Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic ...investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.
Molecular
genetic aberrations and the related phenotypes were investigated in 191
papillary thyroid carcinomas (PTCs) from patients exposed at
young age to radioiodine released from the Chernobyl ...reactor. A high
prevalence of RET gene rearrangements (62.3%) with a
significant predominance of ELE1/RET (PTC3) over
H4/RET (PTC1) rearrangements was found in PTCs of the
first post-Chernobyl decade. NTRK1 rearrangements were
rare (3.3%). In 3.3%, we observed novel types of RET
rearrangements: GOLGA5/RET (PTC5),
HTIF/RET (PTC6), RFG7/RET (PTC7), and an
as yet undefined RFGX/RET . RET
rearrangements, preferentially ELE1/RET , are related to
rapid tumor development. At longer intervals after exposure to ionizing
radiation, the prevalence of RET rearrangements declines
with a shift from ELE1/RET to H4/RET ,
most significantly in female patients. The prevalence of specific types
of rearrangements is independent of age at irradiation. A significantly
higher prevalence of ELE1/RET was observed in the most
heavily contaminated Oblasts, Gomel and Brest, suggesting a
preferential formation of this type of rearrangement after high thyroid
doses. RET rearrangement is related to aggressive
growth: Rearrangement-positive PTCs were in a more advanced pT category
and more frequently in the pN 1 category at presentation
than rearrangement-negative PTCs. ELE1/RET is related to
the solid variant of PTC, H4/RET more frequently to
typical papillary structures. The genotype/phenotype evaluation of
post-Chernobyl PTCs reveals a characteristic spectrum of gene
rearrangements that lead to typical phenotypes with important
biological and clinical implications.
Proteome analysis led to the identification and characterization of tumor‐associated protein variants by two‐dimensional electrophoresis and mass spectrometry. We focused on comparing the influence ...of genotoxic nitroso compounds N‐methyl‐N‐nitrosourea, diethylnitrosamine and N‐nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor‐inducing agents on the protein pattern of rat hepatomas. We found several tumor‐associated variants that represent members of the aldo‐keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor‐associated protein, rat aldose reductase‐like protein‐1 (rARLP‐1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP‐1 were detected in nitroso compound‐induced rat hepatomas, while rat aldo‐keto reductase protein‐c (Rak‐c), a novel tumor‐associated variant (65% sequence identity with 3α‐hydroxysteroid dehydrogenase) was discovered in N‐methyl‐N‐nitrosourea‐induced hepatomas only. 3α‐Hydroxysteroid dehydrogenase and γ4‐3‐ketosteroid‐5β‐reductase, both liver‐specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3α‐hydroxysteroid dehydrogenase (3α‐HSD) and γ4‐3‐ketosteroid‐5β‐reductase (5β‐Red) might be replaced in hepatomas by tumor‐associated proteins that are often present in the embryonal state, like the rARLPs or the Rak‐c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo‐keto reductase superfamily have not been found in Nafenopin‐induced hepatomas. The pattern of tumor‐associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.
A novel type of RET rearrangement, PTC5, was detected in papillary thyroid carcinomas of two patients exposed to radioactive fallout after Chernobyl. Reverse transcription-PCR and rapid amplification ...of 5'-cDNA ends revealed a fusion of the ret tyrosine kinase (TK) domain with a sequence identical to that described previously as ret-II. Ret-II is a transfection artifact in NIH3T3 cells and has not yet been detected in any human tumor. Overlapping sequences found in the expressed sequence tag databases enabled us to sequence the COOH terminus of the ret-fused gene 5 (RFG5). The combined data made it possible to assemble a full-length rfg5 protein sequence. Computer-assisted analysis of this sequence reveals four putative coiled-coil structures, possibly involved in dimerization, but no membrane-binding sequences. Northern blots show a ubiquitous RFG5 expression in various normal tissues, including the thyroid gland. In addition to the RFG5/RET, we also detected the reciprocal RET/RFG5 transcript in both tumor samples, suggesting that the rearrangement is based on a balanced reciprocal translocation. In agreement with other rearranged TKs, it is concluded that the transforming action of the new fusion protein rfg5/ret in thyroid tumors may be due to an activation of the ret TK by constitutive expression and dimerization potential of the 5'-fused rfg5 protein. Ret immunohistochemistry indicates that the fusion protein is expressed in all cells of PTC5 tumors, suggesting that RFG5/RET rearrangement is an early event in thyroid carcinogenesis.
