Automated procedure for candidate compound selection in GC-MS metabolomics based on prediction of Kovats retention index.

MOTIVATION: Matching both the retention index (RI) and the mass spectrum of an unknown compound against a mass spectral reference library provides strong evidence for a correct identification of that compound. Data on retention indices are, however, available for only a small fraction of the compounds in such libraries. We propose a quantitative structure-RI model that enables the ranking and filtering of putative identifications of compounds for which the predicted RI falls outside a predefined window. RESULTS: We constructed multiple linear regression and support vector regression (SVR) models using a set of descriptors obtained with a genetic algorithm as variable selection method. The SVR model is a significant improvement over previous models built for structurally diverse compounds as it covers a large range (360-4100) of RI values and gives better prediction of isomer compounds. The hit list reduction varied from 41% to 60% and depended on the size of the original hit list. Large hit lists were reduced to a greater extend compared with small hit lists. AVAILABILITY: http://appliedbioinformatics.wur.nl/GC-MS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Predicting sub-Golgi localization of type II membrane proteins.

MOTIVATION: Recent research underlines the importance of finegrained knowledge on protein localization. In particular, subcompartmental localization in the Golgi apparatus is important, for example, for the order of reactions performed in glycosylation pathways or the sorting functions of SNAREs, but is currently poorly understood. RESULTS: We assemble a dataset of type II transmembrane proteins with experimentally determined sub-Golgi localizations and use this information to develop a predictor based on the transmembrane domain of these proteins, making use of a dedicated proteinstructure based kernel in an SVM. Various applications demonstrate the power of our approach. In particular, comparison with a large set of glycan structures illustrates the applicability of our predictions on a 'glycomic' scale and demonstrates a significant correlation between sub-Golgi localization and the ordering of different steps in glycan biosynthesis. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Predicting and understanding transcription factor interactions based on sequence level determinants of combinatorial control.

MOTIVATION: Transcription factor interactions are the cornerstone of combinatorial control, which is a crucial aspect of the gene regulatory system. Understanding and predicting transcription factor interactions based on their sequence alone is difficult since they are often part of families of factors sharing high sequence identity. Given the scarcity of experimental data on interactions compared to available sequence data, however, it would be most useful to have accurate methods for the prediction of such interactions. RESULTS: We present a method consisting of a Random Forest-based feature-selection procedure that selects relevant motifs out of a set found using a correlated motif search algorithm. Prediction accuracy for several transcription factor families (bZIP, MADS, homeobox and forkhead) reaches 60-90%. In addition, we identified those parts of the sequence that are important for the interaction specificity, and show that these are in agreement with available data. We also used the predictors to perform genome-wide scans for interaction partners and recovered both known and putative new interaction partners.

Postsymbiotic plasmid acquisition and evolution of the repA1-replicon in Buchnera aphidicola.

Buchnera aphidicola is an obligate, strictly vertically transmitted, bacterial symbiont of aphids. It supplies its host with essential amino acids, nutrients required by aphids but deficient in their diet of plant phloem sap. Several lineages of Buchnera show adaptation to their nutritional role in the form of plasmid-mediated amplification of key-genes involved in the biosynthesis of tryptophan (trpEG) and leucine (leuABCD). Phylogenetic analyses of these plasmid-encoded functions have thus far suggested the absence of horizontal plasmid exchange among lineages of Buchnera. Here, we describe three new Buchnera plasmids, obtained from species of the aphid host families Lachnidae and Pemphigidae. All three plasmids belong to the repA1 family of Buchnera plasmids, which is characterized by the presence of a repA1-replicon responsible for replication initiation. A comprehensive analysis of this family of plasmids unexpectedly revealed significantly incongruent phylogenies for different plasmid and chromosomally encoded loci. We infer from these incongruencies a case of horizontal plasmid transfer in Buchnera. This process may have been mediated by secondary endosymbionts, which occasionally undergo horizontal transmission in aphids.

Structure and evolution of the leucine plasmids carried by the endosymbiont (Buchnera aphidicola) from aphids of the family Aphididae.

