PINT: Pathways INtegration Tool.

New pathway databases generally display pathways by retrieving information from a database dynamically. Some of them even provide their pathways in SBML or other exchangeable formats. Integrating these models is a challenging work, because these models were not built in the same way. Pathways integration Tool (PINT) may integrate the standard SBML files. Since these files may be obtained from different sources, any inconsistency in component names can be revised by using an annotation editor upon uploading a pathway model. This integration function greatly simplifies the building of a complex model from small models. To get new users started, about 190 curated public models of human pathways were collected by PINT. Relevant models can be selected and sent to the workbench by using a user-friendly query interface, which also accepts a gene list derived from high-throughput experiments. The models on the workbench, from either a public or a private source, can be integrated and painted. The painting function is useful for highlighting important genes or even their expression level on a merged pathway diagram, so that the biological significance can be revealed. This tool is freely available at http://csb2.ym.edu.tw/pint/.

SCA db: spinocerebellar ataxia candidate gene database.

UNLABELLED: The positional candidate gene approach accelerates the discovery of genes involved in disease. However, the properties of such disease genes are very diverse and the sample size of known disease genes is too small and does not warrant success by the use of a machine-learning approach. A user-defined scoring system may thus help to determine the priority of candidate genes. Spinocerebellar ataxia (SCA) is a good model to test this approach because most SCA subtypes are caused by an expansion of short tandem repeats (STRs). The SCA db is a candidate gene database for SCA, which collected 3185 genes for 17 types of SCA. Those SCA subtypes that have known disease genes can be used as positive controls to optimize the parameters. The users may browse the candidate genes of a given SCA subtype by using the default parameters. The known disease genes were found to be the top three candidates using the default parameters. Alternatively, the users may score the candidate genes by changing the weight or the scores on the basis of their own working hypothesis. AVAILABILITY: This database is available at http://ymbc.ym.edu.tw/sca/

PALS db: Putative Alternative Splicing database.

PALS db is a collection of Putative Alternative Splicing information from 19 936 human UniGene clusters and 16 615 mouse UniGene clusters. Alternative splicing (AS) sites were predicted by using the longest messenger RNA (mRNA) sequence in each UniGene cluster as the reference sequence. This sequence was aligned with related sequences in UniGene and dbEST to reveal the AS. This information was presented with six features: (i) literature aliases were used to improve the result of a gene name search; (ii) the quality of a prediction can be easily judged from the color-coded similarity and the scaled length of an alignment; (iii) we have clustered those EST sequences that support the same AS site together to enhance the users' confidence on a prediction; (iv) the users can also set up the alignment criteria interactively to recover false negatives; (v) tissue distribution can be displayed by placing the mouse cursor over an alignment; (vi) gene features will be analyzed at foreign sites by submitting the selected mRNA or its encoded protein as a query. Using these features, the users cannot only discover putative AS sites in silico, but also make new observations by combining AS information with tissue distributions or with gene features. PALS db is available at http://palsdb.ym.edu.tw/.

Structure-based mutational analysis of the hepatitis C virus NS3 helicase.

The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3.

Evidence of transmission of hepatitis B virus to spouses from sequence analysis of the viral genome.

Heterosexual contact is one of the common routes of transmission for hepatitis B virus (HBV) among adults in Taiwan, but only a few studies have provided direct evidence at the level of the HBV genome of infected couples with acute non-fulminant hepatitis to document a common source. By cloning and sequencing polymerase chain reaction-amplified HBV-DNA, we analysed the sequences of the conserved region of the surface gene (nucleotide (n.t.) 305-513, representing 6.5% of the viral genome) of HBV in five HBV-infected index patients, their spouses and four randomly selected HBV carriers as controls. Risk factors associated with acute HBV infection in index cases were sexual contact with their spouses within 3 months before the onset of hepatitis. For all five couples, the HBV-infected index patient and the spouse shared a 100% sequence homology of HBV-DNA. In contrast, there was significantly more variation (mean heterogeneity 6.1%, range 1-13.9%) in the amplified region between the five couples and between each couple and the controls (P<0.001). This study demonstrated that sequence analyses can correlate well with epidemiological findings and confirm the value of the molecular approach for linked infections of HBV through heterosexual contact between spouses. Susceptible adults should receive vaccination.

Phosphorylation can modulate the association of different sets of RNA binding proteins with the Vg1 localization signal RNA.

As assayed by both in vivo and in vitro UV-crosslinking techniques, four RNA binding proteins, with apparent molecular weight of 56, 54, 42, and 40 kilodaltons, associated specifically with the Xenopus Vg1 mRNA localization signal (LS) RNA. The 56 and 54 kD proteins were assigned as the masking proteins described previously, on the basis of their thermal stability and the effect of phosphorylation on RNA binding activity. The 42 and 40 kD proteins associated with the LS RNA at a lower extract concentration than the masking proteins did in vitro. Dephosphorylation will eliminate the RNA binding activities of all four proteins. However, either raising the extract concentration or phosphorylating the extract by the catalytic domain of protein kinase A had opposite effects on the crosslinking efficiencies of these two sets of RNA binding proteins. Phosphorylation might regulate this protein exchange process in vitro.

