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J J Dunn

Publications and source records attributed to J J Dunn.

At least 37 records · Page 2Linked to original sources
PubMed1998

Pausing and termination by bacteriophage T7 RNA polymerase.

Two types of sites are known to cause pausing and/or termination by bacteriophage T7 RNA polymerase (RNAP). Termination at class I sites (typified by the signal found in the late region of T7 DNA, TPhi) involves the formation of a stable stem-loop structure in the nascent RNA ahead of the point of termination, and results in termination near runs of U. Class II sites, typified by a signal first identified in the cloned human preproparathyroid hormone (PTH) gene, generate no evident structure in the RNA but contain a conserved sequence ahead of the point of termination, and also contain runs of U. Termination at class I and class II sites may involve non-equivalent mechanisms, as mutants of T7 RNA polymerase have been identified that fail to recognize class II sites yet continue to recognize class I sites. In this work, we have analyzed pausing and termination at several class II sites, and variants of them. We conclude that the 7 bp sequence ATCTGTT (5' to 3' in the non-template strand) causes transcribing T7 or T3 RNA polymerase to pause. Termination 6 to 8 bp past this sequence is favored by the presence of runs of U, perhaps because they destabilize an RNA:DNA hybrid. The effects of T7 lysozyme on pausing and termination are consistent with the idea that termination involves a reversion of the polymerase from the elongation to the initiation conformation, and that lysozyme inhibits the return to the elongation conformation. A kinetic model of pausing and termination is presented that provides a consistent interpretation of our results.

Bacteriophage T7↗
PubMed1998

[Cardiac manifestations of Lyme borreliosis with special reference to contractile dysfunction].

Borrelia burgdorferi infection (BBI) is suggested to be associated with dilated cardiomyopathy (IDC). Stanek et al. were able to cultivate Borrelia burgdorferi (BB) from myocardial biopsy tissue of a patient with longstanding dilated cardiomyopathy. Here we present a study in which we examined the effect of standard antibiotic treatment on the left ventricular ejection fraction (LV-EF) in patients with dilated cardiomyopathy associated with BBI. In this study we assessed the serum (IgG, IgM ELISA; Western Blot) and the history of 46 IDC-patients with specific respect spect to BBI (mean LV-EF: 30.4 +/- 1.3%; measured by cardiac catheterisation and echocardiography--length-area-volume method). All 46 patients received standard treatment for dilated cardiomyopathy: ACE-inhibitors, digitalis and diuretics. 11 (24%) patients showed positive serology and a history of BBI; 9 of these also had a typical history of tick bite and erythema chronicum migrans (ECM) and/or other organ involvement, 2 had no recollection of tick bite or EMC, but showed other BB-associated disorders (neuropathy, oligoarthritis). These 11 patients with BBI received standard antibiotic treatment with intravenous ceftriaxone 2 g bid for 14 days. 6 (55%) recovered completely and showed a normal LV-EF after 6 months, 3 (27%) improved their LV-EF and 2 (18%) did not improve at all. This amounts to 9 (82%) recovery/improvement in the BB-group. The 35 patients who did not show positive serology or a history of BBI did not receive antibiotic treatment. In this group without BBI 12 (26%) showed recovery/improvement following the standard treatment of dilated cardiomyopathy (see above). Our results indicate that BBI could play a decisive role in the development of dilated cardiomyopathy, especially in a geographical region as Graz, where BB is endemic. While aware of the small number of BB-patients in this study, we nevertheless conclude that, in a remarkable number of patients with signs of BBI, dilated cardiomyopathy could be reversed and LV-EF improved upon standard antibiotic treatment.

Adolescent↗
PubMed1997

Mutant bacteriophage T7 RNA polymerases with altered termination properties.

