Exploring an alignment free approach for protein classification and structural class prediction.

Alignment free methods based on Chaos Game Representation (CGR), also known as sequence signature approaches, have proven of great interest for DNA sequence analysis. Indeed, they have been successfully applied for sequence comparison, phylogeny, detection of horizontal transfers or extraction of representative motifs in regulation sequences. Transposing such methods to proteins poses several fundamental questions related to representation space dimensionality. Several studies have tackled these points, but none has, so far, brought the application of CGRs to proteins to their fully expected potential. Yet, several studies have shown that techniques based on n-peptide frequencies can be relevant for proteins. Here, we investigate the effectiveness of a strategy based on the CGR approach using a fixed reverse encoding of amino acids into nucleic sequences. We first explore its relevance to protein classification into functional families. We then attempt to apply it to the prediction of protein structural classes. Our results suggest that the reverse encoding approach could be relevant in both cases. We show that it is able to classify functional families of proteins by extracting signatures close to the ProSite patterns. Applied to structural classification, the approach reaches scores of correct classification close to 84%, i.e. close to the scores of related methods in the field. Various optimizations of the approach are still possible, which open the door for future applications.

Genomic signature: characterization and classification of species assessed by chaos game representation of sequences.

We explored DNA structures of genomes by means of a new tool derived from the "chaotic dynamical systems" theory (the so-called chaos game representation [CGR]), which allows the depiction of frequencies of oligonucleotides in the form of images. Using CGR, we observe that subsequences of a genome exhibit the main characteristics of the whole genome, attesting to the validity of the genomic signature concept. Base concentrations, stretches (runs of complementary bases or purines/pyrimidines), and patches (over- or underexpressed words of various lengths) are the main factors explaining the variability observed among sequences. The distance between images may be considered a measure of phylogenetic proximity. Eukaryotes and prokaryotes can be identified merely on the basis of their DNA structures.

Relationships between colony forming efficiency and parameters of intrinsic radiosensitivity.

PURPOSE: In an attempt to determine whether radiosensitivity is correlated with colony forming efficiency (CFE), a large amount of data have been analysed from the literature. MATERIALS AND METHODS: The survival curves of 446 human cell lines irradiated in exponentially growing phase in vitro are included in this study. Technical factors such as culture type and the use of feeder cells were considered cofactors in addition to the genetic and histological origin of the cells. Intrinsic radiosensitivity is expressed in terms of the parameters of the linear quadratic model and the single-hit multitarget model. RESULTS: It is shown that low CFE is characteristic of cells plated in agar and cells from primary biopsies. Cells plated in the presence of feeder cells have, in general, higher CFE than cells plated without feeder cells. A positive correlation is observed between intrinsic radiosensitivity and CFE: the higher the CFE, the more resistant the cell line. This relationship is particularly obvious when radiosensitivity is expressed in terms of alpha, S2 or D, parameters which essentially characterize the initial part of the survival curve. The correlation is also found within histological or genetic groups of cell lines. However, for a given cell line, there is no relationship between CFE and radiosensitivity among different experiments. Cells irradiated in the presence of feeder cells are less subject to this behaviour. CONCLUSIONS: CFE as well as radiosensitivity are intrinsic properties of a cell line. Experimental conditions determine the quality of the correlation between radiosensitivity and CFE.

DNase-hypersensitive sites in yeast artificial chromosomes containing human DNA.

We have mapped the DNase I-hypersensitive sites (HSs) in Yeast Artificial Chromosomes (YACs) containing segments of human chromosomal DNA. One of the five HSs found in a YAC carrying the beta-globin gene cluster has been localised in the region, termed HS2, that is DNase I hypersensitive in most human cells. We have also identified a class of HSs in YACs containing DNA from the q11.2 band of human chromosome 21, which are located close to, or within, segments of the chromosome that are sensitive to restriction enzymes recognizing CGCG tetranucleotides.

A review of human cell radiosensitivity in vitro.