As part of ongoing studies on the RET rearrangement frequency in children with papillary thyroid carcinoma (PTC) after their exposure to radioactive iodine after the Chernobyl reactor accident, new ...methods for the detection of novel types of RET rearrangements are being developed. In this study, an improved reverse transcription-PCR strategy is used successfully to identify a new type of RET rearrangement. This rearrangement is designated PTC8 and the involved RET-fused gene (RFG) as RFG8. The identification of two reciprocal transcripts coding for the RFG8/RET and RET/RFG8 fusions suggests that the PTC8 rearrangement results from a balanced chromosomal translocation. With a view to clarify its role in tumor induction, we compared the fusion products with those of previously described RET rearrangements. We therefore sequenced and characterized the RFG8 cDNA, which showed no significant similarity to any functional protein described as yet. RFG8 is located on chromosome 18q21-22 and is expressed ubiquitously. Bioinformatic analysis predicts with a high probability that the corresponding rfg8 protein is located in the cytoplasm and is involved putatively in intracellular transport processes. Furthermore, we identified coiled-coil structures upstream of the breakpoint with one of the coiled-coils showing dimerization capability. Thus the rfg8/ret fusion protein exhibits structures for oncogenic activation that are similar to those observed in previously described RET fusions.
23S rRNA genes of 17 strains representing the α, ß, γ, δ and µ subclasses of the
Proteobacteria were completely sequenced. The sequences were aligned to about 120 published as well as unpublished ...complete or almost complete primary structures of 23S rRNAs from other members of the domain
Bacteria representing all known phyla. Primary and higher order structure analyses revealed remarkable differences of predicted 23S rRNA structures from members of the different subclasses. A phylogenetic tree reflecting the relationships among proteobacteria was reconstructed based on 23S rRNA sequence comparison. The topology of the tree is similar to that of a tree based on an equivalent 16S rRNA sequence data set.
Bacteria with specific metabolic capabilities are required for the degradation of industrial wastewater. In reactors with suspended biomass these organisms may easily be washed out. Reactors with ...immobilized biomass appear to be better suited to retain those organisms in the system. In order to monitor immobilization efficiency of such a Biof film reactor, the composition of the biof film grown in the reactor has to be examined. In this study a Membrane Biofilm Reactor (MBR) was inoculated with Alcaligenes eutrophus JMP 134 to yield 2,4-dichlorophenoxyacetic acid (2,4-D) degrading biofilms. In a MBR the biofilm is supplied with oxygen through a membrane from the gas compartment and with substrate from the bulk liquid. In situ hybridization of cross sections of the biofilm with 16S rRNA-targeted oligonucleotide probes revealed the spatial distribution of bacterial cells in the biofilm. An oligonucleotide probe specific for the 2,4-degrading strain A. eutrophus JMP 134 was developed based on comparative sequence analysis. A. eutrophus JMP 134 cells were hardly found directly attached to the membrane, but clusters of them colonized e. g. the testaceous amoebae in the layer close to the membrane. The biofilm itself consisted of three different layers. The bottom layer was characterized by clusters of testaceous amoebae covered with bacterial cells from all groups examined. The base biofilm layer contained organisms of the beta-subclass and - in most cases - fungi. The surface layer exhibited again a higher diversity of bacterial cells and some testaceous amoebae. The overall composition of the biofilm was characterized by a dominance of organisms of the beta-subclass of Proteobacteria. Cells of A. eutrophus JMP 134 were found in all three layers, but in different morphological shapes.
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