We have identified mutants of bacteriophage T7 RNA polymerase (RNAP) that are altered in their ability to pause or terminate at a variety of signals. These signals include a terminator found fortuitously in the human preproparathyroid hormone (PTH) gene, a pause site found in the concatamer junction (CJ) of replicating T7 DNA, and termination signals that are also utilized by Escherichia coli RNAP (e.g. rrnB T1 and T2). Whereas the mutant enzymes terminate normally at the late terminator in T7 DNA (T(phi)) and rrnB T2, they fail to terminate at one of the termination sites of rrnB T1, and also fail to recognize the PTH and CJ signals. The mutant enzymes exhibit normal processivity on linear templates, but show a slightly reduced processivity on supercoiled templates and terminate more efficiently when synthesizing poly(U) tracts. The mutant enzymes also show a decreased tendency to produce aberrant transcription products from DNA templates having protruding 3' ends. T7 lysozyme (an inhibitor of T7 RNAP) has been shown to exert its action by preventing the transition of the RNAP from an unstable initiation complex (IC) to a stable elongation complex (EC). We have found that T7 lysozyme enhances recognition of CJ by wild-type T7 RNAP, and that mutant T7 RNAPs that show increased sensitivity to lysozyme show enhanced recognition of this signal, even in the absence of lysozyme. These results, together with the observation that the mutations that result in the termination-deficient phenotype affect a region of the RNAP that has been implicated in RNA binding and upstream promoter contacts, support the hypothesis that, in some cases, termination represents a reversal of the events that occur during initiation.

Amino Acid Sequence↗
PubMed1997

Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab.

OspA (outer surface protein A) is an abundant immunogenic lipoprotein of the Lyme disease spirochete Borrelia burgdorferi. The crystal structure of a soluble recombinant form of OspA was solved in a complex with the Fab fragment of mouse monoclonal antibody 184.1 and refined to a resolution of 1.9 A. OspA has a repetitive antiparallel beta topology with an unusual nonglobular region of "freestanding" sheet connecting globular N- and C-terminal domains. Arrays of residues with alternating charges are a predominant feature of the folding pattern in the nonglobular region. The 184.1 epitope overlaps with a well conserved surface in the N-terminal domain, and a hydrophobic cavity buried in a positively charged cleft in the C-terminal domain is a potential binding site for an unknown ligand. An exposed variable region on the C-terminal domain of OspA is predicted to be an important factor in the worldwide effectiveness of OspA-based vaccines.

Amino Acid Sequence↗
PubMed1997

Evidence for an uncommon microsatellite instability on mouse chromosomes 2 and 4 and its possible role in radiation leukemogenesis.

Although microsatellite instability (MSI), usually detected by DNA length polymorphisms, has been implicated in the induction of solid tumors in both humans and animals, its role in leukemogenesis is unclear. The goal of this study was to investigate whether there is an association between MSI and radiation leukemogenesis in CBA/Ca mice. Microsatellite lengths at 55 loci, mapped to eight different mouse chromosomes, were examined in two groups of DNA samples: 1) 10 normal DNA samples collected from the bone marrow cells of control male CBA/Ca mice, and 2) 17 DNA samples isolated from the spleens of mice that developed myeloid leukemia (ML) after exposure to neutrons, or X rays, or gamma rays. Microsatellite markers were amplified using the non-radioisotopic multiplex-touchdown PCR protocols developed in our laboratory, and the sizes of amplicons were examined on 6% non-denaturing polyacrylamide gels. Although no correlation between microsatellite length polymorphisms and radiation leukemogenesis was observed at the 55 CBA/Ca mouse loci tested in this study, an uncommon MSI, manifested as the absence of DNA bands after PCR amplification at 2 loci (D2MIT140 and D4MIT104), was observed in both control and ML samples. However, the frequency of ML samples showing this type of MSI is statistically significant (p<0.05). Although there is no direct evidence that this type of MSI predisposes mice to the development of leukemia, the results suggests that genes flanking the D2MIT140 and D4MIT104 are susceptible to spontaneous mutation and perhaps to damage caused by ionizing radiation.

Animals↗
PubMed1997

Genetic divergence among the group B coxsackieviruses.

