Biomedical subjects
Publications and source records attributed to J Mrázek.
In the present paper decays of genetically connected nuclei are considered and equations for their independent cross sections are derived. An optimisation parameter is proposed for an experiment where spectra of the residual nuclei are studied by the induced activity method. This parameter depends on irradiation time, delay time and spectrum measurement time.
Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the SYNECHOCYSTIS: PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In SYNECHOCYSTIS:, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO(2) fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.
Competitive PCR method was developed for the detection and enumeration of Butyrivibrio fibrisolvens. Sequences of 16S rDNA were obtained from our isolates (serving as a source of data for primer design) and were distinguished into nine different groups of butyrivibria. Specific primers for two distinct groups were designed with the help of BioEdit program. These primers were tested with DNA of 20 strains of ruminal B. fibrisolvens isolates. Annealing temperature 58 degrees C showed a little specificity but a better selectivity was found after raising it up to 65 degrees C. A group 1 competitive fragment of 16S rDNA of different length was constructed using restriction cutting with MspI followed by ligation; the size of the resulting fragment was cut down by 75 bp. The fragment worked in the presence of the original 16S rDNA fragment of B. fibrisolvens JK 609.
Predicted highly expressed (PHX) genes are characterized for the completely sequenced genomes of the four fast-growing bacteria Escherichia coli, Haemophilus influenzae, Vibrio cholerae, and Bacillus subtilis. Our approach to ascertaining gene expression levels relates to codon usage differences among certain gene classes: the collection of all genes (average gene), the ensemble of ribosomal protein genes, major translation/transcription processing factors, and genes for polypeptides of chaperone/degradation complexes. A gene is predicted highly expressed (PHX) if its codon frequencies are close to those of the ribosomal proteins, major translation/transcription processing factor, and chaperone/degradation standards but strongly deviant from the average gene codon frequencies. PHX genes identified by their codon usage frequencies among prokaryotic genomes commonly include those for ribosomal proteins, major transcription/translation processing factors (several occurring in multiple copies), and major chaperone/degradation proteins. Also PHX genes generally include those encoding enzymes of essential energy metabolism pathways of glycolysis, pyruvate oxidation, and respiration (aerobic and anaerobic), genes of fatty acid biosynthesis, and the principal genes of amino acid and nucleotide biosyntheses. Gene classes generally not PHX include most repair protein genes, virtually all vitamin biosynthesis genes, genes of two-component sensor systems, most regulatory genes, and most genes expressed in stationary phase or during starvation. Members of the set of PHX aminoacyl-tRNA synthetase genes contrast sharply between genomes. There are also subtle differences among the PHX energy metabolism genes between E. coli and B. subtilis, particularly with respect to genes of the tricarboxylic acid cycle. The good agreement of PHX genes of E. coli and B. subtilis with high protein abundances, as assessed by two-dimensional gel determination, is verified. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are also examined. The spatial distribution of PHX genes within each genome reveals clusters and significantly long regions without PHX genes.
Our approach in predicting gene expression levels relates to codon usage differences among gene classes. In prokaryotic genomes, genes that deviate strongly in codon usage from the average gene but are sufficiently similar in codon usage to ribosomal protein genes, to translation and transcription processing factors, and to chaperone-degradation proteins are predicted highly expressed (PHX). By these criteria, PHX genes in most prokaryotic genomes include those encoding ribosomal proteins, translation and transcription processing factors, and chaperone proteins and genes of principal energy metabolism. In particular, for the fast-growing species Escherichia coli, Vibrio cholerae, Bacillus subtilis, and Haemophilus influenzae, major glycolysis and tricarboxylic acid cycle genes are PHX. In Synechocystis, prime genes of photosynthesis are PHX, and in methanogens, PHX genes include those essential for methanogenesis. Overall, the three protein families-ribosomal proteins, protein synthesis factors, and chaperone complexes-are needed at many stages of the life cycle, and apparently bacteria have evolved codon usage to maintain appropriate growth, stability, and plasticity. New interpretations of the capacity of Deinococcus radiodurans for resistance to high doses of ionizing radiation is based on an excess of PHX chaperone-degradation genes and detoxification genes. Expression levels of selected classes of genes, including those for flagella, electron transport, detoxification, histidine kinases, and others, are analyzed. Flagellar PHX genes are conspicuous among spirochete genomes. PHX genes are positively correlated with strong Shine-Dalgarno signal sequences. Specific regulatory proteins, e.g., two-component sensor proteins, are rarely PHX. Genes involved in pathways for the synthesis of vitamins record low predicted expression levels. Several distinctive PHX genes of the available complete prokaryotic genomes are highlighted. Relationships of PHX genes with stoichiometry, multifunctionality, and operon structures are discussed. Our methodology may be used complementary to experimental expression analysis.
