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PubMed2003

IMGT/PhyloGene: an on-line tool for comparative analysis of immunoglobulin and T cell receptor genes.

IMGT/PhyloGene is an on-line software package for comparative analysis of immunoglobulin (IG) and T cell receptor (TR) variable genes of all vertebrate species, newly implemented in IMGT, the international ImMunoGeneTics information system ((R)). IMGT/PhyloGene is strongly associated with the IMGT gene and allele nomenclature and with the IMGT unique numbering for V-REGION, which directly creates standardized alignments from IMGT reference sequences. IMGT/PhyloGene is the first tool to use the IMGT expertized and standardized data for automated comparative analyses, and the first on-line software package for phylogenetic reconstruction to be integrated to a sequence database. Starting from a standardized alignment of selected sequences, IMGT/PhyloGene computes a matrix of evolutionary distances, builds a tree using the Neighbor-Joining (NJ) algorithm, and outputs various graphical tree representations. The resulting IMGT/PhyloGene tree is then used as a support for studying the evolution of particular subregions, such as the CDR-IMGT (Complementarity Determining Regions) or the V-RS (Variable gene Recombination Signals). IMGT/PhyloGene is freely available at http://imgt.cines.fr.

Databases, Nucleic Acid↗
PubMed2002

Heterogeneous gene distribution reflects human genome complexity as detected at the cytogenetic level.

Human chromosomes are heterogeneous in structure and function and this is the base for the specific banding patterns produced by various chromosome staining techniques. The Human Genome Data Base as of January 2001 was searched for genes mapped to individual chromosomal bands to study the different aspects of human genome organization as they appear at the cytogenetic level of resolution. Genes are unequally distributed both on human chromosomes and chromosome bands. Among more than 5000 genes mapped at individual bands, 81% were located in G-negative bands, which correspond to half of the human genome. The main practical value of having a dense genetic physical map of genes is to accelerate the discovery by positional candidate cloning of human disease genes. Gene content agrees with H3 family isochores and with GC-rich flavors. Interestingly, two G-positive bands, namely 2p chromosome bandings is 12 and 7q35, contain a high number of genes. The finding of heterogeneity in gene content suggests that chromosome banding is not only due to differences in gene content.

Chromosome Banding↗
PubMed2001

Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species.

We have undertaken the first comparative pilot gene discovery analysis of approximately 25,000 random genomic and expressed sequence tags (ESTs) from three species of Plasmodium, the infectious agent that causes malaria. A total of 5482 genome survey sequences (GSSs) and 5582 ESTs were generated from mung bean nuclease (MBN) and cDNA libraries, respectively, of the ANKA line of the rodent malaria parasite Plasmodium berghei, and 10,874 GSSs generated from MBN libraries of the Salvador I and Belem lines of Plasmodium vivax, the most geographically wide-spread human malaria pathogen. These tags, together with 2438 Plasmodium falciparum sequences present in GenBank, were used to perform first-pass assembly and transcript reconstruction, and non-redundant consensus sequence datasets created. The datasets were compared against public protein databases and more than 1000 putative new Plasmodium proteins identified based on sequence similarity. Homologs of previously characterized Plasmodium genes were also identified, increasing the number of P. vivax and P. berghei sequences in public databases at least 10-fold. Comparative studies with other species of Apicomplexa identified interesting homologs of possible therapeutic or diagnostic value. A gene prediction program, Phat, was used to predict probable open reading frames for proteins in all three datasets. Predicted and non-redundant BLAST-matched proteins were submitted to InterPro, an integrated database of protein domains, signatures and families, for functional classification. Thus a partial predicted proteome was created for each species. This first comparative analysis of Plasmodium protein coding sequences represents a valuable resource for further studies on the biology of this important pathogen.

Animals↗
PubMed2003

Distinguishing plant and fungal sequences in ESTs from infected plant tissues.

Expressed sequence tags (ESTs) from fungal-infected plant tissues are composed of a mixture of plant and fungal sequences. Using freely available software and tools, a novel procedure is described for distinguishing plant and fungal DNA sequences. Although the GenBank non-redundant (NR) database is larger and therefore one would presume that BLASTX analysis of it would be more accurate, superior resolution of 700 randomly selected fungal ESTs was found with Standalone TBLASTX analyses with a local matching database composed of a plant and a fungal genome. Standalone TBLASTX analyses of 3,983 ESTs from nine different fungal-infected plant EST libraries also proved to be superior in identifying the origin of sequences as either plant or fungal compared to GenBank BLASTX analysis. Standalone TBLASTX with a matching database comprised of a single plant and a single fungal genome appears to be a faster and more accurate method than BLASTX searches of the GenBank non-redundant database to distinguish fungal and plant sequences in mixed EST collections.

DNA, Fungal↗
PubMed2002

Regulatory context is a crucial part of gene function.

