Sequence analysis of a cryptic plasmid from Flavobacterium sp. KP1, a psychrophilic bacterium

A cryptic plasmid found at high copy number was isolated from Flavobacterium sp. KP1, a psychrophilic Gram-negative bacterium, cloned, and sequenced. The sequence will appear in the DDBJ/EMBL/GenBank databases under the accession number AB007196. The pFL1 plasmid is 2311 nucleotides in length with 32.7% GC content, and shows a distinctive nucleotide sequence without homology to other plasmids of similar length. The plasmid contains two open reading frames of significant length, ORFI and ORFII. ORFI encodes a protein similar to the replication proteins found in Gram-negative bacterial plasmids, Bacteroides fragilis plasmid pBI143 and Zymomonas mobilis plasmid pZM2. The putative translation product of ORFII shows homologies with plasmid recombination proteins found mainly in Gram-positive bacterial plasmids such as Staphylococcus aureus plasmid pT181.     

Functional analysis of pVT745, a plasmid from Actinobacillus actinomycetemcomitans

The expansion of myeloma cells is regulated by cytokines, among which IL-6 is a major growth factor. It has been recently suggested that serum transforming growth factor beta 1 (TGF beta 1), a cytokine found in large amounts in alpha-granules of platelets, might play a role in multiple myeloma (MM). It was the purpose of this study to determine serum TGF beta 1 levels in MM patients and to seek a correlation with disease parameters. Measurements were done by ELISA. We studied 35 MM patients (19 stage II, 16 stage III, 20 IgG, 8 IgA and 6 BJ, 1 IgD) in different phases of the disease, 27 healthy individuals and 17 thrombocytopenic patients with other haematological diseases (three MDS, three congenital thrombocytopenia, 11 ITP). Overall samples from MM patients were included: 10 at diagnosis, 18 in remission and 32 in relapse. In normal controls TGF beta 1 serum levels ranged from 1 to 33 ng/ml (median 16.5 ng/ml). In both thrombocytopenic controls with other diseases and thrombocytopenic MM patients (seven samples), TGF beta 1 serum levels were very low (median 3.2 and 4.5 ng/ml respectively). In MM patients with PLT > 100 x 10(9)/L (53 samples), TGF beta 1 serum levels were in the normal range in patients without immunoparesis (1 to 27 ng/ml, median 16.6 ng/ml), whereas they were higher in patients with immunoparesis (polyclonal immunoglobulins (Igs) below lower normal reference values) ranging from 10.2 to 45 ng/ml (median 26.8 ng/ml) (P < 0.01). Serum TGF beta 1 levels fluctuated in the same patient at different times but not according to relapse or remission. Correlation was found only between serum TGF beta 1 levels and immunoparesis and not between serum TGF beta 1 levels and disease stage or Ig subtype nor with prognostic factors for MM (serum CRP, beta 2M or IL-6). This finding suggests that the remaining normal plasma cells are sensitive to the inhibitory action of TGF beta 1 on Ig production. In conclusion TGF beta 1 serum levels are very low in thrombocytopenic patients confirming that platelets are the major source of this cytokine. Furthermore, a strong correlation was found between TGF beta 1 serum levels and immunoparesis in MM patients.     

Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains are generated in cattle persistently infected with noncytopathogenic (noncp) BVDV.cp BVDV strains are considered crucial for the development of fatal mucosal disease. Comparative analysis of cp and noncp BVDV strains isolated from one animal suffering from mucosal disease revealed that the genomes of the cp BVDV strain (CP7) and the corresponding noncp BVDV strain (NCP7) are highly homologous. However, only the genome of CP7 contains an insertion of 27 nucleotides in the NS2 coding region. The inserted sequence represents a duplication of bases 4064 to 4090 of the viral genome, located between the formerly neighboring nucleotides 4353 and 4354. Parts of the viral polyproteins of CP7 and NCP7 were expressed in the T7 vaccinia virus system. These studies revealed that the insertion identified in the CP7 genome is necessary and sufficient for the induction of NS2-3 cleavage. Since the expression of NS3 is strictly correlated to cp BVDV, the insertion identified in the genome of BVDV CP7 represents most likely the relevant mutation leading to the evolvement of CP7 from NCP7.     

