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Predicting the effect of missense mutations on protein function: analysis with Bayesian networks.

BACKGROUND: A number of methods that use both protein structural and evolutionary information are available to predict the functional consequences of missense mutations. However, many of these methods break down if either one of the two types of data are missing. Furthermore, there is a lack of rigorous assessment of how important the different factors are to prediction. RESULTS: Here we use Bayesian networks to predict whether or not a missense mutation will affect the function of the protein. Bayesian networks provide a concise representation for inferring models from data, and are known to generalise well to new data. More importantly, they can handle the noisy, incomplete and uncertain nature of biological data. Our Bayesian network achieved comparable performance with previous machine learning methods. The predictive performance of learned model structures was no better than a naïve Bayes classifier. However, analysis of the posterior distribution of model structures allows biologically meaningful interpretation of relationships between the input variables. CONCLUSION: The ability of the Bayesian network to make predictions when only structural or evolutionary data was observed allowed us to conclude that structural information is a significantly better predictor of the functional consequences of a missense mutation than evolutionary information, for the dataset used. Analysis of the posterior distribution of model structures revealed that the top three strongest connections with the class node all involved structural nodes. With this in mind, we derived a simplified Bayesian network that used just these three structural descriptors, with comparable performance to that of an all node network.

Algorithms↗

Descriptive functional analysis of behavioral excesses shown by adults with Down syndrome and dementia.

OBJECTIVES: The study examined the hypothesis that a functional relationship exists between social environmental events and behavioral excesses in individuals with Down syndrome and dementia. DESIGN: A case-series design was employed (n = 4) using an direct observation-based descriptive functional assessment procedure. METHODS: Observations were conducted in the natural environments of four participants over periods ranging from 11 to 15.4 hours. Data were collected on non-verbal and verbal behavioral excesses, appropriate engagement and verbal interaction with others. Social environmental events observed including both staff and peer behavior. RESULTS: Analysis of co-occurrence for behavioral excesses and social environmental events indicated significant relationships for some behaviours consistent with operant reinforcement processes. Sequential analysis showed that changes in the probability of social contact occurred in the period directly preceding and following verbal behaviors. CONCLUSIONS: Results support the hypothesis that, consistent with literature for older adults with dementia in the general population, some behavioral excesses were functional in nature and not randomly occurring events. No relationship was found between appropriate engagement and staff contact. Evidence of the functional nature of target behavioral excesses indicates that behavioral interventions have potential for this client group.

Adult↗

[Functional analysis of vernalization-related gene VER17 in flower development using antisense RNA strategy in winter wheat].

To understand the function of vernalization-related gene VER17 in winter wheat (Triticum aestivum L. cv. Jingdong No.1), an antisense RNA strategy was used. The antisense VER17 with a vector pBI121 was constructed and transformed into winter wheat by using the pollen-tube-pathway method. Fourteen independent transgenic plants transformed with antisense VER17 and five control transformants transformed with pBI121 blank vector were obtained and confirmed by GUS histochemical assay and PCR-Southern blot analysis. Phenotypes of T(0) and T(1) transgenic plants showed that the plants of the antisense VER17 transgenic lines degenerated top or basal spikelets and had delayed flowering time, which suggested that the VER17 gene functions in accelerating flowering and the development of the top or basal spikelets and the stamen which were impacted by vernalization treatment.

Blotting, Southern↗

Functional analysis of the pseudoknot structure in human telomerase RNA.

Telomerase is essential for maintaining telomere length and chromosome stability in stem cells, germline cells, and cancer cells. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein component and an RNA molecule that provides the template for telomeric repeat synthesis. A pseudoknot structure in the human telomerase RNA is conserved in all vertebrates and is essential for telomerase activity. It has been proposed that this highly conserved structure functions as a dynamic structure with conformational interchange between the pseudoknot and a hairpin with intraloop base pairings. To examine the structural and functional requirements of the pseudoknot structure, we made mutations in the proposed base-paired regions in the pseudoknot. Although mutations that disrupted the pseudoknot P3 helix abolished activity as predicted, mutations that disrupted the intraloop hairpin base pairings did not reduce telomerase activity, indicating that the intraloop hairpin is not required for telomerase function. This functional study thus provides evidence against the previous proposed molecular-switch model of telomerase pseudoknot function and supports a static pseudoknot structure. The mutational analysis further suggests that telomerase RNA can function independent of the proposed intermolecular pairings between pseudoknot regions on two RNA molecules.

Base Sequence↗

Functional analysis of amino acid residues constituting the dNTP binding pocket of HIV-1 reverse transcriptase.