Our basic observation is that each genome has a characteristic "signature" defined as the ratios between the observed dinucleotide frequencies and the frequencies expected if neighbors were chosen at random (dinucleotide relative abundances). The remarkable fact is that the signature is relatively constant throughout the genome; i.e. , the patterns and levels of dinucleotide relative abundances of every 50-kb segment of the genome are about the same. Comparison of the signatures of different genomes provides a measure of similarity which has the advantage that it looks at all the DNA of an organism and does not depend on the ability to align homologous sequences of specific genes. Genome signature comparisons show that plasmids, both specialized and broad-range, and their hosts have substantially compatible (similar) genome signatures. Mammalian mitochondrial (Mt) genomes are very similar, and animal and fungal Mt are generally moderately similar, but they diverge significantly from plant and protist Mt sets. Moreover, Mt genome signature differences between species parallel the corresponding nuclear genome signature differences, despite large differences between Mt and host nuclear signatures. In signature terms, we find that the archaea are not a coherent clade. For example, Sulfolobus and Halobacterium are extremely divergent. There is no consistent pattern of signature differences among thermophiles. More generally, grouping prokaryotes by environmental criteria (e.g., habitat propensities, osmolarity tolerance, chemical conditions) reveals no correlations in genome signature.
We provide data and analysis to support the hypothesis that the ancestor of animal mitochondria (Mt) and many primitive amitochondrial (a-Mt) eukaryotes was a fusion microbe composed of a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium. The analysis is based on several observations: (i) The genome signatures (dinucleotide relative abundance values) of Clostridium and Sulfolobus are compatible (sufficiently similar) and each has significantly more similarity in genome signatures with animal Mt sequences than do all other available prokaryotes. That stable fusions may require compatibility in genome signatures is suggested by the compatibility of plasmids and hosts. (ii) The expanded energy metabolism of the fusion organism was strongly selective for cementing such a fusion. (iii) The molecular apparatus of endospore formation in Clostridium serves as raw material for the development of the nucleus and cytoplasm of the eukaryotic cell.
We present new methods for calculating codon bias of a group of genes or an individual gene relative to a standard gene class. This method is suitable for identifying alien (e.g., horizontally transferred) and highly expressed genes. In yeast and several bacterial genomes, highly expressed genes typically include ribosomal protein genes, elongation factors, chaperonins (heat shock proteins), and a subset of genes involved in glycolysis generally essential in exponential growth. Highly expressed genes of the Synechocystis genome feature several photosystem II genes, and highly expressed genes in several methanogens (Methanococcus jannaschii, M. thermoautotrophicum) are essential for methanogenesis. Alien genes mostly consist of ORFs of unknown function, transposases, prophage genes, and restriction/modification enzymes. Notably, nuclear ribosomal proteins of yeast are highly expressed, whereas mitochondrial ribosomal protein genes appear to be alien genes. Alien genes often occur in clusters, suggesting in these cases that transfer events entail several genes.
Several bacterial genomes exhibit preference for G over C on the DNA leading strand extending from the origin of replication to the ter-region in the genomes of Escherichia coli, Mycoplasma genitalium, Bacillus subtilis, and marginally in Haemophilus influenzae, Mycoplasma pneumoniae, and Helicobacter pylori. Strand compositional asymmetry is not observed in the cyanobacterium Synechocystis sp. genome nor in the archaeal genomes of Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Archaeoglobus fulgidus. A strong strand compositional asymmetry is observed in beta-type but not alpha- or gamma-type human herpesviruses featuring G > C downstream of oriL and C > G upstream of oriL. Dinucleotide relative abundances (i.e., dinucleotide representations normalized by the component nucleotide frequencies) are consonant with respect to the leading and lagging strands. Strand compositional asymmetry may reflect on differences in replication synthesis of the leading versus lagging strand, on differences between template and coding strand associated with transcription-coupled repair mechanisms, on differences in gene density between the two strands, on differences in residue and codon biases in relation to gene function, expression level, or operon organization, or on differences in single or context-dependent base mutational rates. The absence of strand asymmetry in the archaeal genomes may reflect the presence of multiple origins of replication.