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome

OBJECTIVES: To report the features of malignancies responsible for a chest wall mass and involving the sternum, the sternocostal and/or sternoclavicular joints, the chondrocostal junction and/or the adjacent soft tissues. METHODS: The medical records of patients with a chest wall mass due to malignant disease were reviewed retrospectively. The following data were abstracted from each record: characteristics of the pain and mass, constitutional symptoms, physical findings, laboratory test results, findings from imaging studies (plain radiographs, computed tomography and magnetic resonance imaging of the chest, radionuclide bone scan), histologic features of the biopsy specimen from the chest wall mass and origin of the mass. RESULTS: Seven men and three women with a mean age of 53.1 years were included in the study. A single patient had a history of malignant disease (lymphoma); in the remaining nine patients the chest wall mass was the first manifestation of the malignancy. All ten patients had pain with a mixed time pattern. The mass was located on the sternum in half the patients and in a parasternal location in the other half. Erythrocyte sedimentation rate elevation was found in seven patients, an increased serum level of lactate dehydrogenase in one and a monoclonal immunoglobulin in three. Sternal lesions were visible on plain radiographs in four patients. Computed tomography of the chest consistently disclosed sternal or sternocostal lytic lesions with spread to the adjacent soft tissues; in five cases, enlarged lymph nodes were visible in the anterior part of the mediastinum. Magnetic resonance imaging of the chest did not add to the information provided by computed tomography. Radionuclide uptake on the bone scan was increased, decreased, or normal at the site of the lesion. The cause was Hodgkin's disease in two cases, non-Hodgkin's lymphoma in three, metastatic bone disease in two (from an adenocarcinoma of the lung and a hepatocarcinoma, respectively), multiple myeloma in one, and solitary plasmacytoma in two. CONCLUSION: A chest wall mass can be caused by a known or as yet undiagnosed malignancy. Chest wall involvement due to malignant disease in rare, however. The specific features of sternal metastases, lymphomas involving the sternum, and sternal plasmacytomas are discussed. Nonmalignant chest wall lesions that can manifest as a bulging or swelling of the chest wall are reviewed.     

Sequence analysis of small cryptic plasmids isolated from Selenomonas ruminantium S20

The degradation kinetics of five glutamine dipeptides in aqueous solution, i.e. glycyl-L-glutamine (Gly-Gln), L-alanyl-L-glutamine (Ala-Gln), L-valyl-L-glutamine (Val-Gln), L-leucyl-L-glutamine (Leu-Gln) and L-isoleucyl-L-glutamine (Ile-Gln), were studied. Stability tests were performed using a stability-indicating high-performance liquid chromatographic assay. Two different Ala-Gln degradation routes, i.e. the cleavage of a peptide bond and the deamination of an amide group, were observed. The degradation was adequately described by pseudo-first-order kinetics. The maximum stability of Ala-Gln was obtained at an approximate pH of 6.0. The pH-rate profile described by specific acid-base catalysis and hydrolysis by water molecules agreed with the experimental results. The activation energy of Ala-Gln at pH 6.0 was determined to be 27. 1kcal mol-1, and the shelf-life (90% remaining) at 25 and 40 degrees C was predicted to be 5.3 years and 7.1 months, respectively. The rate constants of the glutamine dipeptides were influenced by the N-terminal amino acid residue and decreased in the order: Gly-Gln, Ala-Gln, Leu-Gln, Val-Gln and Ile-Gln.     

Alternative splicing of a 48-nucleotide exon generates two isoforms of the human calcitonin receptor

A portion of the human calcitonin receptor (hCTR) gene corresponding to the region of the porcine gene at which alternative splicing generates two CTR isoforms was isolated by polymerase chain reaction amplification of placental DNA. In contrast to the porcine CTR gene, in which two acceptor sites in exon 8 are separated by 48 nucleotides, we found a distinct 48-nucleotide exon in the hCTR gene that is present approximately 6400 basepairs from the up-stream exon, which corresponds to porcine exon 7, and approximately 1100 basepairs from the down-stream exon, which corresponds to porcine exon 8. Splicing of this exon accounts for the two isoforms of hCTR, containing or not containing a 16-amino acid insertion in the first putative intracellular loop. A region similar to the intron 7-exon junction in the porcine CTR gene is present in the human gene, but contains four extra nucleotides that shift the reading frame. Using probes derived from these introns in somatic cell and in situ hybridization analyses, we assigned the CTR gene to human chromosome band 7q21.2-q21.3. Thus, human and porcine species have evolved distinct mechanisms to generate two similar CTR isoforms.     

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.     

Isolation and characterization of a cryptic plasmid from Erwinia citreus ATCC 31623

Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of phospholamban, which is associated with mutating Cys41 to Phe41, was shown to be as effective as pentameric phospholamban in inhibiting Ca2+ transport in expression systems. To determine whether this monomeric form of phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys41-->Phe41) phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac phospholamban protein levels compared with wild-type controls, with approximately equal to 50% of phospholamban migrating as monomers and approximately 50% as pentamers upon SDS-PAGE. The mutant-phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca(2+)-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32 +/- 0.01 mumol/L in hearts overexpressing monomeric phospholamban, 0.49 +/- 0.05 mumol/L in hearts overexpressing wild-type phospholamban, and 0.26 +/- 0.01 mumol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inhibitory effects of mutant-phospholamban overexpression (mt) were less pronounced than those of wild-type phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov) (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2+ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac, myocyte mechanics and Ca2+ transients among the animal groups overexpressing monomeric or wild-type phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.     

