Single cell analysis of cancer cells using an improved RT-MLPA method has potential for cancer diagnosis and monitoring.

Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.

The adhesin related 30-kDa protein of Mycoplasma pneumoniae exhibits size and antigen variability.

Mycoplasma pneumoniae is a pathogenic bacterium colonizing epithelial cells of the human respirator tract. Using an erythrocyte binding assay we isolated a cytadsorption negative mutant designated M7 which has lost 12 of a total of 13 repetitive sequences of a proline rich C-terminal region of the adhesin related 30-kDa protein. The truncated adhesin related protein of 22 kDa showed reduced antigenicity compared to the corresponding wild-type protein. Moreover, the mutant M7 proved incapable of adhering to erythrocytes and to a human colon carcinoma cell line indicating that the repetitive C-terminal region of the 30-kDa protein is essential for effective cytadherence. The adhesin related 30-kDa protein as well as the truncated forms of the corresponding protein were accessible to carboxypeptidase Y which clearly shows surface exposure of the C-terminus of this protein.

The thioredoxin reductase system of mycoplasmas.

Representative species of the Mollicutes possess a thioredoxin reductase system (NTS) composed of a low-molecular-mass thioredoxin (TRX) and NADPH-binding thioredoxin reductase (NTR). The TRXs of Mycoplasma pneumoniae and M. capricolum have molecular masses of 11-2 and 12 kDa, respectively, and are stable at 90 degree C for 10 min. Both TRXs reacted with monospecific polyclonal antibodies generated against the Bacillus subtilis TRX, but not with anti-Escherichia coli TRX antisera. The M. capricolum and M. pneumoniae NTRs were partially purified and were found to be active with the homologous TRX, but not with the TRX of B. subtilis or E. coli. The NTS activity had an optimal pH of 6.5-7.5 and was dependent on NADPH as an election donor, a requirement which could not be fulfilled by NADH. The genes encoding the TRX and NTR (trxA and trxB) or M. pneumoniae were cloned and sequenced. The comparative analysis of the predicted amino acid sequence of trxA showed that the 11.2 kDa protein (102 aa) shared 26-68% sequence similarity with products of other known trxA genes and contained the conserved active site Cys-Gly-Pro-Cys. The predicted amino acid sequence of trxB contained 315 residues with a conserved NADPH binding domain and FAD binding domains I and II. The cysteine dithiol redox active region had isoleucine rather than threonine at the active site, as compared with other NTRs. The high activity of the NTS in mycoplasmas suggests that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge.

Comparative analysis of the genomes of the bacteria Mycoplasma pneumoniae and Mycoplasma genitalium.

The sequenced genomes of the two closely related bacteria Mycoplasma genitalium and Mycoplasma pneumoniae were compared with emphasis on genome organization and coding capacity. All the 470 proposed open reading frames (ORFs) of the smaller M.genitalium genome (580 kb) were contained in the larger genome (816 kb) of M.pneumoniae. There were some discrepancies in annotation, but inspection of the DNA sequences showed that the corresponding DNA was always present in M. pneumoniae. The two genomes could be subdivided into six segments. The order of orthologous genes was well conserved within individual segments but the order of these segments in both bacteria was different. We explain the different organization of the segments by translocation via homologous recombination. The translocations did not disturb the continuous bidirectional course of transcription in both genomes, starting at the proposed origin of replication. The additional 236 kb in M.pneumoniae,compared with theM.genitalium genome, were coding for 209 proposed ORFs not identified in M.genitalium. Of these ORFs, 110 were specific to M.pneumoniae exhibiting no significant similarity to M.genitalium ORFs, while 76 ORFs were amplifications of ORFs existing mainly as single copies in M. genitalium. In addition, 23 ORFs containing a copy of either one of the three repetitive DNA sequences RepMP2/3, RepMP4 and RepMP5 were annotated in M.pneumoniae but not in M.genitalium,although similar DNA sequences were present. TheM.pneumoniae-specific genes included a restriction-modification system, two transport systems for carbohydrates, the complete set of three genes coding for the arginine dihydrolase pathway and 14 copies of the repetitive DNA sequence RepMP1 which were part of several different translated genes with unknown function.

Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae.

The entire genome of the bacterium Mycoplasma pneumoniae M129 has been sequenced. It has a size of 816,394 base pairs with an average G+C content of 40.0 mol%. We predict 677 open reading frames (ORFs) and 39 genes coding for various RNA species. Of the predicted ORFs, 75.9% showed significant similarity to genes/proteins of other organisms while only 9.9% did not reveal any significant similarity to gene sequences in databases. This permitted us tentatively to assign a functional classification to a large number of ORFs and to deduce the biochemical and physiological properties of this bacterium. The reduction of the genome size of M. pneumoniae during its reductive evolution from ancestral bacteria can be explained by the loss of complete anabolic (e.g. no amino acid synthesis) and metabolic pathways. Therefore, M. pneumoniae depends in nature on an obligate parasitic lifestyle which requires the provision of exogenous essential metabolites. All the major classes of cellular processes and metabolic pathways are briefly described. For a number of activities/functions present in M. pneumoniae according to experimental evidence, the corresponding genes could not be identified by similarity search. For instance we failed to identify genes/proteins involved in motility, chemotaxis and management of oxidative stress.

Sequence analysis of 56 kb from the genome of the bacterium Mycoplasma pneumoniae comprising the dnaA region, the atp operon and a cluster of ribosomal protein genes.

To sequence the entire 800 kilobase pair genome of the bacterium Mycoplasma pneumoniae, a plasmid library was established with contained the majority of the EcoR1 fragments from M.pneumoniae. The EcoR1 fragments were subcloned from an ordered cosmid library comprising the complete M.pneumoniae genome. Individual plasmid clones were sequenced in an ordered fashion mainly by primer walking. We report here the initial results from the sequence analysis of -56 kb comprising the dnaA region as a potential origin of replication, the ATPase operon and a region coding for a cluster of ribosomal protein genes. The data were compared with the corresponding genes/operons from Bacillus subtilis, Escherichia coli, Mycoplasma capricolum and Mycoplasma gallisepticum.

Sequence divergence in the ORF6 gene of Mycoplasma pneumonia.

The ORF6 gene product of Mycoplasma pneumoniae is involved in a yet-unknown manner in the adhesion of the bacterium to its host cell. Part of the ORF6 gene is a repetitive DNA sequence (RepMP5), about 1,900 bp long. Seven additional similar copies of RepMP5 are dispersed on the genome. In the independently isolated strains M. pneumoniae M129 and FH, the RepMP5 copies residing in the ORF6 gene are not identical. Two conserved regions, ranging from nucleotides 1 to 799 and from nucleotide 1795 to the end of the gene, border a variable region, ranging from nucleotides 800 to 1794. This variable region differs in DNA sequence and by 201 bp. Analysis of RepMP5 copies outside the ORF6 gene showed that both M. pneumoniae M129 and M. pneumoniae FH carry a RepMP5 copy on a 6-kbp EcoRI fragment which has the same DNA sequence as the variable region of RepMP5 in the M. pneumoniae FH ORF6 gene. According to these data, a switch from the M. pneumoniae M129 ORF6 gene to the M. pneumoniae FH ORF6 gene could take place by gene conversion.

Degradation of compounds containing carbon atoms by photooxidation in the presence of water.

It was shown that all compounds containing carbon atoms, tested in this study, were degraded by the coincident influence of light, oxygen (air), and water. By this photooxidation, saturated as well as unsaturated aliphatic and aromatic compounds, monomeric, polymeric and high polymeric compounds were degraded and the carbon atoms of these compounds as well as carbon itself in form of amorphous carbon or diamond, were converted to carbon dioxide, if dispersed in water or together with water as very small solid, liquid, or gaseous particles. As such conditions occur extensively in nature, it was concluded that this degradation of organic substances is of great importance in nature, in addition to microbial and enzymatic degradation. This photooxidation further represents a possibility for pollution control to prevent damage to environment by degrading organic substances which are either slightly soluble or practically insoluble in water, as well as compounds which will not be destroyed by microbial and enzymatic degradation.