As documented in the preceding discussion, the noncoding regions, and in particular the 5' NTR, of the CVB are tolerant of a substantial degree of nucleotide diversity while still being capable of fulfilling the life cycle requirements for these viruses. While diversity among the CVB is observed in the sequences encoding for the capsid proteins, it tends to involve predominantly those regions coding for amino acids located at the surface of the virus and not those responsible for the structural integrity of the mature virion, i.e., beta-barrels and alpha-helices. It is these capsid surface differences that define the six serotypes of the CVB and subdivide them antigenically into strains. Additionally, these proteins most likely play the major role in determining host and cellular tropism. The most conserved of the CVB proteins and, therefore those with the least diversity in their coding sequences, appear to be the nonstructural proteins. Perhaps, as speculated earlier, it is a conformational requirement imposed by the necessity to interact with host or viral substrates that maintains the high degree of amino acid identity of this group of viral proteins.

Amino Acid Sequence↗
PubMed1996

N-ras mutations in radiation-induced murine leukemic cells.

N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron-induced and 12 cases of photon- (3 gamma-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of the N-ras gene: (i) a 120 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of the N-ras gene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GAC-->GAT), 62 (CAA-->CAC), or 70 (CAG-->CAA) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CAA-->CAC) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency of N-ras mutations in neutron-induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N-ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence of N-ras mutations. Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.

Animals↗
PubMed1995

Ligation of hexamers on hexamer templates to produce primers for cycle sequencing or the polymerase chain reaction.

A method is described for the ordered ligation of hexanucleotides (hexamers) in solution to produce unique longer oligonucleotides. To form an 18-mer, for example, six different hexamers are selected that can base pair unambiguously to form a double-stranded complex of indefinite length. In the most efficient arrangement, each hexamer forms three complementary base pairs with two other hexamers, generating complementary chains of contiguous hexamers with strand breaks staggered by three bases. Two adjacent hexamers in one chain contain 5' phosphate groups and the others are unphosphorylated. Both T4 and T7 DNA ligase can ligate the phosphorylated hexamers to their neighbors in such a complex at hexamer concentrations in the 50-100 microM range, producing an 18-mer and leaving three unphosphorylated hexamers. Twenty-nine of 34 complexes that satisfied the requirements for unambiguous ligation generated the desired 18-mers, which could be used directly for cycle sequencing or, after removal of the unreacted hexamers, for polymerase chain reactions (PCR). Comparable ligation reactions also produced 12-, 24-, and 30-mers. With a library of all 4096 possible hexamers, unambiguous ligation has the potential to produce more than 82% of all possible 18-mers and could readily supply the oligonucleotides needed for DNA sequencing by primer walking, for PCR, or for gene synthesis.

Animals↗
PubMed1995

Incomplete protection of hamsters vaccinated with unlipidated OspA from Borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mAb LA-2.

Efforts to develop a recombinant vaccine for Lyme disease have focused on using the outer surface protein A (OspA) of Borrelia burgdorferi as an immunogen. We evaluated the effectiveness of an unlipidated recombinant OspA as a vaccine in hamsters. This molecule is soluble and can be produced in high yield in Escherichia coli, characteristics that permit simple and relatively low cost production. Vaccination with unlipidated OspA protected a substantial portion of animals--59-79%, depending on the challenge strain and route--against moderate doses of spirochetes delivered either by injection or by bite of infected nymphal ticks (Ixodes scapularis). The instances of vaccine failure were associated with development of low levels of antibody to a particular OspA epitope, one defined by mAb LA-2. At least 50 ng ml-1 of LA-2 equivalent antibody was necessary for protection of hamsters. Lower LA-2 equivalent antibody concentrations occurred in unprotected animals in the presence of high-titered polyclonal antibody to native OspA. A competitive binding assay to quantitate this serum fraction is described that should be of use in monitoring the quality of the antibody response to OspA in vaccine trials. Concentrations of LA-2 equivalent antibody parallel the ability of the serum specimens to inhibit the growth of B. burgdorferi in culture.