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 microF, and 600 omega. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10(1) transformants/microgram DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained.     

Sequence analysis of the AAA protein family

The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.     

Sequence analysis of porcine gut GLI-1

A protein from porcine gut with 100 amino acid residues (porcine gut GLI-1) and having glucagon-like immunoreactivity has been characterized by partial sequences. The sequence of the C-terminal amino acid residues is -Met-Asn-Thr-Lys-Arg-Asn-Lys-Asn-Asn-Ile-Ala and includes the C-terminal amino acid residue sequence (-Met-Asn-Thr) of porcine glucagon. Evidence is presented that the glucagon sequence -Thr-Ser-Asp-Tyr-Ser-Lys-Tyr- is found in the gut GLI-1 as well. The data support the theory that gut GLI-1 contains the full glucagon sequence and that gut GLI-1 and glucagon are formed from a common precursor.     

Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis

Because certain antiganglioside monoclonal antibodies can facilitate antibody-dependent cellular cytotoxicity against GD2+ ganglioside-bearing human and canine tumor cells, we wished to determine if clinically relevant antiganglioside monoclonal antibodies (Mabs) could also fix canine complement to lyse tumor cells in vitro. Using flow cytometry, human tumor cell lines (M21 melanoma and OHS osteosarcoma) were shown to highly express ganglioside GD2 and, to a lesser degree, GD3. In 51Cr release assays, M21 cells were lysed with canine serum, as a source of complement, plus either Mab 14.G2a or its mouse-human chimera, ch 14.18, specific for GD2. Heating canine serum abrogated its lytic activity and addition of rabbit complement reconstituted M21 lysis. Similar results were obtained with M21 cells when Mab R24 (against GD3) and canine serum were used. OHS cells were also lysed with canine serum plus Mab 14.G2a and lytic activity was abolished by heating canine serum but reconstituted with rabbit complement. Alone, canine serum or Mabs were not lytic to M21 or OHS cells. Conversely, human neuroblastoma (LAN-5) and K562 erythroleukemia cells were lysed by canine serum alone which was shown by flow cytometry to contain naturally occurring canine IgM antibodies that bound LAN-5 and K562 cells. The lytic activity of canine serum for LAN-5 or K562 cells was abolished by heating and restored by addition of either human or rabbit complement. Thus, human tumor cell lines can be lysed with antiganglioside Mabs through fixation and activation of canine complement-dependent lytic pathways. Canine xenoantibodies also mediate complement-dependent cytotoxicity of some human tumor cell lines. Together, these results are significant because they demonstrate an antitumor effect of the canine immune system which is of potential importance for cancer immunotherapy in a promising animal model.     

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar

Cyclic nucleotide-gated channels are composed of a core transmembrane domain, structurally homologous to the voltage-gated K+ channels, and a cytoplasmic ligand-binding domain. These two modules are joined by approximately 90 conserved amino acids, the C-linker, whose precise role in the mechanism of channel activation by cyclic nucleotides is poorly understood. We examined cyclic nucleotide-gated channels from bovine photoreceptors and Caenorhabditis elegans sensory neurons that show marked differences in cyclic nucleotide efficacy and sensitivity. By constructing chimeras from these two channels, we identified a region of 30 amino acids in the C-linker (the L2 region) as an important determinant of activation properties. An increase in both the efficacy of gating and apparent affinity for cGMP and cAMP can be conferred onto the photoreceptor channel by the replacement of its L2 region with that of the C. elegans channel. Three residues within this region largely account for this effect. Despite the profound effect of the C-linker region on ligand gating, the identity of the C-linker does not affect the spontaneous, ligand-independent open probability. Based on a cyclic allosteric model of activation, we propose that the C-linker couples the opening reaction in the transmembrane core region to the enhancement of the affinity of the open channel for agonist, which underlies ligand gating.     

Conjugative mobilization of a vancomycin resistance plasmid by a putative Enterococcus faecium sex pheromone response plasmid

The purpose of this study was to develop a model of gastrointestinal carcinogenesis using C57BL/6 mice. Treatment regimens consisted of one control group and 2 groups which received N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in drinking water: 50 micrograms/ml x 52 weeks and 100 micrograms/ml x 27 weeks. In addition, 2 protocols using adjuvant agents intended to increase tumor formation were used: MNNG (100 micrograms/ml) x 27 weeks + 0.2% taurocholic acid added to the diet from weeks 13-52, and MNNG (50 micrograms/ml) x 33 weeks+caerulein (10 micrograms/kg) subcutaneously 3 times/week from weeks 21-52. High-grade dysplasia was observed in the duodenum of 1/13 mice treated with MNNG (50 micrograms/ml). The combination of the latter and caerulein did not augment tumorigenesis. Mice treated with MNNG (100 micrograms/ml) frequently developed neoplasia in the duodenum and upper jejunum. Foci of low-grade and high-grade dysplasia alone were found in 3/12 (25%) mice; and intramucosal and invasive adenocarcinoma were found in 7/12 (58.3%) mice. The addition of taurocholic acid significantly increased the number and histological stages of the tumors (adenocarcinoma occurred in 100%, P = 0.03) and decreased the time for tumor formation.     