Information about the time and place of gene transcription, which until recently was only possible by extensive experimental analysis, can now be predicted through in silico analysis. Using the human RANTES/CCL5 promoter, we show that organizational features of promoters derived from promoter sequences contain information about the spatial and temporal 'functional context' of expression.

Animals↗
PubMed2002

Antisense transcripts in the human genome.

By a systematic search of vertebrate mRNA sequences, we have identified a surprisingly large number of human antisense transcripts. These data suggest that regulation of gene expression by antisense and double-stranded RNAs could be a common phenomenon in mammalian cells.

Algorithms↗
PubMed2004

[Multilocus sequence typing: the molecular marker of the Internet era].

Global or longer term epidemiology track the spread of clonal lineages, associated with hipervirulence or resistance or multi-resistance to antimicrobial agents. Therefore, the application of a molecular typing system for this purpose should produce data easily shared by different and geographically distant laboratories, as well as distinguish those clonal lineages even with low levels of variability accumulated in the genome.A marker based on the DNA sequence will produce objective results easily organized in data bases accessible by Internet. The application of a similar strategy that was used in the analysis of isoenzymes, by sequencing variable fragments of selected housekeeping genes, will allow obtaining a general view of the distribution of the clonal lineages and tracking their spread.

Bacterial Infections↗
PubMed2002

Reexamining the polyadenylation signal: were we wrong about AAUAAA?

Polyadenylation is the process by which most eukaryotic mRNAs form their 3' ends. It was long held that polyadenylation required the sequence AAUAAA and that 90% of mRNAs had AAUAAA within 30 nucleotides of the site of poly(A) addition. More recent studies, aided by computer analysis of sequences made available in GenBank and expressed sequence tag (EST) databases, have suggested that the actual incidence of AAUAAA is much lower, perhaps as low as 50-60%. Reproductive biologists have long recognized that a large number of mRNAs in male germ cells of mammals lack AAUAAA but are otherwise normally polyadenylated. Recent research in our laboratory has uncovered a new form of an essential polyadenylation protein, tauCstF-64, that is most highly expressed in male germ cells, and to a smaller extent in the brain, and which we propose plays a significant role in AAUAAA-independent mRNA polyadenylation in germ cells.

Animals↗
PubMed2002

Using expressed sequence tag databases to identify ovarian genes of interest.

GenBank contains 4879 expressed sequence tags (EST) derived from four non-normalized human ovarian cDNA libraries. Of these EST, 2646 are contributors to UniGene clusters and have UniGene numbers. The EST map to 1206 distinct UniGenes. A gene expression profile was established for the human ovary by identifying the abundance of each UniGene cluster and its corresponding annotation. The most highly expressed transcripts were for proteins associated with protein synthesis (ribosomal proteins, elongation factors, thymosins, etc.). However, there are also transcripts for genes of unknown function that are ovary-specific. This ovarian gene expression profile provides useful data for the design of DNA microarrays targeted at ovarian function and highlights novel sequences that warrant further investigation.

Databases, Nucleic Acid↗
PubMed2001

Construction and characterization of a cDNA library from 4-week-old human embryo.

Development and differentiation studies of early human embryos have been severely impeded by general difficulties in obtaining suitable samples. In order to isolate and identify new genes expressed during early human development, we constructed and characterized a PCR-based cDNA library using a 4-week-old chorion-free human embryo. The constructed cDNA library contained 6.3 x 10(6) directional recombinants, and its insert size ranged from 0.4 to 1.8 kb. The cDNA library proportionally represents the mRNA population, containing beta-actin, tPA and LINE1 repetitive sequences at the expected frequencies as in other conventionally constructed and PCR-based cDNA libraries. PCR analyses of the library for specific genes have also revealed the presence of cDNAs for developmentally important genes such as CD59, MCP, Quox-1 and ZNF268. Among the 70 randomly selected cDNA clones, 53% encoded previously known genes, 26% matched with anonymous sequences, and 17% showed no sequence similarity and were designated as human early embryo-specific ESTs. These results demonstrate the sequence complexity and relatively low redundancy of our cDNA library. Furthermore, approximately 40% of those randomly analyzed clones contained full-length encoding regions. To our knowledge, this is the first description of the PCR-based cDNA library from a 4-week-old chorion-free human embryo, and the presence of novel sequences within this library makes it a valuable and unique resource for studying gene expression and regulatory mechanisms that underlie the early process of human embryogenesis.

Cloning, Molecular↗
PubMed2002

Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of Staphylococcus aureus 8325.