Animals↗
PubMed1995

Identification of an immunologically important hypervariable domain of major outer surface protein A of Borrelia burgdorferi.

The gene for the major outer surface protein A (OspA) from several clinically obtained strains of Borrelia burgdorferi, the cause of Lyme disease, has been cloned, sequenced, and expressed in Escherichia coli by using a T7-based expression system (J. J. Dunn, B. N. Lade, and A. G. Barbour, Protein Expr. Purif. 1:159-168, 1990). All of the OspAs have a single conserved tryptophan at residue 216 or, in some cases, 217; however, the region of the protein flanking the tryptophan is hypervariable, as determined by a moving-window population analysis of ospA from 15 European and North American isolates of B. burgdorferi. Epitope-mapping studies using chemically cleaved OspA and a TrpE-OspA fusion have indicated that this hypervariable region is important for immune recognition. Biophysical analysis, including fluorescence and circular dichroism spectroscopy, have indicated that the conserved tryptophan is buried in a hydrophobic environment. Polar amino acid side chains flanking the tryptophan are likely to be exposed to the hydrophilic solvent. The hypervariability of these solvent-exposed amino acid residues may contribute to the antigenic variation in OspA. To test this, we have performed site-directed mutagenesis to replace some of the potentially exposed amino acid side chains in the B31 protein with the analogous residues of a Borrelia garinii strain, K48. The altered proteins were then analyzed by Western blot (immunoblot) with monoclonal antibodies which bind OspA on the surface of the intact B31 spirochete. Our results indicate that specific amino acid changes near the tryptophan can abolish the reactivity of OspA to these monoclonal antibodies, which is an important consideration in the design of vaccines based on recombinant OspA.

Amino Acid Sequence↗
PubMed1994

Characterization of two types of termination signal for bacteriophage T7 RNA polymerase.

The late bacteriophage T7 terminator (T7-T phi) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; termination occurs at a 3' G residue just downstream of the U run. In this work, we have explored the features of this signal that are required for efficient termination by T7 RNA polymerase. Whereas replacement of the template-encoded 3' G residue with A, C, or U by site-directed mutagenesis had little effect, removal of the U-tract prevented termination. Deletion analysis indicates that the stem-loop and U-tract are not sufficient for termination, and that sequences upstream from the terminator have marked effects on the position and efficiency of termination. A sequence within the human preproparathyroid hormone (PTH) gene that encodes an interrupted run of six U residues, but lacks an apparent stem-loop structure, also serves as an efficient terminator for T7 RNA polymerase. We have mapped the primary site of termination in the PTH signal to a G residue that lies downstream of the U-rich run (UUUUCUUG). Deletion analysis indicates that the minimal region required for PTH terminator function extends only 23 bp upstream from the termination site, and subcloning of a 31 bp fragment that includes this region of the PTH signal provides efficient termination. A modified form of T7 RNA polymerase resulting from a single proteolytic cleavage between residues 178 and 179, or mutant polymerases that are altered in this region of the enzyme, fail to recognize the PTH signal while still terminating at T7-T phi.

Amino Acid Sequence↗
PubMed1994

Induction of experimental allergic arthritis with outer surface proteins of Borrelia burgdorferi.

OBJECTIVE: The arthritogenic potential of the cationic outer surface proteins (Osp) from Borrelia burgdorferi was tested in rats. METHODS: Water-soluble Osps were prepared by butanol extraction and were administered by intraarticular injection. Tissue injury was assessed by scintigraphy and histology. RESULTS: A mild arthritis was seen in naive rats. Preimmunized animals had more severe, longer lasting bouts of inflammation. CONCLUSION: The Osps of Borrelia burgdorferi are potent arthritogens in rats. These immunodominant antigens may play a role in the development of Lyme arthritis in humans.

Animals↗
PubMed1994

Mice deficient for the CD40 ligand.