The survival curves of 694 human cell lines irradiated in exponentially growing phase in vitro were collected from the literature. Among them, 271 were derived from tumors, 423 were nontransformed fibroblasts and other normal cell strains from healthy people or people with some genetic disorders. Seventy-six different cell types are identified, and a specific radiosensitivity could be associated with each, using D and surviving fraction at 2 Gy. Technical factors such as culture medium, feeder cells, and scoring method were found to affect intrinsic radiosensitivity. In particular, the cell type is not a discriminating factor when cells are studied in agar. Results obtained with cells irradiated in agar must be used cautiously, depending on how the cells were prepared for the experiments. The use of feeder cells narrows the range of radiosensitivity of human cells. For cells irradiated as monolayer, it was possible to build a scale of radiosensitivity according to cell type, ranging, in terms of D from 0.6 Gy for the most sensitive cell lines to more than 4 Gy for the most resistant. Considering that, in most cases, we could estimate the variation of radiosensitivity within each cell type, our classification among cell types can be used by researchers to place their results in the context of the literature.

New approaches to the mapping of chromosomal domains.

Although it is generally accepted that the chromosome is divided into elementary subunits, the structural and functional domains, the organisation of these structures at the molecular level is not well understood. In particular, the domain boundaries are not easily identifiable. Several possible candidates such as MARs/SARs, insulators, LCRs, palindromic sequences, or easily melting sequences have been found in the regions having properties one would except for boundaries. None of these elements, however, has been found in all of the constructs functioning as boundaries in tests in vivo. Recent work suggests that the common denominator might be the presence og GC-rich oligonucleotide stretches and the formation of the chromatin hypersensitive sites. A model is discussed in which "unusual" structures, in particular the four-stranded DNA sequence elements containing unpaired bases, play the role of domain boundaries.

Loop-size spacings between CGCG clusters in long segments of human DNA.

The CGCG tetranucleotides are clustered inside the CpG islands in the genomes of vertebrates. In order to study the distribution of the islands in the human chromosome we have mapped the loci sensitive to the CGCG specific restriction nuclease, in a 1.5 Mb long DNA segment cloned as Yeast Artificial Chromosome (YAC). The sites most sensitive to Bsh 1236 I nuclease show chromosomal loop-size spacing. This result, as well as the result of nucleotide sequence analysis of long genomic segments, suggests that the CGCG are organised in clusters (not always undermethylated) which are coincident with GC peaks on the sine wave-like curve representing DNA composition along the mammalian chromosome.

Correlation of GC content with replication timing and repair mechanisms in weakly expressed E.coli genes.

Regional variations of DNA GC content are observed in species as different as S.cerevisiae and humans. In vertebrates and yeast they are correlated with replication timing; late replicating chromosomal regions are more AT-rich than early replicating regions. We show here that gene composition in E.coli also has long range variations which are similarly correlated with replication timing. We suggest that the enrichment in AT base pairs in late replicating DNA reflects differences in DNA repair modes. These sequences, which are in single copy for a greater part of the cell cycle than origin-linked genes, have less opportunity to engage in repair via homologous recombination and therefore may resort more often to translesion synthesis involving the misincorporation of adenine opposite modified nucleotides.

A benchmark of cell survival models using survival curves for human cells after completion of repair of potentially lethal damage.

Six models of radiation action (the linear-quadratic model, the multitarget model with initial slope, the repair-misrepair model, the lethal-potentially lethal model, the cybernetic model, the saturable repair model) were tested for their goodness of fit to survival curves for human cells. Fifty-three survival curves for human cells irradiated in plateau phase and after completion of repair of potentially lethal damage (PLD) provided the experimental basis for the tests. Three criteria were considered. The capacity to describe the survival data was estimated, using the error left unexplained by the model. A validation of models was achieved by consideration of the mean residual squared errors. The ability of the parameters to characterize survival curves was investigated, studying their variation within and among curves. The models were not equivalent, whatever the test. The saturable repair model and the multitarget with initial slope model gave the most accurate description of survival data. The linear-quadratic model had the most reliable parameters, so that comparisons of the cell survival curves could be made advantageously. The cybernetic model and the lethal-potentially lethal model were found inappropriate for the analysis of survival curves for human cells after completion of PLD repair.

The Rad3 protein from Saccharomyces cerevisiae: a DNA and DNA:RNA helicase with putative RNA helicase activity.

