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G Christiansen

Publications and source records attributed to G Christiansen.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular biology of Mycoplasma.

Mycoplasmas are the smallest free living microorganisms with the smallest genome. The G+C content is in general low (25-33%) and the coding capacity is about 600 proteins. Mycoplasma species are phylogenetically related, they use the genetic codon UGA for tryptophan, and show rapid evolution, with a high rate of divergence. The genomes of Mycoplasma genitalium and Mycoplasma pneumoniae have been fully sequenced. Striking features of the M. genitalium sequencing project are the presence of a high number of membrane proteins with no resemblance to previously sequenced genes and the presence of repeated fragments of the gene encoding the tip-localized 140 kDa adhesin (MgPa). Many Mycoplasma species display a high frequency of antigenic variation, both as phase and size variation of individual antigens. Mycoplasma hominis isolates are known to be antigenic heterogeneous, as reflected in the reactivity with monoclonal antibodies (MAbs). The genetics of the antigenic variation has been studied for three different surface exposed antigens: P120, Lmp, and P50/Vaa. The gene encoding P120 had a hyper-variable region in the N-terminal region. In addition, a second gene with homology to p120 was identified. The gene encoding Lmp, a 135 kDa protein is repeated and both genes are translated and both contain internal repeated sequences. Deletion mutants in the lmp gene were obtained by cultivation of M. hominis PG21 with MAb 552 specific for the repeated part of Lmp. One of the lmp genes had deletions of from four to eight repeats. The other gene was left unaltered. The genes encoding P50/Vaa show a different form of variability where domains of the genes seem to be exchangeable. The genomic maps of five M. hominis strains showed that even though the size of the genomes varied the position of the different genes were in general conserved.

Animals↗
PubMed1997

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection.

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.

Animals↗
PubMed1997

Molecular biology of the Chlamydia pneumoniae surface.

Chlamydia pneumoniaeis a fastidious microorganism with a characteristic biphasic lifecycle causing a variety of human respiratory tract infections. There is limited knowledge about the molecular biology of C. pneumoniae, and only a few genes have been sequenced. The structure of the chlamydial surface differs from that of Chlamydia trachomatis. In order to study the surface of C. pneumoniae we generated monoclonal antibodies (MAbs) against C. pneumoniae strain VR-1310 and selected 14 MAbs that reacted with the surface of C. pneumoniae. All MAbs reacted in immunoelectron microscopy with the surface of both whole C. pneumoniae VR-1310 elementary bodies and with purified sarcosyl extracted outer membrane complexes. However, only 2 of the MAbs reacted in immunoblotting with C. pneumoniae proteins and only with antigen that had not been heat treated in SDS-sample buffer. This indicates the dominance of conformational epitopes at the C. pneumoniae surface.

Antibodies, Monoclonal↗
PubMed1996

Mapping of Chlamydia trachomatis proteins by immobiline-polyacrylamide two-dimensional electrophoresis: spot identification by N-terminal sequencing and immunoblotting.

Proteins from purified elementary bodies of Chlamydia trachomatis were separated by two-dimensional gel electrophoresis on nonlinear wide-range immobilized pH gradients in the first dimension and polyacrylamide gradient gels in the second dimension. The maps obtained with this system are highly reproducible and resolve ca. 600 spots. By using immunoblot analysis with specific antibodies and/or N-terminal amino acid sequencing, we established the map positions of a number of described chlamydial proteins, such as the major outer membrane protein (MOMP) the 60 kDa cystein-rich outer membrane protein (OMP2), the DnaK-like, GroEL-like, and macrophage infectivity potentiator (MIP)-like proteins, the plasmid-encoded pgp3 protein, two ribosomal proteins (S1 and L7/L12), and the protein-elongation factor EF-Tu. Other proteins, for which gene assignment was not possible, have been identified by three parameters (Mr, pI and N-terminal sequence). This work provides a preliminary basis for a future and progressive compilation of a genome-linked database of chlamydial proteins.

Acrylamides↗
PubMed1996

Involvement of N-acyl-L-hormoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens.

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer-cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high-performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N-butanoyl-L-homoserine lactone (BHL) and N-hex anoyl-L-homoserine lactone (HHL) in cell-free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrl (swarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the LuxI family of putative N-acyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

4-Butyrolactone↗
PubMed1996

Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1.

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro. Our results imply that DNA condensation is not the only function of Hc2.

Antibodies, Bacterial↗
PubMed1996

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.

Adenosine Triphosphatases↗
PubMed1996

Analysis of 0.5-kilobase-pair repeats in the Mycoplasma hominis lmp gene system and identification of gene products.

