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Query: UNIPROT:P06889 (Mol)
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Laboratory diagnosis of mycoplasma infections is hampered by the difficulty or total failure to cultivate the organisms in vitro, and by the frequently weak and poorly specific serological response of the host. DNA probes consisting of cloned ribosomal RNA genes, cDNA to mycoplasmal rRNA, synthetic 16S rRNA oligonucleotide sequences, or cloned mycoplasmal protein genes, have been developed and applied as diagnostic tools in a variety of human and animal mycoplasma infections. These included primary atypical pneumonia caused by Mycoplasma pneumoniae, urogenital infections associated with M. genitalium and Ureaplasma urealyticum, and infections with M. fermentans, M. penetrans or M. pirum--mycoplasmas recently incriminated as cofactors in AIDS. DNA probes were also designed to aid in diagnosis of mycoplasma diseases of farm and laboratory animals, and the hard-to-diagnose mycoplasma infections of cell cultures. Sensitivity of mycoplasma detection by the different probes ranged between 10(3) and 10(6) colony-forming units, a level which may not be sufficiently high for use in a clinical laboratory. The introduction of PCR has pushed aside the previously developed DNA probes, by providing faster and much more sensitive tests. The sensitive level of a PCR test can be as low as a single organism, enabling detection of mycoplasmas in patients treated with antibiotics and in asymptomatic patients. PCR becomes positive prior to serological response and is also effective in immunocompromised hosts. PCR was shown to be most valuable in detection and identification of the non-culturable plant and insect mycoplasma-like organisms (MLOs). Nevertheless, false-negative PCR results are rather common due to inhibitors of the PCR reaction in the clinical specimen, while false-positive results may occur due to contamination of the reagents with target DNA. In conclusion, the PCR procedure is still too complex to be carried out in a routine diagnostic laboratory. PCR prepackaged quality-controlled diagnostic kits are now in the process of rapid development. Once these kits become available, and at a reasonable cost, PCR will certainly take its place as a major diagnostic tool in the routine diagnosis of mycoplasma infections.
Mol Cell Probes 1994 Dec
PMID:DNA probes and PCR in diagnosis of mycoplasma infections. 770 Feb 72

Using an in vitro protein-synthesizing system that allowed us to monitor separately the reading of each glycine codon, we have previously shown, that in constructs based on glycine tRNA1 from Escherichia coli the nature of the nucleotide in position 32 determines the ability of the anticodon UCC to discriminate between the glycine codons. Thus, with a U in position 32 the anticodon UCC discriminated according to the wobble rules, but with a C in this position it had lost its ability to discriminate. In the present paper we show that the same is true also for constructs based on mycoplasma glycine tRNA. When C32 in the wild type was changed to U32, the anticodon UCC discriminated between the glycine codons, while in wild type mycoplasma glycine tRNA it did not. Furthermore, when U32 was changed to C32 in glycine tRNA1(CCC), the anticodon CCC loses its ability to discriminate. We therefore conclude that the nature of the nucleotide in position 32 determines the discriminatory ability of both anticodons UCC and CCC in the glycine tRNA1 structural background, and that the same is true for the anticodon UCC in the mycoplasma glycine tRNA background.
J Mol Biol 1995 Mar 24
PMID:Glycine codon discrimination and the nucleotide in position 32 of the anticodon loop. 770 68

We report that the human gene SB1.8 (DXS423E) encodes a protein of 1233 amino acids that is highly homologous (30% identity) to the essential yeast protein SMC1 which is required for the segregation of chromosomes at mitosis. Both SB1.8 and SMC1 contain an N-terminal NTP binding site, a central coiled-coil region and a C-terminal helix-loop-helix domain, and have structural features in common with the force generating proteins myosin and kinesin. SB1.8 also exhibits regions of homology and overall structural similarity to the prokaryote (Mycoplasma hyorhinis) protein 115p. Thus SB1.8 and SMC1 are members of a highly conserved and ubiquitous family of proteins that appear to have a fundamental role in cell division. In addition we show that SB1.8 (DXS423E) maps to a cosmid contig that lies centromeric to the OATL2 locus at chromosome Xp11.2.
Hum Mol Genet 1995 Feb
PMID:The human SB1.8 gene (DXS423E) encodes a putative chromosome segregation protein conserved in lower eukaryotes and prokaryotes. 775 74

We developed a microtitre hybridization assay for the detection of polymerase chain reaction (PCR) amplified sequences. For this, cloned Mycoplasma pneumoniae DNA containing a sequence complementary to the PCR products is first covalently bound to microtitre wells. These coated microplates can be stored for several months. Then, an aliquot of the PCR product, labelled with digoxigenin-dUTP during its synthesis is hybridized to the immobilized DNA. The use of a rapid hybridization buffer makes this step very short (5 min). Finally, the hybridization signal is detected by an anti-digoxigenin antibody conjugated with alkaline phosphatase. Compared to Southern or other microplate hybridization techniques, this method is cheaper, involved fewer steps and allows easy handling of a large number of samples. This method was used for detection of M. pneumoniae in a series of clinical specimens.
Mol Cell Probes 1995 Feb
PMID:Improved microplate immunoenzymatic assay of PCR products for rapid detection of Mycoplasma pneumoniae. 776 Aug 56