The Rad3 protein from Saccharomyces cerevisiae is a DNA helicase which participates in the repair of ultraviolet-irradiated DNA and is inhibited in the presence of DNA containing thymine dimers. This protein is also involved in mitotic recombination and spontaneous mutagenesis and is essential for cell viability in the absence of DNA damage. Furthermore, the Rad3 protein also exhibits a DNA:RNA helicase activity in which there is a significant preference for a partial DNA:RNA hybrid rather than a partial duplex DNA substrate, which suggests that this protein might be involved in DNA repair within transcriptionally active genes. Finally, the Rad3 protein contains the DEAH motif and shares high amino acid sequence similarity with the DEAD family of RNA helicase proteins, suggesting that it might also possess an RNA helicase activity.

Counterselection of GATC sequences in enterobacteriophages by the components of the methyl-directed mismatch repair system.

Weak to severe deficit of GATC sequences in the DNA of enterobacteriophages appears to be correlated with their undermethylation during growth in dam+ (GATC ade-methylase) bacteria. This observation is corroborated by the sequence analysis showing no evidence for site-specific mutagenicity of 6meAde. The MutH protein of the methyl-directed mismatch repair system recognizes and cleaves the undermethylated GATC sequences in the course of mismatch repair. To enquire whether the MutH function of the methyl-directed mismatch repair system participates in counterselection of GATC sequences in enterobacteriophages, we have studied the yield of bacteriophage phi X174 containing either 0, 1, or 2 GATC sequences, in wild type, dam, and mut (H, L, S, U) Escherichia coli. Following transfection with unmethylated DNA containing two GATC sequences, a net decrease in the yield of infective particles was observed in all bacterial mutH+ dam- strains, whereas no detectable decrease was observed in bacteria infected by DNA without GATC sequence. This effect of the MutH function is maximum in wild type and mutL and mutS bacteria whereas the effect is not significant in mutU bacteria, suggesting an interaction of the helicase II with the MutH protein. However, in dam+ bacteria, the presence of GATC sequences leads to an increased yield of infective particles. The effect of GATC sequence and its Dam methylation system on phage yield in mutH- bacteria reveals that methylated GATC sequences are advantageous to the phage.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between radiosensitivity and repair of potentially lethal damage in human tumor cell lines with implications for radioresponsiveness.

The relationship between intrinsic radiosensitivity and repair capacity was studied for 22 human tumor cell lines in vitro. The experimental material was taken from 19 published papers. Parameters from three radiobiological models were used to assess this relationship: the one-hit multitarget model (D0 and n), the linear-quadratic model (alpha and beta), and the mean inactivation dose (D). Data were obtained for cells in three stages: exponentially growing cells (exp), plateau-phase cells plated immediately after irradiation (ip), and plateau-phase cells plated after completion of PLD repair (dp). No significant difference was found between radiosensitivity of exp and ip cells. There was no correlation between repair capacity and intrinsic radiosensitivity assessed with plateau-phase cells plated immediately after irradiation. The correlation studies between intrinsic radiosensitivity or repair capacity and clinical responsiveness were achieved by assigning cell lines to one of three groups of decreasing in vivo radioresponsiveness: highly, medium, and poorly responsive. There was a significant correlation between radiosensitivity and radioresponsiveness, but no correlation between repair capacity and radioresponsiveness. The average repair capacity was about 0.6 Gy, in terms of D. Three parameters, the mean inactivation dose of exponentially growing cells, of plateau-phase cells plated immediately after irradiation, and of plateau-phase cells plated after completion of PLD repair, could be used equally to assess the relationship between in vitro data and radioresponsiveness. The present results are compared to those obtained in a similar study on a group of 48 nontransformed fibroblast cell strains.

The relationship between potentially lethal damage repair and intrinsic radiosensitivity of human cells.

