[Recombinant small heat shock protein from Acholeplasma laidlawii increases the Escherichia coli viability in thermal stress by selective protein rescue].

In both prokaryotes and eukaryotes, the survival at temperatures considerably exceeding the optimum is supported by intense synthesis of the so-called heat shock proteins (HSPs), which act to overcome the adverse effects of heat stress. Among mycoplasmas (class Mollicutes), which have significantly reduced genomes, only some members of the Acholeplasmataceae family possess small HSPs of the α-crystallin type. Overproduction of a recombinant HSP IbpA (Hsp20) from the free-living mycoplasma Acholeplasma laidlawii was shown to increase the resistance of Escherichia coli to short-term heat shock. It has been long assumed that IbpA prevents protein aggregation and precipitation thereby increasing viability of E. coli cells. Several potential target proteins interacting with IbpA under heat stress were identified, including biosynthetic enzymes, enzymes of energy metabolism, and components of the protein synthesis machinery. Statistical analysis of physicochemical properties indicated that IbpA interaction partners significantly differ in molecular weight, charge, and isoelectric point from other members of the E. coli proteome. Upon shortterm exposure to increased temperature, IbpA was found to preferentially interact with high-molecular weight proteins having a pI of about 5.1, significantly lower than the typical values of E. coli proteins.

CELL CULTURES PURITY CONTROL BY METHODS OF CLINICAL DIAGNOSTICS.

Cell cultures of higher organisms, especially cultures of human cells, are increasingly used in medical, pharmaceutical and scientific research. The main problem of cell cultures ­ non-lethal hidden contamination by mycoplasmas, viruses and outsider cell lines. As an available and reliable method for monitoring the purity of the cell cultures, we offer to use PCR kits designed and officially used in clinical diagnostics. We have tested 50 human cell lines using commercial diagnostic systems for detection of papilloma viruses, herpes viruses, adenoviruses, Mycoplasma hominis and total bacterial mass. Contamination in tested cell lines was not found. In the case of cell lines that contain integrated parts of viral genomes, the presence of the respective DNA sequences was confirmed. The proposed diagnostic systems can be effectively used to control the purity of cell lines, for qualitative detection of possible contamination, as well as for quantitative evaluations with calculation of viral load like it is practiced in clinical diagnostics.

Characterization of the Mycoplasma hominis ftsZ gene and its sequence variability in mycoplasma clinical isolates.

We cloned and sequenced Mycoplasma hominis chromosomal fragment containing ftsZ gene. The wild-type expression of the gene was shown at RNA level by reverse transcription followed by PCR amplification. We revealed that M. hominis FtsZ had a comparatively low similarity to proteins of Mycoplasma genitalium and Mycoplasma pneumoniae. After full ftsZ gene sequencing for 14 clinical isolates of M. hominis, single-nucleotide substitutions were found in 21 positions, 6 of them being common for almost all isolates. This ftsZ gene polymorphism may be used for subtyping of M. hominis in clinical samples. Expression of the M. hominis ftsZ gene in different Escherichia coli strains was also demonstrated, and M. hominis FtsZ protein was purified from E. coli cells transformed with recombinant expression plasmid. Complementation between the M. hominis FtsZ and E. coli FtsZ could be shown. The comparison of FtsZ protein structures may also be used for investigation of bacterial phylogenetic relationships.

Characterization of Acholeplasma laidlawii ftsZ gene and its gene product.

The ftsZ gene was found among representatives of all bacterial groups. FtsZ protein is an essential component of cell division ring. Contraction of this cytoskeleton-like ring is believed to be the universal way of bacterial division. Acholeplasma laidlawii possesses all features of the minimal mycoplasma cell and some traits of cell-wall bacteria and seems to be a promising object for study of basic principles of the bacterial division process. We cloned an A. laidlawii chromosomal fragment containing ftsZ gene and two flanking orf which also were identified. A. laidlawii FtsZ protein has been determined with polyclonal antibodies raised in rabbit. It was demonstrated that ftsZ gene of A. laidlawii could be expressed in E. coli cells. We also revealed that A. laidlawii FtsZ had a low similarity to proteins of Mycoplasma genitalium and M. pneumoniae. The comparison of FtsZ structures may be used for investigation of bacterial phylogenetic relations.

Tubular structures of Mycoplasma gallisepticum and their possible participation in cell motility.

In five strains of Mycoplasma gallisepticum, a protein with a molecular mass of about 40 kDa was detected by immunoblotting with anti-pig brain tubulin polyclonal and monoclonal antibodies. In eight other mycoplasma species similarly tested no reaction was observed. Thin serial sections of M. gallisepticum and Acholeplasma laidlawii cells examined by transmission electron microscopy revealed a submembrane system of tubules in M. gallisepticum but not in A. laidlawii. The intracellular spatial distribution of the tubular structures was reconstructed. Thin sections of M. gallisepticum treated with anti-tubulin antibodies and colloidal gold particles (immunogold labelling) revealed distinct labelling of the tubular system. Analysis of the tubular structures by high resolution electron microscopy and optical diffraction showed their helical organization to be: diameter 40 nm, helix pitch approximately 20 nm and electron-transparent core 10 nm in diameter. A possible involvement of the tubular system in mycoplasma motility is suggested.

Continuous distribution of Mycoplasma genome sizes.

Genome sizes of eleven strains of eight species of Mollicutes Mycoplasmataceae were investigated by pulsed-field gel electrophoresis. Mycoplasma genomic sizes were determined from the sum of the sizes of fragments obtained after digestion of genomic DNA with restriction endonucleases. The sizes of the fragments were determined by comparison of their electrophoretic mobilities with those of lambda DNA concatemers. Specific restriction endonucleases were chosen so that after digestion three to ten fragments were obtained. The values for genome size derived by this method showed a continuous distribution that ranged from approximately 650 kb for Mycoplasma hyorhinis BTS-7 to 1600 kb for Acholeplasma laidlawii FHM.

Genome structure of Tetrahymena pyriformis.

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0 X 10(8) base pairs (or 1.2 X 10(11) daltons). About 90% of the macronuclear DNA fragments 200-300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7-9% at a rate corresponding to moderate repetitive sequences; 3-4% of such DNA fragments reassociate at C0t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with S1-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200-300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called "zero time binding" or "foldback" DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to S1-nuclease treatment. The S1-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper- and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.

[Distribution of repeating nucleotide sequences in the macronucleus DNA of Tetrahymena infusoria]. / Raspredelenie povtoriaiushchikhsia nukleotidnykh posledovatel'nostei v DNK makronukleusa infuzorii Tetrahymena.

The genome of Tetrahymena pyriformis macronucleus contain less than 10% of repetitive sequences. About half of them consist of long, more than 2000 base pairs, moderately repeated sequences. Short repetitive sequences (200--300 base pairs) represent 4--5% of the genome. We assume, that the short repetitive sequences are interspersed with single copy sequences of about 1000 base pairs. In this case less than 15% of the Tetrahymena DNA should consist of interspersed repetitive and single copy sequences.