To study the potential roles of CD40L in immune responses, we generated CD40L-deficient mice by gene targeting. Similar to the effects of CD40L mutations in humans (hyper-IgM syndrome), CD40L-deficient mice have a decreased IgM response to thymus-dependent antigens, fail altogether to produce an antigen-specific IgG1 response following immunization, yet respond normally to a T-independent antigen, TNP-Ficoll. Moreover, these mice do not develop germinal centers in response to thymus-dependent antigens, suggesting an inability to develop memory B cell responses. Although CD40L-deficient mice have low levels of most circulating immunoglobulin isotypes, they do not exhibit the spontaneous hyper-IgM syndrome seen in humans, at least up to 12 weeks of age. In summary, our study confirms the important role of CD40-CD40L interactions in thymus-dependent humoral immune responses and germinal center formation.

Animals↗
PubMed1994

Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains.

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.

Amino Acid Sequence↗
PubMed1994

Complete nucleotide sequence of a circular plasmid from the Lyme disease spirochete, Borrelia burgdorferi.

We have determined the complete nucleotide sequence of a small circular plasmid from the spirochete Borrelia burgdorferi Ip21, the agent of Lyme disease. The plasmid (cp8.3/Ip21) is 8,303 bp long, has a 76.6% A+T content, and is unstable upon passage of cells in vitro. An analysis of the sequence revealed the presence of two nearly perfect copies of a 184-bp inverted repeat sequence separated by 2,675 bp containing three closely spaced, but nonoverlapping, open reading frames (ORFs). Each inverted repeat ends in sequences that may function as signals for the initiation of transcription and translation of flanking plasmid sequences. A unique oligonucleotide probe based on the repeated sequence showed that the DNA between the repeats is present predominantly in a single orientation. Additional copies of the repeat were not detected elsewhere in the Ip21 genome. An analysis for potential ORFs indicates that the plasmid has nine highly probable protein-coding ORFs and one that is less probable; together, they occupy almost 71% of the nucleotide sequence. Analysis of the deduced amino acid sequences of the ORFs revealed one (ORF-9) with features in common with Borrelia lipoproteins and another (ORF-2) having limited homology with a replication protein, RepC, from a gram-positive plasmid that replicates by a rolling circle (RC) mechanism. Known collectively as RC plasmids, such plasmids require a double-stranded origin at which the Rep protein nicks the DNA to generate a single-stranded replication intermediate. cp8.3/Ip21 has three copies of the heptameric motif characteristically found at a nick site of most RC plasmids. These observations suggest that cp8.3/Ip21 may replicate by an RC mechanism.

Amino Acid Sequence↗
PubMed1994

Early and specific antibody response to OspA in Lyme Disease.

Borrelia burgdorferi (Bb), the cause of Lyme disease, has appeared not to evoke a detectable specific antibody response in humans until long after infection. This delayed response has been a biologic puzzle and has hampered early diagnosis. Antibody to the abundant organism-specific outer surface proteins, such as the 31-kD OspA, has rarely been detected less than 6 mo after infection. Antibody to a less organism-specific 41-kD flagellin protein, sharing common determinants with other bacteria and thus limiting its diagnostic potential, may appear after 4 to 6 wks. To investigate our hypothesis that specific antibody to OspA may actually be formed early but remain at low levels or bound in immune complexes, we analyzed serum samples from patients with concurrent erythema migrans (EM). This is the earliest sign of Lyme disease and occurs in 60-70% of patients, generally 4-14 d after infection. We used less conventional but more sensitive methods: biotin-avidin Western blots and immune complex dissociation techniques. Antibody specificity was confirmed with recombinant OspA. Specific complexed antibody to whole Bb and recombinant OspA was detected in 10 of 11 of the EM patients compared to 0 of 20 endemic area controls. IgM was the predominant isotype to OspA in these EM patients. Free IgM to OspA was found in half the EM cases. IgM to OspA was also detected in 10 of 10 European patients with EM who also had reactive T cells to recombinant OspA. In conclusion a specific antibody response to OspA occurs early in Lyme disease. This is likely to have diagnostic implications.

Antibodies, Bacterial↗