In all examined species of the family Aphididae, the bacterial endosymbiont Buchnera aphidicola carries a plasmid encoding the genes leuABCD (involved in leucine biosynthesis) along with repA1, repA2 and ORF1. The gene organisation of the leucine plasmids was conserved, except in Buchnera isolated from Pterocomma populeum, where ORF1 was located in a different position. An inverted repeat (LIR1) located between repA2 and leuA is found in all of the Buchnera leucine plasmids examined. The predicted secondary structure of the LIR1 transcript conforms to a long hairpin loop, suggesting an involvement in transcription termination or messenger stability. Phylogenetic reconstruction based on repA2 sequences suggests that horizontal transfer of Buchnera leucine plasmids has not occurred.

Putative evolutionary origin of plasmids carrying the genes involved in leucine biosynthesis in Buchnera aphidicola (endosymbiont of aphids).

An 8.5-kb plasmid encoding genes (leuABCD) involved in leucine biosynthesis and a small plasmid of 1.74 kb of yet unknown function were found in the intracellular symbiont, Buchnera aphidicola, of two divergent aphid species, Thelaxes suberi and Tetraneura caerulescens, respectively. The leuABCD-carrying plasmid (pBTs1) was amplified from total aphid DNA by inverse long PCR, using outwardly oriented oligonucleotide primers specific to leuA. The resulting 8.2-kb PCR fragment as well as the 1.74-kb plasmid (pBTc1) were cloned and sequenced. pBTs1 differed from a previously described B. aphidicola plasmid (pRPE) of the aphid Rhopalosiphum padi by the presence of a small heat shock gene (ibp) and in the order of the leuABCD and repA genes. Comparison of both leucine plasmids to the small plasmid pBTc1 revealed extensive similarity with respect to putative replication functions as well as in the presence of a highly conserved open reading frame that was found to be homologous to Escherichia coli YqhA and Haemophilus influenzae HI0507 and which may encode an integral membrane protein. The three B. aphidicola plasmids most likely evolved from a common ancestral replicon, which in turn may be distantly related to IncFII plasmids. Phylogenetic affiliations of the B. aphidicola strains of the two aphid species were assessed by sequencing of their 16S rRNA genes. Evaluation of the distribution of the leuABCD-encoding plasmids within a phylogenetic framework suggests independent origins for pBTs1 and pRPE from an ancestral replicon resembling pBTc1. The implications for symbiotic essential amino acid biosynthesis and provisioning are discussed.

Isolation and identification of the human homolog of a new p53-binding protein, Mdmx.

We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a protein of 490 amino acids, 90% similar to mouse Mdmx. The homology between Mdmx and Mdm2 is most prominent in the p53-binding domain and the putative metal-binding domains. The Mdmx protein, which, based on SDS-PAGE, has a MW of 80 kDa, can bind p53 in vitro. The human MDMX gene is transcribed in all tissues tested, with high levels in thymus. By fluorescence in situ hybridization analysis we mapped the mouse mdmx gene to chromosome 1 (region F-G) and the human MDMX gene to chromosome 1q32.

Wilms' tumor 1-KTS isoforms induce p53-independent apoptosis that can be partially rescued by expression of the epidermal growth factor receptor or the insulin receptor.

The Wilms' tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides that differ in the presence or absence of a stretch of 17 amino acids just NH2 terminal of the four zinc fingers and a stretch of three amino acids (+/-KTS) between zinc fingers 3 and 4. In this study, cDNA constructs encoding the four human Wilms' tumor 1 splice variants were transiently transfected into the p53-negative Hep3B and the p53-positive HepG2 hepatoma cell lines. Morphological assessment of the WT1-expressing cells showed that the WT1(-KTS) splice variants induced apoptosis in both cell lines, whereas the WT1(+KTS) isoforms did not. The induction of apoptosis by the WT1(-KTS) isoforms appears to be p53 independent in the hepatoma cell lines. Furthermore, it was found that the WT1(-KTS)-induced apoptosis could not be suppressed by coexpression of either the Mr 21,000 E1B, the Bcl-2, or the BAG-1 protein. Coexpression of either the epidermal growth factor receptor or the insulin receptor, however, partially rescued the cells from apoptosis.

MDMX: a novel p53-binding protein with some functional properties of MDM2.

