlike particles had a long branch 20.1 nm in diameter with a length of 189 nm, and a short branch 21.4 nm in diameter with a length of 112 nm. The ratio of Dane particles and cobra- and horn-shaped particles in the supernatants was 5 : 4 : 1. After ultracentrifugation, the cobra- and horn-shaped particles completely disappeared; there were only Dane particles together with spheres of 22 nm and filaments. In conclusion, this study showed for the first time that the native replicative form of HBV is cobra- and horn-shaped.

Cell Line↗

A modified primer extension procedure for specific detection of DNA-RNA hybrids on nylon membranes.

We have developed a modified primer extension procedure for specific detection of mRNA. Alkali-fragmented total cellular RNA or some RNA fraction is hybridized to single-stranded or double-stranded M13 DNA containing the insert of interest which is immobilized on nylon membranes. Hybridized RNA is then detected by incubation of membranes with Escherichia coli RNase H and DNA polymerase I. RNase H is used for nicking the RNA in the hybrids. The resulting 3'-OH groups can subsequently be used by DNA polymerase I to synthesize a labeled complementary strand. The method described is both relatively fast and sensitive and particularly useful for screening large numbers of DNA clones for their representation in RNA populations. Using total cellular RNA as hybridization probe and single-stranded M13 DNA as template as low as 0.25 ng of a specific mRNA was detected (2.5-fold background) when adding 1 microCi [3H]dCTP or 2.5 microCi [32P]d-CTP alternatively as radioactive precursor for the labeling reaction. The detection limit increased to 1 ng (2-fold background) with denatured replicative form double-stranded M13 DNA as template.

Animals↗

Inactivation of biologically active DNA by gamma-ray-induced superoxide radicals and their dismutation products singlet molecular oxygen and hydrogen peroxide.

Since superoxide radicals are involved in many metabolically important as well as in some other, detrimental cellular processes, the reactivity of gamma-ray-induced superoxide radicals and its dismutation products singlet molecular oxygen and hydrogen peroxide with DNA have been studied. Superoxide dismutase which removes superoxide radicals and inhibits the formation of singlet oxygen in the solution protects the biologically active replicative form of DNA (from bacteriophage theta X174) against inactivation by ionizing radiation. Catalase which removes hydrogen peroxide also protects the DNA. Attempts with various chemical sources of singlet oxygen to determine whether this species inactivates DNA did not give an unequivocal answer. It is concluded from the presented experiments that a combination of the protonated form of the superoxide radical (HO-2) and H2O2 do inactivate DNA.

Binding Sites↗

Action of exonuclease V (the recBC enzyme) on ultraviolet-irradiated DNA.

Exonuclease V (the recBC enzyme) of Escherichia coli can release pyrimidine dimers from ultraviolet-irradiated linear duplex DNA though it acts more slowly on irradiated DNA than on non-irradiated DAN. However, close circular lambda-dv DNA or phi X174 replicative form I DNA is not attacked by exonuclease V even though the DNA has been irradiated and treated with T4 endonuclease V to produce single-stranded breaks at the 5'-side of pyrimidine dimers. When irradiated circular DNA, previously nicked by T4 endonuclease V, is briefly exposed to elevated temperature, the DAN becomes susceptible to the action of exonuclease V, and pyrimidine dimers are selectively released. The increased susceptibility to exonuclease V may be resulted from locarized denaturation, or "fraying" of the 5'-termini at the nicks. The preferential release of pyrimidine dimers was observed when irradiated DNA, treated with T4 endonuclease V, was incubated with crude extracts of Escherichia coli. The activity was found in various strains defective in exonuclease V and/or DNA polymerase I.

Coliphages↗

Saltatory thermal denaturation of double-stranded viral RNAs.