The intrinsic radiosensitivity of exponentially growing cells (exp) was compared to that of immediately plated plateau phase cells (ip) using published data on 60 human cell lines (27 fibroblast lines and 33 tumour cell lines). The values for alpha, D and S2 are not significantly different for the two groups; beta is significantly higher in ip cells. This produces a smaller alpha/beta ratio in ip cells than in exp cells. The influence of potentially lethal damage (PLD) repair was assessed by comparing the radiosensitivities of ip cells and plateau phase cells with delayed plating (dp). The published data for 81 human cell lines (48 fibroblasts and 33 tumour lines) were used. PLD repair was found to lead to a decrease in alpha and an increase in D and S2, whereas neither beta nor the alpha/beta ratio changed significantly. The relationship between PLD repair and intrinsic radiosensitivity was assessed by repair capacity and the repair ratio. The fitted relationship is a bell-shaped curve with a maximum at 2.2 Gy for repair capacity. The fitted curve predicts that repair capacity is zero at a D up of 0.28 Gy and at 4 Gy. Thus, PLD repair is a reasonable reflection of intrinsic radiosensitivity up to 2.2 Gy. Above 2.2 Gy, the relationship is reversed: the greater the radioresistance, the lower the PLD repair.

Correlation between PLD repair capacity and the survival curve of human fibroblasts in exponential growth phase: analysis in terms of several parameters.

Published data on the in vitro radiosensitivity of 46 nontransformed fibroblasts of different genetic origins studied in plateau phase with immediate or delayed plating were used to investigate to what extent potentially lethal damage repair capacity is related to intrinsic radiosensitivity (i.e., irradiated in exponential growth phase). While most of the survival curve analysis is conducted in terms of D0, Dq, and the mean inactivation dose D, some of the data are also discussed in terms of the linear-quadratic model parameter alpha. Using D it is shown that: (i) the radiosensitivity of human fibroblasts in exponential growth phase does not significantly differ from that of plateau-phase fibroblasts with immediate plating; (ii) the radiosensitivity of plateau-phase cells with delayed plating is correlated to the radiosensitivity of cells with immediate plating: the more radioresistant the cell strain in exponential growth phase, the higher its repair capacity; (iii) the repair capacity of the cell strains is related to their genetic origin. In conclusion, we suggest that the survival curve of growing cells depends on the repair capacity of the cells.

Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts.

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the gamma-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of gamma-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.

Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts.

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.

Re-evaluation of in vitro radiosensitivity of human fibroblasts of different genetic origins.

A statistical analysis of the radiosensitivity of 204 different survival curves of nontransformed human fibroblast cell strains of different genetic origins was made using three criteria: the multi-target one-hit model (characterized by parameters n and D0), the surviving fraction for a 2 Gy dose (S2) and the mean inactivation dose (D). D is found to be the best parameter for characterization of anomalous radiosensitivity linked to a genetic disorder and for discrimination between groups of cell strains of differing radiosensitivity. Its use allows the description of a range of 'normal' radiosensitivity for control fibroblasts and the classification of the various genetic disorders as a function of their mean radiosensitivity expressed in terms of D. Nine groups of cell strains appear to exhibit radiosensitivity which differs significantly from that of the controls: seven groups are hypersensitive (ataxia-telangiectasia homozygotes and heterozygotes, Cockayne's syndrome, Gardner's syndrome, 5-oxoprolinuria homozygotes and heterozygotes, Fanconi's anaemia) and two groups are more radioresistant (fibroblasts from retinoblastoma patients and from individuals with chromosome 13 anomalies). Since the coupled parameter n and D0 failed to discriminate between the radiosensitivity of the different genetic groups, we recommend the use of D to make an intercomparison of intrinsic radiosensitivity of nontransformed human fibroblasts.

Reduced repair of potentially lethal radiation damage in glutathione synthetase-deficient human fibroblasts after X-irradiation.

Using a human fibroblast strain deficient in glutathione synthetase and a related proficient control strain, the role of glutathione (GSH) in repair of potentially lethal damage (PLD) has been investigated in determining survival by plating cells immediately or 24 h after irradiation. After oxic or hypoxic irradiation, both cell strains repair radiation-induced damage. However, under hypoxic conditions, the proficient cells repair PLD as well as under oxic conditions while the deficient cells repair less PLD after irradiation under hypoxic than under oxic conditions. Therefore, the oxygen enhancement ratio (o.e.r.) for proficient cells is similar whether the cells are plated immediately or 24 h later (2.0 and 2.13, respectively). In contrast, the o.e.r. for deficient cells is lower when the cells are plated 24 h after irradiation than when they are plated immediately thereafter (1.16 as compared to 1.55). The results indicate that GSH is involved in PLD repair and, in particular, in the repair of damage induced by radiation delivered under hypoxic conditions.