Repetitive elements in the third intron of murine apolipoprotein A-I gene.

Genomic DNAs containing the gene encoding apolipoprotein A-I (apoA-I) from four strains of mice and three strains of rats have been sequenced. Some peculiar repetitive sequences were found in the third intron of apoA-I of the murine species. The striking features include regular tandem repeats of C(A)4 and C(A)6 in mice and long A-tracts in rats. Not completely identical but very similar motifs were found in mice or rats belonging to the same species while repetitive elements from different species show some variation from their species-specific consensus sequences. These repetitive motifs are very similar to the sequences flanking human Alu and rodent B1 repetitive elements, although no evidence for the existence of Alu or B1 was found near the peculiar repetitive DNA sequences in apoA-I gene.

mRNA export correlates with activation of transcription in human subgroup C adenovirus-infected cells.

To investigate the mechanisms by which viral mRNA species are distinguished from their cellular counterparts for export to the cytoplasm during the late phase of subgroup C adenovirus infection, we have examined the metabolism of several cellular and viral mRNAs in human cells productively infected by adenovirus type 5 (Ad5). Several cellular mRNAs that were refractory to, or could escape from, adenovirus-induced inhibition of export of mRNA from the nucleus have been identified. This group includes Hsp70 mRNAs synthesized upon heat shock of Ad5-infected 293 or HeLa cells during the late phase of infection. However, successful export in Ad5-infected cells is not a specific response to heat shock, for beta-tubulin and interferon-inducible mRNAs were also refractory to virus-induced export inhibition. The export of these cellular mRNAs, like that of viral late mRNAs, required the E1B 55-kDa protein. Export to the cytoplasm during the late phase of Ad5 infection of several cellular mRNAs, including members of the Hsp70 family whose export was inhibited under some, but not other, conditions, indicates that viral mRNA species cannot be selectively exported by virtue of specific sequence or structural features. Cellular and viral late mRNAs that can be exported from the nucleus to the cytoplasm were expressed from genes whose transcription was induced or activated during the late phase of Ad5 infection. Consistent with the possibility that successful export is governed by transcriptional activation in the late phase of adenovirus infection, newly synthesized viral early E1A mRNA was subject to export inhibition during the late phase of infection.

Rapid high-performance liquid chromatography of nucleic acids with polystyrene-based micropellicular anion exchangers.

Nucleic acids were separated by ion-exchange chromatography on 30 x 4.6 and 100 x 4.6 mm columns packed with a micropellicular anion exchanger made of 3-microns rigid polystyrene-based non-porous microspheres with a covalently bound hydrophilic layer and DEAE functional groups at the surface. The stationary phase particles showed negligible swelling in methanol according to permeability measurements with water and methanol. Nucleic acids and their fragments including synthetic single-stranded oligonucleotides, linear, nicked and supercoiled DNAs as well as DNA restriction fragments were separated in less than 5 min, a time scale that is much smaller than that of conventional high-performance liquid chromatographic analysis for such samples. When only buffer and sodium chloride were used in the eluent for the separation of double-stranded DNA restriction fragments pGEM-3Z/Taq I, electrophoretic analysis of the effluent revealed the presence of smaller fragments in the bands of the larger ones. Upon addition of ethylenediaminetetraacetic (EDTA) salt to the eluent, however, such contamination by shorter fragments was no longer observed. In the absence of EDTA, magnesium chloride in the eluent at a concentration of 1 mM precluded the separation of the restriction fragments under otherwise identical chromatographic conditions.

Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA.

The interactions of the major core protein of adenovirus type 2 (Ad2) protein VII, and its precursor, protein pre-VII, with viral DNA, were studied using UV light induced crosslinking of 32P-labelled oligonucleotides to the proteins. Proteolytic fragments of these two proteins that contain DNA-binding domains were identified by virtue of their covalently attached, alkali-resistant 32P-radioactivity. The overall efficiency of crosslinking of protein pre-VII to DNA, in H2ts1 virions assembled at 39 degrees C, was comparable to that of the crosslinking of protein VII to DNA in Ad2 virions. However, a protease V8 fragment comprising the N-terminal half of protein pre-VII crosslinked to DNA at least ten times more efficiently than the corresponding N-terminal fragment of protein VII, which is truncated by the removal of 23 amino acids from the N-terminus of protein pre-VII during virion maturation.

Effect of adenovirus infection on expression of human histone genes.

The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.