Here we report the isolation of a cDNA encoding a new p53-associating protein. This new protein has been called MDMX on the basis of its structural similarity to MDM2, which is especially notable in the p53-binding domain. In addition, the putative metal binding domains in the C-terminal part of MDM2 are completely conserved in MDMX. The middle part of the MDMX and MDM2 proteins shows a low degree of conservation. We can show by co-immunoprecipitation that the MDMX protein interacts specifically with p53 in vivo. This interaction probably occurs with the N-terminal part of p53, because the activity of the transcription activation domain of p53 was inhibited by co-transfection of MDMX. Northern blotting showed that MDMX, like MDM2, is expressed in all tissues tested, and that several mRNAs for MDMX can be detected. Interestingly, the level of MDMX mRNA is unchanged after UV irradiation, in contrast to MDM2 transcription. This observation suggests that MDMX may be a differently regulated modifier of p53 activity in comparison with MDM2. Our study indicates that at least one additional member of the MDM protein family exists which can modulate p53 function.

Wilms' tumor 1 splice variants have opposite effects on the tumorigenicity of adenovirus-transformed baby-rat kidney cells.

The Wilms' Tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides which differ in the presence or absence of two stretches of amino acids: one of 17 residues (17aa) just N-terminal of the four zinc-fingers and of three residues (K-T-S) between zinc finger 3 and 4. In this study, four human cDNA constructs encoding the Wilms' tumor 1 splice variants were stably transfected into adenovirus-transformed baby rat kidney (Ad-BRK) cells. The in vivo produced WT1 proteins that lacked the KTS residues were found to bind efficiently to both the Egr-1 consensus sequence and the recently described WTE DNA sequence, as determined by electrophoretic mobility shift assays. Our studies show distinct effects of the different WT1 isoforms. Expression of the WT1 (-/+) protein, lacking the 17aa insert, strongly suppressed the tumorigenic phenotype of the Ad-BRK cells. Intriguingly, expression of the WT1 (-/-) protein, lacking both inserts, increased the tumor growth rate. In contrast to the growth in vivo, the growth rate of the transfectants in tissue culture is not influenced by any of the WT1 isoforms. However, the suppression of tumorigenicity appears to be correlated with a reduced ability of the cells to grow in serum-free medium.

Human chromosome 11 suppresses the tumorigenicity of adenovirus transformed baby rat kidney cells: involvement of the Wilms' tumor 1 gene.

Human chromosome 11 was introduced into adenovirus-transformed baby rat kidney (BRK) cells by microcell-mediated chromosome transfer. The resulting microcell hybrids (MCHs) showed a reduced ability to form tumors upon s.c. injection into athymic mice. Further analysis, with the use of defined deletion chromosomes of 11p, indicated that the presence of region 11p13-p12 is necessary for the suppression of tumorigenicity. In contrast, the presence of region 11p15-14.1 appeared to increase the rate of tumor growth. Expression studies on the human Wilms' tumor I (WTI) and the insulin-like growth factor II (IGF-II) genes, which lie in regions 11p13 and 11p15, respectively, suggested the involvement of both genes in determining the degree of suppression of tumorigenicity. Finally, stable expression of a murine WTI protein in the adenovirus-transformed cells resulted in almost complete suppression of tumorigenicity, establishing the WTI protein as a tumor suppressor in this cell system.

Molecular evolution of noncoding regions of the chloroplast genome in the Crassulaceae and related species.

Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA (cpDNA) in order to study sequence-length variation in the Crassulaceae and in related species. Several length mutations were observed that are of diagnostic value for evolutionary relationships in the Crassulaceae and the Saxifragaceae. Length variation and sequence divergence in the intergenic spacer between the trnL (UAA) 3' exon and the trnF (GAA) gene among 15 species were studied in detail by nucleotide-sequence analysis. A total of 50 insertion/deletion mutations were observed, accounting for a spacer-length variation in the range of 228-360 bp. Eighteen short direct repeat motifs (4-11 bp) and two inverted repeat motifs (7-11 bp) were found to be associated with length variation. Phylogenetic analysis of the sequence data indicated a pattern of relationships that was largely consistent with a previous analysis of cpDNA restriction-site variation. Evaluation of the level of homoplasy in insertion/deletion mutations within a phylogenetic framework revealed that only 1 out of 34 length mutations longer than 2 bp must have had multiple origins. The feasibility of the noncoding chloroplast DNA regions for molecular evolutionary studies is discussed.