A new measure for assessing codon bias of one group of genes with respect to a second group of genes is introduced. In this formulation, codon bias correlations for Escherichia coli genes are evaluated for level of expression, for contrasts along genes, for genes in different 200 kb (or longer) contigs around the genome, for effects of gene size, for variation over different function classes, for codon bias in relation to possible lateral transfer and for dicodon bias for some gene classes. Among the function classes, codon biases of ribosomal proteins are the most deviant from the codon frequencies of the average E. coli gene. Other classes of 'highly expressed genes' (e.g. amino acyl tRNA synthetases, chaperonins, modification genes essential to translation activities) show less extreme codon biases. Consistently for genes with experimentally determined expression rates in the exponential growth phase, those of highest molar abundances are more deviant from the average gene codon frequencies and are more similar in codon frequencies to the average ribosomal protein gene. Independent of gene size, the codon biases in the 5' third of genes deviate by more than a factor of two from those in the middle and 3' thirds. In this context, there appear to be conflicting selection pressures imposed by the constraints of ribosomal binding, or more generally the early phase of protein synthesis (about the first 50 codons) may be more biased than the complete nascent polypeptide. In partitioning the E. coli genome into 10 equal lengths, pronounced differences in codon site 3 G+C frequencies accumulate. Genes near to oriC have 5% greater codon site 3 G+C frequencies than do genes from the ter region. This difference also is observed between small (100-300 codons) and large (>800 codons) genes. This result contrasts with that for eukaryotic genomes (including human, Caenorhabditis elegans and yeast) where long genes tend to have site 3 more AT rich than short genes. Many of the above results are special for E. coli genes and do not apply to genes of most bacterial genomes. A gene is defined as alien (possibly horizontally transferred) if its codon bias relative to the average gene exceeds a high threshold and the codon bias relative to ribosomal proteins is also appropriately high. These are identified, including four clusters (operons). The bulk of these genes have no known function.
We review concepts and methods for comparative analysis of complete genomes including assessments of genomic compositional contrasts based on dinucleotide and tetranucleotide relative abundance values, identifications of rare and frequent oligonucleotides, evaluations and interpretations of codon biases in several large prokaryotic genomes, and characterizations of compositional asymmetry between the two DNA strands in certain bacterial genomes. The discussion also covers means for identifying alien (e.g. laterally transferred) genes and detecting potential specialization islands in bacterial genomes.
Development of the internet network at the 2nd Faculty of Medicine, Charles University from one of the first nodes of Prague Academic Network up to a complicated infrastructure with many client computers is accompanied with a corresponding development of information services available in this network. For today's users, infrastructure of the network is hidden behind the services whose technical details are not significant. The paper deals with history, current state and possible perspectives of information services (both basic as e-mail, WWW and more advanced like specialized database server, proxy, etc.) available to users at the 2nd Faculty of Medicine--taking into account the development of information technologies, networking infrastructure and the possibilities and limitations of co-operation between the faculty and its teaching hospital.
Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences. Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is rho*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement). Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size >/=50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms. On this basis we refer to the collection (rho*XY) as the genome signature. This paper identifies rho*XY extremes and compares genome signature differences for a diverse range of eukaryotic species. Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases. It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures. The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.
We compare and contrast genome-wide compositional biases and distributions of short oligonucleotides across 15 diverse prokaryotes that have substantial genomic sequence collections. These include seven complete genomes (Escherichia coli, Haemophilus influenzae, Mycoplasma genitalium, Mycoplasma pneumoniae, Synechocystis sp. strain PCC6803, Methanococcus jannaschii, and Pyrobaculum aerophilum). A key observation concerns the constancy of the dinucleotide relative abundance profiles over multiple 50-kb disjoint contigs within the same genome. (The profile is rhoXY* = fXY*/fX*fY* for all XY, where fX* denotes the frequency of the nucleotide X and fY* denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complementary sequence.) On the basis of this constancy, we refer to the collection [rhoXY*] as the genome signature. We establish that the differences between [rhoXY*] vectors of 50-kb sample contigs of different genomes virtually always exceed the differences between those of the same genomes. Various di- and tetranucleotide biases are identified. In particular, we find that the dinucleotide CpG=CG is underrepresented in many thermophiles (e.g., M. jannaschii, Sulfolobus sp., and M. thermoautotrophicum) but overrepresented in halobacteria. TA is broadly underrepresented in prokaryotes and eukaryotes, but normal counts appear in Sulfolobus and P. aerophilum sequences. More than for any other bacterial genome, palindromic tetranucleotides are underrepresented in H. influenzae. The M. jannaschii sequence is unprecedented in its extreme underrepresentation of CTAG tetranucleotides and in the anomalous distribution of CTAG sites around the genome. Comparative analysis of numbers of long tetranucleotide microsatellites distinguishes H. influenzae. Dinucleotide relative abundance differences between bacterial sequences are compared. For example, in these assessments of differences, the cyanobacteria Synechocystis, Synechococcus, and Anabaena do not form a coherent group and are as far from each other as general gram-negative sequences are from general gram-positive sequences. The difference of M. jannaschii from low-G+C gram-positive proteobacteria is one-half of the difference from gram-negative proteobacteria. Interpretations and hypotheses center on the role of the genome signature in highlighting similarities and dissimilarities across different classes of prokaryotic species, possible mechanisms underlying the genome signature, the form and level of genome compositional flux, the use of the genome signature as a chronometer of molecular phylogeny, and implications with respect to the three putative eubacterial, archaeal, and eukaryote domains of life and to the origin and early evolution of eukaryotes.