Previously, we described the identification of a novel Mycoplasma pneumoniae M129 protein, named P65 because of its apparent molecular mass of 65 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (T. Proft and R. Herrmann, Mol. Microbiol. 13:337-348, 1994). DNA sequence analysis of the P65 open reading frame (orfp65), however, revealed an ORF encoding a protein with a molecular weight of 47,034. This discrepancy can be explained by the unusual amino acid composition of this protein. According to the deduced amino acid sequence, the N-terminal half of P65 contains several penta- and hexapeptides (DPNAY and DPNQAY) forming a proline-rich acidic domain. Secondary-structure predictions indicated beta-sheets and turns within that region, suggesting an extended and rigid conformation. Near the C terminus of P65 the tripeptide Arg-Gly-Asp (RGD) was found. This motif is known to play an important role in binding of extracellular matrix proteins to integrins. P65 could be located exclusively to the Triton X-100-insoluble cell fraction. The results of immunofluorescence microscopy and of immunoadsorption experiments indicated that P65 carries surface-exposed regions. Mild treatment of whole cells with proteases resulted in cleavage of a limited amount of P65 molecules, suggesting either that only a small percentage of P65 molecules are exposed on the surface or that protease cleavage is hampered by a compact protein conformation or by binding of an unknown component to P65. P65 exhibits size polymorphism in M. pneumoniae M129 and FH. This is caused by an intragenetic duplication of a 54-bp sequence within the FH orfp65. As a consequence, the number of DPNAY pentapeptides increased from 9 to 12 repeats in the FH strain.
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PMID:The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH. 776 45

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.
Mol Microbiol 1994 Sep
PMID:DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC. 781 43

We demonstrate here that the arbitrary primer polymerase-chain-reaction-based DNA fingerprinting method (also termed random amplified polymorphic DNA or RAPD) can be used to distinguish among strains of the avian pathogen Mycoplasma gallisepticum. Ten base oligonucleotide primers were used individually to prime DNA synthesis from genomic DNAs. Strain-specific arrays of DNA fragments were generated, which allowed us to identify and group isolates. Isolates of M. synoviae, M. gallinarum and M. iners yielded arrays of DNA fragments that differed markedly from those generated from the M. gallisepticum isolates using the same arbitrary primers. These results show that the RAPD fingerprinting method distinguishes genetically different strains of M. gallisepticum and indicates that it should be valuable for monitoring transmission of this pathogen.
Mol Cell Probes 1994 Aug
PMID:Mycoplasma gallisepticum strain differentiation by arbitrary primer PCR (RAPD) fingerprinting. 787 72

The heterogeneity among Mycoplasma gallisepticum strains was characterized by comparing genomic DNA from different strains. Two criteria were used for the comparison. 1. Analysis of SmaI and BglI restriction patterns. A method using a dialysis sack for preparation of mycoplasma DNA suitable for pulsed-field gel electrophoresis is described. 2. Comparison of "fingerprinting" patterns obtained as a result of Southern transfer of HindIII-digested DNA followed by hybridization with cloned repetitive M. gallisepticum sequences. It was shown that both types of patterns analyzed are strain-specific; moreover, we were able to identify as a A5969 the strain of M. gallisepticum which was wrongly referred to as S6 in our previous works (Mol. Biol. 1991. V. 25. P. 1643-1649; FEBS Lett. 1991. V. 291. P. 71-74).
Mol Biol (Mosk)
PMID:[Identification of a Mycoplasma strain using fingerprinting of restriction enzyme hydrolysates of chromosomal DNA]. 788 30

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.
Mol Microbiol 1994 May
PMID:Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis. 793 78

The detection limits of the polymerase chain reaction (PCR) for Mycoplasma pneumoniae were determined using specimens from persons known to have had M. pneumoniae pneumonia. Four primers were selected from the known sequence of the P1 gene. The primer pair (P1-178 and P1-809) which generates a 631 fragment gave the lowest detection limit. Nineteen of 21 throat swabs, which contained between 0.06 and 2 colony-forming units (CFU) per microlitre, from culture positive patients, were positive by PCR. The fact that M. pneumoniae grows in broth culture in spherules causes problems for determining the number of CFU detected in PCR. Filtering broth cultures through a 0.6 micron polycarbonate filter increased the number of CFUs two-to-ten-fold compared to unfiltered cultures. The lysis method needed to assay throat swabs differed from that necessary for broth cultures in that proteinase K treatment for 18 h increased the detection limit 10- to 100-fold when compared to NaOH digestion.
Mol Cell Probes 1994 Apr
PMID:Evaluation of the detection limits of PCR for identification of Mycoplasma pneumoniae in clinical samples. 793 10


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