MOSAIC: segmenting multiple aligned DNA sequences.

UNLABELLED: MOSAIC is a set of tools for the segmentation of multiple aligned DNA sequences into homogeneous zones. The segmentation is based on the distribution of mutational events along the alignment. As an example, the analysis of one repeated sequence belonging to the subtelomeric regions of the yeast genome is presented. AVAILABILITY: Free access from ftp://ftp.biomath.jussieu.fr/pub/papers/MOSAIC

A strategy for finding regions of similarity in complete genome sequences.

MOTIVATION: Complete genomic sequences will become available in the future. New methods to deal with very large sequences (sizes beyond 100 kb) efficiently are required. One of the main aims of such work is to increase our understanding of genome organization and evolution. This requires studies of the locations of regions of similarity. RESULTS: We present here a new tool, ASSIRC ('Accelerated Search for SImilarity Regions in Chromosomes'), for finding regions of similarity in genomic sequences. The method involves three steps: (i) identification of short exact chains of fixed size, called 'seeds', common to both sequences, using hashing functions; (ii) extension of these seeds into putative regions of similarity by a 'random walk' procedure; (iii) final selection of regions of similarity by assessing alignments of the putative sequences. We used simulations to estimate the proportion of regions of similarity not detected for particular region sizes, base identity proportions and seed sizes. This approach can be tailored to the user's specifications. We looked for regions of similarity between two yeast chromosomes (V and IX). The efficiency of the approach was compared to those of conventional programs BLAST and FASTA, by assessing CPU time required and the regions of similarity found for the same data set. AVAILABILITY: Source programs are freely available at the following address: ftp://ftp.biologie.ens. fr/pub/molbio/assirc.tar.gz CONTACT: vincens@biologie.ens.fr, hazout@urbb.jussieu.fr

Computational method to predict mitochondrially imported proteins and their targeting sequences.

Most of the proteins that are used in mitochondria are imported through the double membrane of the organelle. The information that guides the protein to mitochondria is contained in its sequence and structure, although no direct evidence can be obtained. In this article, discriminant analysis has been performed with 47 parameters and a large set of mitochondrial proteins extracted from the SwissProt database. A computational method that facilitates the analysis and objective prediction of mitochondrially imported proteins has been developed. If only the amino acid sequence is considered, 75-97% of the mitochondrial proteins studied have been predicted to be imported into mitochondria. Moreover, the existence of mitochondrial-targeting sequences is predicted in 76-94% of the analyzed mitochondrial precursor proteins. As a practical application, the number of unknown yeast open reading frames that might be mitochondrial proteins has been predicted, which revealed that many of them are clustered.

Computer analysis of two-dimensional electrophoresis gels as a tool in cell biology: study of the protein expression of human keratinocytes from normal to tumor cells.

The usefulness of computer analysis of two-dimensional electrophoresis gels has been investigated on the example of human keratinocytes transformation. For this purpose, the protein expression of various keratinocytes strains from normal to tumor cells has been analysed by two-dimensional electrophoresis. The resulting gels have been submitted to computer analysis, including various data analysis techniques allowing to select spots on the gels or to classify the gels themselves. The latter techniques appeared very useful, since they demonstrated that the major transition in words of variation of the protein expression lies at the normal cell/transformed cell transition rather than at the transformed cell/tumorigenic cell transition.

Characterization of chilling-acclimation-related proteins in soybean and identification of one as a member of the heat shock protein (HSP 70) family.

Through a 5-d exposure at 14 degrees C/8 degrees C (day/night), soybean (Glycine max [L.] Merr.) was acclimated to a lower temperature of 8 degrees C. In order to assess changes in protein synthesis related to chilling acclimation, proteins were labeled in vivo with [35S]methionine, separated by two-dimensional gel electrophoresis, and the derived autoradiograms were subjected to computer analysis. Two sets of chilling-acclimation-related proteins were characterized following exposure and labeling at 8 degrees C. One set corresponded to proteins whose synthesis was stimulated in acclimated plants in comparison with non-acclimated plants after transfer to 8 degrees C for 2 d. The other set also displayed an enhanced synthesis in the acclimated plants versus the non-acclimated plants but after 7 d of exposure at 8 degrees C. Most of these chilling-acclimation-related proteins were not increased during the acclimation period at 14 degrees C. Using microsequence analysis, one of these proteins was shown to have a high sequence homology with members of the heat-shock protein (HSP 70) family.

Striking differences in cellular catalase activity between two DNA repair-deficient diseases: xeroderma pigmentosum and trichothiodystrophy.

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are two recessively transmitted human diseases characterized by DNA repair deficiency. While XP is associated with a very high incidence of cancer on skin exposed to sunlight, TTD is not a cancer-prone disease. Therefore, unrepaired UV-induced DNA lesions do not appear to be enough to give rise to tumors. In order to understand the differences between these two syndromes, we measured catalase activity in cellular extracts, UV irradiated or not, and quantified H2O2 production following in vitro UV irradiation. We confirmed on 21 different XP diploid fibroblast lines that catalase activity was decreased on average by a factor of five as compared to controls, while XP heterozygote lines exhibited intermediary responses. All seven TTD lines we tested were deficient in UV-induced lesion repair and exhibited a high level of catalase activity. However, molecular analysis of catalase transcription showed no difference between normal, XP and TTD cell lines. This was confirmed by Western blots where the amount of catalase subunits was identical in all cell lines studied. Finally, UV irradiation induces five and three times more H2O2 production in XP lines compared with TTD or controls respectively. These striking differences between TTD and XP indicate that UV light, directly or indirectly, together with defective oxidative metabolism may increase the initiation and/or the progression steps in the XP environment compared to TTD. This may partly explain the different tumoral phenotype observed between the two diseases.

Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid.

Primary sequences of cholinesterases and related proteins have been systematically compared. The cholinesterase-like domain of these proteins, about 500 amino acids, may fulfill a catalytic and a structural function. We identified an aspartic acid residue that is conserved among esterases and lipases (Asp-397 in Torpedo acetylcholinesterase) but that had not been considered to be involved in the catalytic mechanism. Site-directed mutagenesis demonstrated that this residue is necessary for activity. Analysis of evolutionary relationships shows that the noncatalytic members of the family do not constitute a separate subgroup, suggesting that loss of catalytic activity occurred independently on several occasions, probably from bifunctional molecules. Cholinesterases may thus be involved in cell-cell interactions in addition to the hydrolysis of acetylcholine. This would explain their specific expression in well-defined territories during embryogenesis before the formation of cholinergic synapses and their presence in noncholinergic tissues.

Stage transitions in B-lymphocyte differentiation correlate with limited variations in nuclear proteins.

Total nuclear proteins extracted from cell lines representing various stages of differentiation of mouse B lymphocytes were studied by computer analysis of two-dimensional gels. Of the 1438 spots present on the gels, 55 varied significantly in intensity during differentiation. The variations occurred most often in steps correlating with those classically defined for B-cell differentiation. Seventeen spots were not detectable in at least one of the stages (qualitative variations) and could represent switching on or off of genes coding for nuclear proteins. Detailed analysis of the 55 variable spots showed that they fall into small sets characterized by similar expression profiles, which argues for a combinatorial, multistep control mechanism of gene expression. In addition, analysis of the expression of all the nuclear proteins resolved on the gels clearly differentiated B-lineage cells from myeloid cells and suggested that the most important transition in B-cell differentiation occurs between the resting B cell and plasmocyte stages.

Catalase-associated abnormalities and H2O2 increase in pre-neoplastic and neoplastic lesions of the human lower female genital tract and their near adjacent epithelia.

We report that an internal and non-UV-dependent type of neoplasia, the human cervical intraepithelial neoplasia (SIL), is also deficient in catalase activity, like the UV-induced tumors in the autosomal recessive human epithelial disease, xeroderma pigmentosum (XP). Whether or not the lesions are papillomavirus (HPV) positive in the different categories of preneoplastic and neoplastic extracts, the following parameters are affected: i), catalase activity level; ii), kinetic profile of catalase activity; iii), H2O2 increase. Mathematical treatment of these parameters (CONSTEL-Program), unambiguously distinguishes between normal and pathological cases. Such analyses make it possible to grade the pathological samples into 4 classes, depending on their deviance from normality. These classes may be correlated with the gradual steps in the process of malignant transformation defined by histological and clinical diagnosis. We found conformity between catalase activity and histological analyses in 66 biopsies, out of a total of 100 biopsies (35 patients). Moreover, 23 patients presenting decreased catalase activities in 31 biopsies showed disease progression after 3 to 6 months contrary to surgery histological data. We show that ATP synthesis in the presence of catalase and H2O2 (further aspect of catalase function), may occur in neoplastic extracts at much lower concentrations of H2O2 than in normal extracts. Thus, the catalase abnormality seems to be a good tool to study pre-neoplastic to neoplastic evolution of lesions and their adjacent tissues of the lower female genital tract; furthermore, i) it provides an earlier, more powerful means of detecting micro-SIL in progression to squamous cell carcinoma, than combined clinical and histological examinations; ii) model for investigating drugs such as in situ H2O2 scavengers or agents increasing glutathione peroxidase activity (GSH).

Specific modulation by ethanol of the protein synthesis pattern in the C2 rat hepatoma cell line.

The effect of ethanol on protein synthesis in the C2 rat hepatoma cell line was analyzed by two-dimensional gel electrophoresis after the labeling with [35S]methionine of cells that were untreated or had been treated with 180 mM ethanol. In this cell line, this concentration of ethanol is known to induce gamma-glutamyl transpeptidase, a marker of alcoholism in man (Barouki et al., Hepatology 1983; 3: 323-329). In the present work we demonstrate that ethanol, besides causing a slight decrease in overall protein synthesis (less than 25%), primarily regulates the expression of two unique proteins among 1500 labeled products that were analyzed: one of these was induced and did not correspond to gamma-glutamyl transpeptidase, and one was repressed after 20 h of ethanol treatment. We conclude that the set of hepatic proteins altered by ethanol is likely to be very limited in number, which reflects the specificity of alcohol action on protein synthesis in the C2 cell line.

Two-dimensional electrophoresis computerized processing.

This paper describes various methods suitable for implementation of two-dimensional processing software. The different steps leading to a complete processing are described, from the digitalization of the image to the processing of the resulting data. The characteristics of a convenient digitalization system are discussed. The different software devoted to spot detection is reviewed with respect to the presence or otherwise of a spot model and its characteristics. The major techniques for gel matching are compared as are designs for database structures suitable for tabulation of measurements. Finally, the need for a sophisticated system of data processing is stressed and its main requirements are described.

Changes in nuclear proteins induced by heat shock in Drosophila culture cells.

Nuclear proteins of normal and heat-shocked Drosophila cells were analysed by two-dimensional electrophoresis. The computerized processing of the gels allowed us to detect 6 proteins strongly induced by the heat treatment, but which were different from the usually described heat-shock proteins. The possible role of these proteins in genetic regulation is discussed, as is the value of this type of approach for the study of other genetic regulation phenomena.