In order to understand the functional implication of residues constituting the dNTP-binding pocket of human immunodeficiency virus type 1 reverse transcriptase, we performed site-directed mutagenesis at positions 65, 72, 113, 115, 151, 183, 184, and 219, and the resulting mutant enzymes were examined for their biochemical properties and nucleotide selectivity on RNA and DNA templates. Mutations at positions 65, 115, 183, 184, and 219 had negligible to moderate influence on the polymerase activity, while Ala substitution at positions 72 and 151 as well as substitution with Ala or Glu at position 113 severely impaired the polymerase function of the enzyme. The K219A, Y115F, and Q151M mutants had no influence on the fidelity; Y183A, Y183F, K65A, and Q151N mutants exhibited higher fidelity on both RNA and DNA templates, while Y115A was less error-prone selectively on a DNA template. Analysis of the three-dimensional model of the enzyme-template primer-dNTP ternary complex suggests that residues Tyr-183, Lys-65, and Gln-151 may have impact on the flexibility of the dNTP-binding pocket by virtue of their multiple interactions with the dNTP, template, primer, and other neighboring residues constituting the pocket. Recruitment of the correct versus incorrect nucleotides may be a function of the flexibility of this pocket. A relatively rigid pocket would provide greater stringency, resulting in higher fidelity of DNA synthesis in contrast to a flexible pocket. Substitution of a residue having multiple interactions with a residue having reduced interaction capability will alter the internal geometry of the pocket, thus directly influencing the fidelity.

Amino Acids↗

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences.

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10(-5.7)). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.

Alternative Splicing↗

Molecular and functional analysis of the type III secretion signal of the Salmonella enterica InvJ protein.

Central to the pathogenicity of Salmonella enterica is the function of a type III secretion system (TTSS) encoded within a pathogenicity island at centisome 63 (SPI-1). An essential component of this system is a supramolecular structure termed the needle complex. Proteins to be delivered into host cells possess specific signals that route them to the type III secretion pathway. In addition, some bacterial proteins have signals that deliver them to the secretion complex to either become their structural components or exert their function at that location. One of these proteins is InvJ, which controls the length of the needle substructure of the needle complex. In this study, we have analysed the signal that targets InvJ to the TTSS. We found that amino acid residues 4 to 7 of InvJ are necessary and sufficient to mediate secretion of InvJ or a reporter protein in a TTSS-dependent manner. InvJ secretion was found to be essential for its function in needle length determination, effector protein secretion and bacterial invasion of epithelial cells. Frameshift mutagenesis analysis indicated that the InvJ type III secretion signal sequence tolerates significant alterations in its amino acid sequence without affecting InvJ secretion. Introduction of silent mutations in the secretion signal coding sequence that result in drastically different predicted mRNA folds had no effect on InvJ secretion or expression.

Amino Acid Sequence↗

Functional analysis of mutations in the OCTN2 transporter causing primary carnitine deficiency: lack of genotype-phenotype correlation.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease is caused by mutations in the novel organic cation transporter OCTN2 (SLC22A5 gene). The disease can present early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. To determine whether the variation in phenotypic severity is due to mutations retaining residual function, we extended mutational analysis of OCTN2 to four additional European families with primary carnitine deficiency. Three patients were homozygous for novel missense mutations (R169W, G242V, A301D). The fourth patient was compound heterozygous for R169W and W351R substitutions. Stable expression of all the mutations in CHO cells confirmed that all mutations abolished carnitine transport, with the exception of the A301D mutation in which residual carnitine transport was 2-3% of the value measured in cells expressing the normal OCTN2 cDNA. Analysis of the patients characterized in molecular detail by our laboratory failed to indicate a correlation between residual carnitine transport and severity of the phenotype or age at presentation.

Animals↗

A strategy for fine-structure functional analysis of a 6- to 11-centimorgan region of mouse chromosome 7 by high-efficiency mutagenesis.

A refined functional map of a 6- to 11-centimorgan region surrounding the albino (c) locus in mouse chromosome 7 is being generated by N-ethyl-N-nitrosourea (EtNU) "saturation" mutagenesis of stem-cell spermatogonia. In the first phase of an experiment that will eventually test at least 3000 gametes, we screened 972 mutagenized gametes for the induction of both lethal and visible mutations with a two-cross breeding protocol. Thirteen mutations mapping within the limits of a segment corresponding to the cytologically visible Df(c Mod-2 sh-1)26DVT deletion were recovered. They represented three phenotypic groups: prenatal lethality (six mutations); a fitness/runting syndrome (three mutations, provisionally designated as fit variants); and a neurological/balance-defect abnormality (four mutations). Complementation analysis provided evidence for a true repeat mutation at the sh-1 (shaker-1) locus (for the neurological mutations) and another at the here defined fit-1 (fitness-1) locus. In addition, four complementation groups were defined by induced lethal mutations; the two other lethal mutations were each part of a cluster. The recovery of the repeat mutations suggests that the EtNU-induced mutation rate, estimated from specific-locus tests, should make it possible to achieve saturation mutagenesis of a chromosomal region. This experiment is providing basic logistical and statistical information on which to base strategies for expanding the functional map of larger segments of the mouse genome by experimental mutagenesis. It is also yielding additional mutations useful in dissecting the functional and molecular complexity of this segment of chromosome 7.