Mycoplasma hominis, an opportunistic pathogenic bacterium of humans, has a small genome of 700 kb. Despite this, multiple copies of gene sequences with similarities to the structural gene (lmp1) of a 135-kDa surface-located membrane protein (Lmp1) have been identified on the genome of M. hominis PG21 (lmp2, lmp3, and lmp4). The distance between the lmp1-lmp2 region and the lmp3-lmp4 region was more than 110 kb. lmp3-lmp4 of M. hominis PG21 was sequenced and found to contain two putative genes. The gene region of 6.5 kb contained a 5' unique region and a 3' unique region separated by 9 0.5-kb repeats with 51 to 90% similarity to 10 similar repeats found in the lmp1-lmp2 region. The 0.5-kb DNA repeats thus comprised about 1% of the entire genome. In both regions, a base change in one of the repeats gave rise to a stop codon, and thereby lmp2 and lmp4 occurred. By PCR amplification of reverse-transcriptase-generated cDNA it was shown that all four genes were transcribed. By use of Lmp-specific antibodies we showed that both lmp1 and lmp3 were translated into proteins (Lmp1 and Lmp3). Each of the four lmp genes represented by their unique cloned segments was used as a probe to analyze the presence, distribution, and organization of the genes within the genome in 13 M. hominis isolates. The repetitive element was detected at one or two locations on the chromosome for all isolates. The lmp3-specific element was present in all isolates, and lmp1- and lmp2-specific elements were present in all but one isolate. The lmp4-specific element was present in about half the isolates tested. For five M. hominis isolates the chromosomal location of the lmp genes was mapped.

Amino Acid Sequence↗
PubMed1996

Differentiation of Serratia liquefaciens into swarm cells is controlled by the expression of the flhD master operon.

The velocity with which a swarming colony of Serratia liquefaciens colonizes the surface of a suitable solid substratum was controlled by modulating the expression of the flhD master operon. In liquid medium, the stimulation of flhD expression resulted in filamentous, multinucleate, and hyperflagellated cells that were indistinguishable from swarm cells isolated from the edge of a swarm colony. Thus, expression of the flhD master operon appears to play a central role in the process of swarm cell differentiation.

Bacterial Proteins↗
PubMed1996

The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.

Amino Acid Sequence↗
PubMed1995

Rescue of measles viruses from cloned DNA.

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.

Base Sequence↗
PubMed1995

Analysis of RET protooncogene point mutations distinguishes heritable from nonheritable medullary thyroid carcinomas.

BACKGROUND: The distinction of sporadic from inherited medullary thyroid carcinomas (MTCs) is of clinical importance because of the differences in prognosis, and the need for family screening for genetic counseling required in the latter. Germline mutations in the RET protooncogene are associated with multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Somatic point mutations in the same gene have been identified in a subset of sporadically occurring medullary thyroid carcinomas. METHODS: A nonisotopic polymerase chain reaction-(PCR) based single strand conformation polymorphism (SSCP) analysis and heteroduplex gel electrophoresis method was used to screen DNA extracted from 32 formaldehyde fixed and paraffin embedded MTC specimens and normal tissue or blood of the same patient for point mutations in RET exons 10, 11, and 16. Point mutations were identified by nonisotopic cycle sequencing of PCR-products using an automated DNA-sequencer. Results were compared with the disease phenotype, clinical findings, and follow-up. RESULTS: Six different missense germline mutations were identified at cysteine residues 618, 630, and 634 of the cysteine-rich extracellular RET domain encoded by exons 10 and 11 in all patients with FMTC and MEN 2A. The frequency of mutations at codon 634 was higher in patients with MEN 2A than with FMTC and a 634 Cys-->Arg mutation was associated with parathyroid disease in three patients. A germline Met-->Thr point mutation at codon 918 of the RET tyrosine kinase domain was identified in all three patients with MEN 2B. Two patients with clinically sporadic MTCs and negative family history exhibited a RET germline mutation at codon 634, indicating the presence of an nonpredicted inherited MTC. Furthermore, one patient had a 618 Cys-->Ser mutation in the tumor and nontumorous thyroid DNA but not in blood DNA, indicating a mosaic mutation affecting thyroid tissue but not blood cells. Tumor specific (somatic) Met-->Thr point mutations at codon 918 were identified in 5 of 13 sporadic MTCs. The remaining eight sporadic MTCs lacked mutations in all three RET exons tested. CONCLUSIONS: This study demonstrates that (1) the molecular methods are not only suitable to identify asymptomatic individuals at risk for MEN 2A, FMTC, and MEN 2B but also to distinguish heritable from nonheritable MTCs using archival tissue specimens, and (2) that more MTCs than clinically expected are heritable, indicating the need for genetic analysis of all patients with MTC.

Adult↗
PubMed1995

Chimeric FimH adhesin of type 1 fimbriae: a bacterial surface display system for heterologous sequences.