The complete Haemophilus influenzae genome (1.83 Mb, Rd strain) provides opportunities for characterizing global genomic inhomogeneities and for detecting important sequence signals. Along these lines, new methods for identifying frequent words (oligonucleotides and/or peptides) and their distributions are applied to the H.influenzae genome with some comparisons and contrasts made with frequent words of other bacterial genomes. Three major classes of frequent oligonucleotides stand out: (i) oligos related to the familiar uptake signal sequences (USSs), AAGTGCGGT (USS+) and its inverted complement (USS-), (ii) multiple tetranucleotide iterations and (iii) intergenic dyad sequences (ISDs) found as AAGCCCACCCTAC and its dyad form. The USS+ and USS- occur in almost equal counts, are remarkably evenly spaced around the genome, and appear predominantly in the same reading frame of protein coding domains (USS+ translated to Ser-Ala-Val, USS- translated to Thr-Ala-Leu). These observations suggest that USSs contribute to global genomic functions, for example, in replication and/or repair processes, or as membrane attachment sites, or as sequences helping to pack DNA. The long tetranucleotide iterations, virtually unique to H.influenzae (i.e., unknown in other prokaryotes), through polymerase slippage during replication and/or homologous recombination may produce subpopulations expressing alternative proteins. The 13 bp frequent IDS words, invariably intergenic, occur mostly in clusters and provide potential for complex secondary structures suggesting that these sequences may be important signals for regulating the activity of their flanking genes. The frequent oligopeptides of H.influenzae are principally of two kinds--those induced by oligonucleotide frequent words (USSs, tetranucleotide iterations), and those associated with ATP or GTP binding sites that are generally composed of three motifs: the A-box which contributes to delineating the binding pocket; the B-box which functions in hydrolysis; and the C-box whose function is unknown. The A-box occurs fairly universally in prokaryotes and eukaryotes. The B- and C-motifs appear to be specialized to various functional groups (e.g., transport, recombination, chaperone activity). Other putative motifs correspond to homologs of Escherichia coli motifs, for example, are associated with proteins of transcriptional processing, aminoacyl-tRNA synthetases and proteins functioning in electron transfer.
Synonymous codon usage is based and the bias seems to be different in different organisms. Factors with proposed roles in causing codon bias include degree and timing of gene expression, codon-anticodon interactions, transcription and translation rate and fidelity, codon context, and global and local G + C content. We offer a new perspective and new methods for elucidating codon choices applied especially to the human genome. We present data supporting the thesis that codon choices for human genes are largely a consequence of two factors: (1) amino acid constraints, (2) maintaining DNA structures dependent on base-step conformational tendencies consistent with the organism's genome signature that is determined by genome-wide processes of DNA modification, replication and repair. The related codon signature defined as the dinucleotide relative abundances at the distinct codon positions (1,2), (2,3), and (3,4) (4 = 1 of the next codon) accommodates both the global genome signature and amino acid constraints. In human genes, codon positions (2,3) and (3,4) containing the silent site have similar codon signatures reflecting DNA symmetry. Strong CG and TA dinucleotide underrepresentation is observed at all codon positions as well as in non-coding regions. Estimates of synonymous codon usage based on codon signatures are in excellent agreement with the actual codon usage in human and general vertebrate genes. These properties are largely independent of the isochore compartment (G + C content), gene size, and transcriptional and translational constraints. We hypothesize that major influences on codon usage in human genes result from residue preferences and diresidue associations in proteins coupled to biases on the DNA level, related to replication and repair processes and/or DNA structural requirements.
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We propose a novel, transparent and very simple algorithm to analyze middle-range correlations in genomic nucleotide sequences. Analysis by this algorithm of the EMBL Nucleotide Sequence Database demonstrates that all four nucleotides cluster in the genomic nucleotide sequences of eukaryotes on the scale of several hundred base pairs. In prokaryotes, the clustering is weak but still evident. The non-dominant three bases are deficient in the clusters, while A is the most deficient nucleotide in the clusters of C, and vice versa, and G is the most deficient nucleotide in the clusters of T, and vice versa. The algorithm also detects CG islands, extending over 1 kb, in vertebrate sequences. In plants, the CG islands are shown to be much smaller, if they exist at all. A clustering tendency is also exhibited by the TA doublet. Other doublets do not cluster. We observe no strong correlation between nucleotides separated in genomes by > 1 kb.