The genomes of the three temperate bacteriophages contained in the chromosome of Staphylococcus aureus 8325 have been extracted from the sequence database and analyzed. phi 11, phi 12 and phi 13 are members of the same lytic group but different serogroups and consequently co-habitate the same host cell. Their genomes are approximately 42 kb to 45 kb and contain about 90 ORFs of at least 50 codons. Of these, about 50 have similarities to known genes or to genes of other staphylococcal phages. Each of the phages clusters within a homology group that share large regions of sequence identity while intergroup homology is comparatively low. The arrangement of genes on the chromosomes of the three phages is similar and consistent with current modular theory of phage gene organization. The replicated genomes appear to be packaged by different mechanisms. Phage phi 11 and phi 12 have been found to contain sequences consistent with pac-site phages while phi 13 has sequences consistent with cos-site phages. The attBsite for phi 11 is located in an intergenic region of the S. aureus chromosome while phi 12 and phi 13 integrate into specific genes. The phi 12 att-site is within an unknown gene, but the phi 13 att-site is within the beta-toxin gene. In contrast to the other two phages, phi 13 also introduces the staphylokinase gene (sak) and a second gene related to expression of fib.

Base Composition↗
PubMed2002

A computerized database-scan to identify c-MYC targets.

The c-MYC oncogene plays a pivotal role in the malignant transformation of various types of human cancer. It is also a key regulator of cellular proliferation, embryonic differentiation and apoptosis. c-MYC encodes a transcription factor that activates target genes in a sequence specific manner through heterodimerization with the ubiquitously expressed factor MAX. Identifying c-MYC target genes is therefore crucial for elucidating the molecular pathways that are downstream of MYC. Most of the c-MYC targets isolated to date as well as targets of other transcription factors have been identified by differential expression or the candidate gene approach. In this paper, we outline a computer-based scan that allows us to create a pool of putative target genes for a transcription factor. The scan is based on a set of criteria including sequence specificity of the c-MYC transcription factor, sequence location and evolutionary conservation of these regulatory elements. Using this procedure, we have identified 12 putative targets for c-MYC. Expression analyses, DNA binding assays and chimeric promoter-reporter experiments suggest that two genes, NM23-H2 and N-RAS, may indeed be direct targets for c-MYC activation. This type of computer-based scan may have a general use to identify targets for other transcription factors.

3T3 Cells↗
PubMed2002

Identification of cadmium-sensitive genes in the Antarctic fish Chionodraco hamatus by messenger RNA differential display.

To investigate the ability of cadmium to affect gene transcription in fish, the messenger RNA (mRNA) differential display technique was used to analyze gene expression in the Antarctic icefish Chionodraco hamatus exposed to sublethal doses of cadmium salt. Seven DNA complementary to RNA (cDNA) bands whose steady-state levels of expression significantly changed in response to cadmium exposure were identified. The results obtained show that two groups of genes are affected by cadmium in icefish liver. The first group comprises genes that are up-regulated by the metal: in particular, a gene encoding the heat-shock protein HSP70 and another encoding a protein homologous to GP49 of Sparus aurata egg envelope. The other group comprises genes down-regulated by cadmium. These are the transferrin gene and a gene encoding a protein presenting homology to mouse T2K, a kinase having a role in the prevention of apoptosis. Three cDNAs had no homology to known gene sequences, thus suggesting that may either encode not yet identified proteins, or correspond to untranslated regions of mRNA molecules.

Animals↗
PubMed2002

On biased distribution of introns in various eukaryotes.

We conducted comprehensive analyses on intron positions in the Mus musculus genome by comparing genomic sequences in the GenBank database and cDNA sequences in the mouse cDNA library recently developed by Riken Genomic Sciences Center. Our results confirm that introns have a tendency to be located toward the 5' end of the gene. The same type of analysis was conducted in the coding region of seven eukaryotes (Saccharomyces cerevisiae, Plasmodium falciparum, Caenorhabditis elegans, Drosophila melanogaster, M. musculus, Homo sapiens, Arabidopsis thaliana). Introns in genes with a single intron have a locational bias toward the 5' end in all species except A. thaliana. We also measured the distance from the start codon to the position of the intron, and found that single introns prefer the location immediately after the start codon in S. cerevisiae and P. falciparum. We discuss three possible explanations for these findings: (1) they are the consequence of intron loss by reverse-transcriptase; (2) they are necessary to accommodate the function; and (3) they are concerned with the mechanism of pre-mRNA splicing.

Animals↗
PubMed2002

A simple and species-independent coding measure.

We present a coding measure which is based on the statistical properties of the stop codons, and that is able to estimate accurately the variation of coding content along an anonymous sequence. As the stop codons play the same role in all the genomes (with very few exceptions) the measure turns out to be species-independent. We show results both for prokaryotic and for eukaryotic genomes, indicating, first, the accuracy of the measure, and, second, that better prediction is achieved if the measure is applied on homogeneous, isochore-like sequences than if it is applied following the standard moving window approach. Finally, we discuss on some of the possible applications of the measure.

Animals↗