The double-stranded RNAs from bacteriophage phi6 and the replicative form of mengovirus denature upon heating in a series of abrupt steps which resemble the subtransitions (thermalites) observed within the high resolution profiles of small, naturally occurring DNA molecules. Such RNA thermalites are approximately an order of magnitude narrower than typical thermal subtransitions of nominally single-stranded RNA. We conclude that the same features of nucleotide sequence that give rise to cooperative denaturation in DNA genomes are to be found also in RNA genomes. Thus, high resolution thermal denaturation profiles are useful for characterizing double-stranded RNA molecules as well as native DNA in the size range of common viruses. A medium containing dimethylsulfoxide was required to lower the Tm of the RNA samples to a satisfactory temperature range. For double-stranded RNA in 50% dimethylsulfoxide, the dependence of Tm on G . C composition was greater than that of DNA in the same medium and also greater than that of double-stranded RNA in an aqueous medium. The fact that RNA thermalites are broader than DNA thermalites and that the melting temperature of double-stranded RNA has a greater dependence on base composition than that of DNA, indicates that at least one of the thermodynamic parameters for double helix formation in RNA is different from that in DNA.

Bacteriophages↗

Location of the cooperative melting regions in bacteriophage fd DNA.

Differential melting profiles of the linear replicative form (RF-III) DNA of bacteriophage fd, of the fragments obtained by the restriction endonuclease R.HinHI and of those obtained by R.Hga were investigated. With these results a physical map which locates the cooperative melting regions on the DNA was constructed, and compared with the genetic map.

Base Sequence↗

Induction of mutation in vitro in phage phi X174 am3 by N4-aminodeoxycytidine triphosphate.

When phi X174 am3-phage-infected E. coli is treated with N4-aminocytidine, reversion of the phage to the wild type is efficiently induced. The mechanism of this reversion is considered to consist of metabolic conversion of N4-aminocytidine into its deoxynucleoside 5'-triphosphate followed by incorporation of the nucleotide into the replicating phage DNA, thereby causing AT-to-GC transition at the am3 locus. The second half of this mechanism has now been experimentally proved, using an in vitro mutagenesis system. Thus, by nick-translation, N4-aminodeoxycytidine 5'-triphosphate was incorporated into the replicative form of phi X174 am3 DNA, and the DNA was used to transfect CA++-treated E. coli HF4714 (sup+). The reversion frequency of the phage produced was up to one-order of magnitude greater than that of the control in which the nick-translation had been done without the addition of N4-aminodeoxycytidine triphosphate. This nucleotide analog may be useful as a reagent for in vitro site-directed mutagenesis.

Bacteriophage phi X 174↗

Induction of breaks in deoxyribonucleic acid by photoexcited promazine derivatives.

Near-u.v. photoexcited promazine and three of its derivatives are shown to induce single-strand breaks in phi X174-DNA replicative form. The mechanisms of this DNA breakage depend upon the various photochemical properties of the promazine derivatives. Chlorpromazine is shown to act predominantly via the photodechlorination reaction both in aerobic and anaerobic conditions. The three other promazine derivatives (promazine, trifluopromazine and methoxypromazine) display two mechanisms for DNA breakage. One of them occurs through the cation radical, which is formed during near-u.v. irradiation of promazine derivatives. The second mechanism is demonstrated to act via an hydroxyl radical-dependent pathway. Acepromazine is without photoactivated action. EPR-spin-trapping studies of irradiated mixtures, containing the drugs and 5,5-dimethyl-1-pyrroline-N-oxide (as spin trap), suggest the production of superoxide radical by photoexcited promazines. When DNA is present in the irradiation mixture, this superoxide radical is converted into hydroxyl radical probably via a Haber Weiss-type reaction, catalysed by DNA-iron complexes.

DNA↗

Formation of different reaction products with single- and double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213).