Characterization of the mouse homolog of the XPBC/ERCC-3 gene implicated in xeroderma pigmentosum and Cockayne's syndrome.

The human XPBC/ERCC-3 DNA repair gene specifically corrects the repair defect of xeroderma pigmentosum (XP) complementation group B and rodent repair mutant cell lines of group 3. The gene encodes a presumed DNA- and chromatin-binding helicase involved in early steps of the excision repair pathway. To study the evolution of this gene, its expression in different tissues and stages of development and to permit the generation of a mouse model of XP by targeted gene replacement in mouse embryonal stem cells, we have isolated the mouse XPBC/ERCC-3 homolog. Sequence comparison of the predicted protein revealed a 96% amino acid identity with the human gene product. Notably, all postulated functional domains were strictly conserved. The mouse XPBC/ERCC-3 promoter is--like its human counterpart--devoid of classical promoter elements such as TATA and CAAT boxes and contains several conserved segments with unknown function. One of these conserved regions, consisting in part of a polypyrimidine track, is also present in the ERCC-1 promoter. The mouse XPBC/ERCC-3 gene is expressed constitutively at low levels in all tissues examined except for testis, where its expression is significantly enhanced.

Structure and expression of the human XPBC/ERCC-3 gene involved in DNA repair disorders xeroderma pigmentosum and Cockayne's syndrome.

The human XPBC/ERCC-3 was cloned by virtue of its ability to correct the excision repair defect of UV-sensitive rodent mutants of complementation group 3. The gene appeared to be in addition implicated in the human, cancer prone repair disorder xeroderma pigmentosum group B, which is also associated with Cockayne's syndrome. Here we present the genomic architecture of the gene and its expression. The XPBC/ERCC-3 gene consists of at least 14 exons spread over approximately 45 kb. Notably, the donor splice site of the third exon contains a GC instead of the canonical GT dinucleotide. The promoter region, first exon and intron comprise a CpG island with several putative GC boxes. The promoter was confined to a region of 260 bp upstream of the presumed cap site and acts bidirectionally. Like the promoter of another excision repair gene, ERCC-1, it lacks classical promoter elements such as CAAT and TATA boxes, but it shares with ERCC-1 a hitherto unknown 12 nucleotide sequence element, preceding a polypyrimidine track. Despite the presence of (AU)-rich elements in the 3'-untranslated region, which are thought to be associated with short mRNA half-life actinomycin-D experiments indicate that the mRNA is very stable (t 1/2 greater than 3h). Southern blot analysis revealed the presence of XPBC/ERCC-3 cross-hybridizing fragments elsewhere in the genome, which may belong to a related gene.

A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome.

The human gene ERCC-3 specifically corrects the defect in an early step of the DNA excision repair pathway of UV-sensitive rodent mutants of complementation group 3. The predicted 782 amino acid ERCC-3 protein harbors putative nucleotide, chromatin, and helix-turn-helix DNA binding domains and seven consecutive motifs conserved between two superfamilies of DNA and RNA helicases, strongly suggesting that it is a DNA repair helicase. ERCC-3-deficient rodent mutants phenotypically resemble the human repair syndrome xeroderma pigmentosum (XP). ERCC-3 specifically corrects the excision defect in one of the eight XP complementation groups, XP-B. The sole XP-B patient presents an exceptional conjunction of two rare repair disorders: XP and Cockayne's syndrome. This patient's DNA contains a C----A transversion in the splice acceptor sequence of the last intron of the only ERCC-3 allele that is detectably expressed, leading to a 4 bp insertion in the mRNA and an inactivating frameshift in the C-terminus of the protein. Because XP is associated with predisposition to skin cancer, ERCC-3 can be considered a tumor-preventing gene.

Molecular cloning and biological characterization of the human excision repair gene ERCC-3.

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion (L. H. Thompson, A. V. Carrano, K. Sato, E. P. Salazar, B. F. White, S. A. Stewart, J. L. Minkler, and M. J. Siciliano, Somat. Cell. Mol. Genet. 13:539-551, 1987).