Sequence and phylogenetic analyses of a new STLV-I from a naturally infected tantalus monkey from Central Africa

Simian T-cell leukemia virus (STLV-I) is an oncovirus highly related to human T-cell leukemia virus type I (HTLV-I). To further examine the extent of variability, dissemination patterns, phylogeny, and evolution of these viruses, we analyzed a new STLV-I variant from a naturally infected Cercopithecus aethiops var. tantalus from the Central African Republic. Sequence analyses of its LTR, gag, pol, env, and pX (OrfII) genes indicated that this isolate, STLV-I (Tan 90), is 6% divergent from the prototype HTLV-I (ATK) and is the most divergent African STLV-I characterized to date. Our phylogenetic data indicate that southeast Asian and African STLV-I and HTLV-I strains segregated from each other thousands of years ago and that Japanese HTLV-I strains represent a relatively recent introduction of African or New World isolates. The data also indicate that interspecies transmission occurred several times on different continents over prolonged periods of time.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Sequence analysis of the 'Goodpasture antigen' of mammals

BACKGROUND: Autoimmunity to the NC1 domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), the Goodpasture antigen, is the cause of spontaneous human antiglomerular basement membrane (anti-GBM) disease, and of anti-GBM nephritis in several animal models. METHODS: We have derived amino acid sequences from alpha3(IV)NC1 for a number of mammalian species (monkey, sheep, pig, dog, rabbit, and rat) by RT-PCR and cDNA cloning. The GBM of some species was studied comparatively for binding to Goodpasture autoantibodies. RESULTS: From this work and other data the sequences of nine mammalian species can be aligned. Regions and residues that may be functionally important are identified. Alpha3(IV)NC1 sequences were found to be less closely conserved across species than alpha1 and alpha2(IV)NC1, 91 to 99% in comparison to a minimum of 97% for alpha1, but these differences were unevenly distributed along the molecule. There was a particularly striking homology between rodent and human sequences in the carboxyl-terminal region. Binding of Goodpasture autoantibodies to rat alpha3(IV)NC1 was poor in comparison with other species. CONCLUSIONS: Comparison of sequences and binding casts doubt on the importance of the carboxyl-terminal region for antibody binding, a region identified as a potential major epitope in previous studies. Sequence comparisons suggest possible reasons for the nephritogenicity of alpha3(IV)NC1 in active models of anti-GBM disease.     

Sequence and transcriptional analysis of groES and groEL genes from the thermophilic bacterium Clostridium thermocellum

Ca ++/cAMP response element binding protein (CREB), phosphoCREB, and c-Fos-like (c-FL) immunoreactivity (IR) were examined in the nucleus of the solitary tract (NTS) and parabrachial nucleus (PBN) after peripheral cholecystokinin (CCK). c-FLIR was observed only after CCK, but CCK did not alter high basal levels of CREB-IR and phosphoCREB-IR. PhosphoCREB may be necessary but is not sufficient to induce c-Fos after CCK injection.     

Sequence specificity of illegitimate plasmid recombination in Bacillus subtilis: possible recognition sites for DNA topoisomerase I

Previous work in our group indicated that structural plasmid instability in Bacillus subtilis is often caused by illegitimate recombination between non-repeated sequences, characterized by a relatively high AT content. Recently we developed a positive selection vector for analysis of plasmid recombination events in B. subtilis which enables measurement of recombination frequencies without interference of selective growth differences of cells carrying wild-type or deleted plasmids. Here we have used this system to further analyse the sequence specificity of illegitimate plasmid recombination events and to assess the role of the host-encoded DNA topoisomerase I enzyme in this process. Several lines of evidence suggest that single-strand DNA nicks introduced by DNA topoisomerase I are a major source of plasmid deletions in pGP100. First, strains overproducing DNA topoisomerase I showed increased levels of plasmid deletion. Second, these deletions occurred predominantly (>90% of the recombinants) between non-repeated DNA sequences, the majority of which resemble potential DNA topoisomerase I target sites. Sequence alignment of 66 deletion end-points confirmed the previously reported high AT content and, most importantly, revealed a highly conserved C residue at position -4 relative to the site of cleavage at both deletion termini. Based on these genetic data we propose the following putative consensus cleavage site for DNA topoisomerase I of B.subtilis: 5'-A/TCATA/TTAA/TA/TA-3'.