In this report, the types of DNA damage introduced by the ortho-quinone and the semiquinone free radical of 4'-demethylepipodophyllotoxin-9-(4-6-O-ethylidene-beta-D- glucopyranoside) (etoposide) and their relevance for the inactivation of single-stranded (ss) and double-stranded (ds) replicative form (RF) phi X174 DNA have been examined in vitro. The ortho-quinone yielded in both ss and ds DNA only chemical adducts, of which on the average about 1 out of 3 and 1 out of 12 per DNA molecule led to inactivation of ss or RF phi X174 DNA, respectively. The semi-quinone free radical, on the other hand, generated both frank and alkali-labile strand-breaks in ss and in ds DNA which, however, did not contribute significantly to DNA inactivation. The radical introduced, in addition, chemical DNA adducts. Unlike the ortho-quinone adducts, however, each of the semi-quinone adducts was lethal in ss phi X174 DNA, while more than 40 were required for the inactivation of RF DNA. The excision repair system of Escherichia coli did not operate on semi-quinone-modified RF DNA but removed about half of the ortho-quinone adducts [van Maanen JMS, Lafleur MVM, Mans DRA, van den Akker E, de Ruiter C, Koostra PR, Pappie D, de Vries J, Retèl J and Pinedo HM, Biochem Pharmacol 37: 3579-3589, 1988]. When ortho-quinone-modified ss or ds DNA was subjected to a post-alkaline treatment, the adducts remained stably bound to the DNA and the degree of biological inactivation was not influenced. In contrast, post-alkaline treatment removed about 70 and 60% of the semi-quinone adducts from ss and ds DNA, respectively, which, in the case of ss phi X174 DNA, resulted in a partial restoration of the biological activity. It is concluded that the ortho-quinone and the semi-quinone free radical of etoposide produce different types of damage in DNA which have different effects on the biological activity.

DNA↗

The bond in the bacteriophage phi X174 gene A protein--DNA complex is a tyrosyl-5'-phosphate ester.

The bacteriophage phi X174 gene A protein cleaves the viral strand of the double-stranded replicative form (RF) DNA of the phage at a specific site, the origin. It leaves a free 3'-OH at nucleotide 4305 (G) of the phi X DNA sequence and binds covalently to the DNA. The nature and position of the covalent bond have been determined using the octadecadesoxyribonucleotide CAACTTG[32P]ATATTAATAAC. This octadecamer, which corresponds to nucleotides 4299-4316 of phi X viral DNA, is cleaved by gene A protein. Gene A protein is bound to the labelled phosphate via a tyrosyl residue, indicating that binding occurs to the nucleotide corresponding to 4306 (A) of the phi X viral DNA strand.

Bacteriophage phi X 174↗

Sizing of single-stranded regions in double-stranded DNA by preparative benzoylated DEAE-cellulose chromatography.

The concentration of caffeine required to elute wholly single-stranded DNA from benzoylated DEAE-cellulose is proportional to the polynucleotide length. The use of benzoylated DEAE-cellulose chromatography for isolating and sizing single-stranded regions in double-stranded DNA has been examined using a series of hybrid molecules. Restriction fragments of the replicating form of bacteriophage luminal diameter X174 were hybridized to the intact 'plus' strand, thereby forming hybrids having single- and/or double-stranded regions in the kilobase range. A series of such hybrid preparations were subject to caffeine concentration gradient elution from benzoylated DEAE-cellulose. After logarithmic transformation, a linear relationship (R = 0.94) could be demonstrated between eluting caffeine concentration and single-stranded length, irrespective of the length of associated double-stranded regions or the location, within a given fragment, of unpaired nucleotides. Benzoylated DEAE-cellulose chromatography may therefore be used to separate and characterize, on a preparative scale, double-stranded DNA containing single-stranded regions.

Bacteriophage phi X 174↗

Foscarnet decreases serum and liver duck hepatitis B virus DNA in chronically infected ducks.

Foscarnet (trisodium phosphonoformate) is a new antiviral compound with in vitro inhibitory effects against the DNA polymerases of hepadna viruses. To study the effects of the drug in chronic hepadna virus infection, we treated ducks chronically infected with duck hepatitis B virus for 10 days with either low-dose foscarnet (50 mg/kg i.p. b.i.d.), high-dose foscarnet (250 mg/kg i.p. b.i.d.), or sterile water injections. Serum duck hepatitis B virus DNA and intrahepatic replicative forms of the virus were measured using molecular biological techniques with both a double-stranded radiolabeled DNA probe and a plus-strand (noncoding) specific RNA probe. We found a dose-related decrease in serum and intrahepatic duck hepatitis B virus DNA during treatment, with a rapid return toward baseline values after the cessation of treatment. There was a disproportionate decrease in the plus strand of viral DNA with treatment. We conclude that foscarnet exerts its effect in hepadna virus infection through inhibition of viral DNA polymerase. Further study is necessary to determine whether foscarnet, by itself or in combination with other treatment modalities, has a role to play in the treatment of chronic hepatitis B infections in humans.

Animals↗