The FimH adhesin of type 1 fimbriae has been tested as a display system for heterologous protein segments on the surface of Escherichia coli. This was carried out by introduction of restriction site handles (BglII sites) in two different positions in the fimH gene, followed by in-frame insertion of heterologous DNA segments encoding two reporter sequences. In the selected positions such insertions did not significantly alter the function of the FimH protein with regard to surface location and adhesive ability. The system seemed to be quite flexible, since chimeric versions of the FimH adhesin containing as many as 56 foreign amino acids were transported to the bacterial surface as components of the fimbrial organelles. Furthermore, the foreign protein segments were recognized by insert-specific antibodies when expressed within chimeric proteins on the surface of the bacteria. The results from this feasibility study point to the possibility of using the FimH adhesin as a general surface display system for sizeable protein segments.

Adhesins, Bacterial↗
PubMed1995

Induction of phospholipase- and flagellar synthesis in Serratia liquefaciens is controlled by expression of the flagellar master operon flhD.

When a liquid culture of Serratia spp. reaches the last part of the logarithmic phase of growth it induces the synthesis of several extracellular hydrolytic enzymes. In this communication we show that synthesis and secretion of the extracellular phospholipase is coupled to expression of flagella. Expression of flagella is demonstrated to follow a growth-phase-dependent pattern. Cloning, complementation studies and DNA-sequencing analysis has identified a genetic region in Serratia liquefaciens which exhibits extensive homology to the Escherichia coli flhD flagellar master operon. Interruption of the chromosomal flhD operon in S. liquefaciens results in non-flagellated and phospholipase-negative cells, but the synthesis of other exoenzymes is not affected. By placing the flhD operon under the control of a foreign inducible promoter we have shown that increased transcription through the flhD operon leads to induction of flagellar synthesis and phospholipase expression.

Amino Acid Sequence↗
PubMed1995

A 135-kilodalton surface antigen of Mycoplasma hominis PG21 contains multiple directly repeated sequences.

A monoclonal antibody was used to characterize a 135-kDa surface-located membrane protein (Lmp1) generally present in Mycoplasma hominis strains. The monoclonal antibody, 552, was applied to identify the corresponding gene in an expression library of M. hominis PG21 DNA. The M. hominis PG21 lmp1 gene was sequenced, and its gene product was characterized with the goal of elucidating the structure and function of Lmp1. A total of 7,196 bp in the lmp1 region was sequenced. An open reading frame of 4,032 bp, encoding a protein of 1,344 amino acids with a calculated molecular weight of 147,000, was identified. Analysis of the deduced amino acid sequence predicted a hydrophilic protein with a basic pI (10.0). The N-terminal 24 amino acids were a typical leader sequence. Downstream from the first 726 nucleotides, six similar direct repeats of 471 nucleotides were found. In repeat 7, a single-base substitution, C-->A, gave rise to the stop codon of lmp1. Thus, the C-terminal 945 amino acids were encoded by the 471-bp direct repeats. As evidenced by Southern blot analysis, the gene encoding the 135-kDa antigen is part of a multigene family. One of the genes, lmp2, was situated directly downstream from lmp1 where the direct repeats continued.

Amino Acid Sequence↗
PubMed1995

Selection of Mycoplasma hominis PG21 deletion mutants by cultivation in the presence of monoclonal antibody 552.

Three mutants of Mycoplasma hominis PG21 were isolated and shown to contain alterations in the size of a repeat-containing gene encoding a surface-localized 135-kDa antigen designated Lmp1. The mutants were isolated by cultivating M. hominis for a 3-month period in the presence of Lmp1-specific monoclonal antibody (MAb) 552. The epitope for MAb 552 was localized at the repeated part of the protein. The gene encoding Lmp1 is part of a transcriptional complex that contains 9.5 direct repeats of 471 bp each. Pure cultures of mutant strains were obtained by subcloning, and three mutants were characterized. The mutants showed deletions of a various number of repeats. The deletions were accompanied by a decrease in size of the proteins. With increasing size of deletions, agglutination and growth inhibition by MAb 552 became less pronounced. Spontaneous aggregation of the mutant M. hominis cells in culture medium was, however, increased, indicating that the repeated elements may be of importance for repulsion of the cells.

Agglutination Tests↗
PubMed1995

The export systems of type 1 and F1C fimbriae are interchangeable but work in parental pairs.

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found, among others, on strains associated with urinary tract infections. Biosynthesis of type 1 and F1C fimbrial organelles requires individual, specialized two-component assembly systems. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structure of the organelles, is very similar; however, the actual sequence homology of the structural elements is not remarkable (34 to 60%). Both gene clusters encode a periplasmically located chaperone and an usher protein, located in the outer membrane, required for organelle biogenesis. Deletion of either element causes abolishment of fimbriation. The present report addresses the question of promiscuity in fimbrial biogenesis. Our data indicate that the two-component export systems of the two organelle systems are reciprocally interchangeable; however, they seem to function only in parental pairs.

Amino Acid Sequence↗