Albinism↗

Functional analysis and regulation of nuclear import of dorsal during the immune response in Drosophila.

In addition to its function in embryonic development, the NF-kappa B/rel-related gene dorsal (dl) of Drosophila is expressed in larval and adult fat body where its RNA expression is enhanced upon injury. Injury also leads to a rapid nuclear translocation of dl from the cytoplasm in fat body cells. Here we present data which strongly suggest that the nuclear localization of dl during the immune response is controlled by the Toll signaling pathway, comprising gene products that participate in the intracellular part of the embryonic dorsoventral pathway. We also report that in mutants such as Toll or cactus, which exhibit melanotic tumor phenotypes, dl is constitutively nuclear. Together, these results point to a potential link between the Toll signaling pathway and melanotic tumor induction. Although dl has been shown previously to bind to kappa B-related motifs within the promoter of the antibacterial peptide coding gene diptericin, we find that injury-induced expression of diptericin can occur in the absence of dl. Furthermore, the melanotic tumor phenotype of Toll and cactus is not dl dependent. These data underline the complexity of the Drosophila immune response. Finally, we observed that like other rel proteins, dl can control the level of its own transcription.

Animals↗

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development.

The physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1'-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.

Base Sequence↗

Expression patterns of purple acid phosphatase genes in Arabidopsis organs and functional analysis of AtPAP23 predominantly transcribed in flower.

Purple acid phosphatases (PAPs) are metallo-phosphoesterases. Their expression and function have not been systematically investigated in higher plants. In this work, we compared the transcript levels of 28 Arabidopsis PAP (AtPAP) genes in five Arabidopsis organs. The 28 members, although differed in their expression patterns in vegetative organs, were all transcribed in flower. Furthermore, the transcription of seven members (AtPAPs 6, 11, 14, 19, 23, 24 and 25) occurred predominantly in the flower. To begin dissecting the role of AtPAP genes in flower development, further expression and functional analyses were conducted using AtPAP23. Histochemical staining of transgenic plants expressing AtPAP23 promoter-beta-glucuronidase (GUS) gene construct revealed that AtPAP23 transcription was strong in flower apical meristems, but became restricted to petals and anther filaments in fully developed flower. A GST (glutathione S-transferase) fusion protein of AtPAP23 (GST:AtPAP23) was expressed in bacterial cells, and was found to contain significant amounts of Fe and Mn (whereas the control GST protein contained none). In biochemical tests, GST:AtPAP23 showed typical acid phosphatase activities. The fusion protein was also highly active on phosphoserine, but not phosphotyrosine. Despite its highly specific expression pattern and the demonstrated biochemical function of its protein product, the RNAi (RNA interference), T-DNA knock-out and overexpression lines of AtPAP23 were indistinguishable from wild type plants in the development of flower (or other organs). Interestingly, the Fe and Mn contents were found significantly increased in AtPAP23 overexpression lines, which may offer a new direction for further functional studies of AtPAPs in Arabidopsis.

Acid Phosphatase↗

Sequence and functional analysis of the gene encoding Vibrio cholerae cAMP receptor protein.

We describe here the cloning, nucleotide sequence, and functional expression of the crp gene of Vibrio cholerae (Vc) encoding the cyclic AMP receptor protein (CRP). The Vc crp gene shows 81% identity with the crp gene from Escherichia coli (Ec) and its deduced amino acid sequence shows 95% identity with the Ec protein. When expressed from inducible promoters, the cloned Vc gene produced an approximately 20-kDa protein which complemented the carbohydrate-negative and growth-defective phenotypes of both Ec and Vc crp mutants. In the Vc crp mutant, the cloned crp gene also restored the normal repression of ToxR-regulated virulence genes which occurs under certain environmental conditions.

Amino Acid Sequence↗

Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44.

ERM (ezrin, radixin and moesin) proteins function as linkers between the actin cytoskeleton and the plasma membrane. In addition to this structural role, these proteins are highly regulatable making them ideal candidates to mediate important physiological events such as adhesion and membrane morphology and to control formation and breakdown of membrane-cytoskeletal junctions. Recently, a direct interaction in vitro has been demonstrated between ERM proteins and the hyaluronan receptor, CD44. We have mapped the ezrin-binding site to two clusters of basic amino acids in a membrane-proximal 9 amino-acid region within the CD44 cytoplasmic domain. To investigate the functional importance of this interaction in vivo, we created a number of mutations within full-length CD44 and expressed these mutants in human melanoma cells. We demonstrate here that mutations within the ezrin-binding site do not disrupt the plasma membrane localization of CD44 and, in addition, that this region is not required to mediate efficient hyaluronan binding. These studies suggest that ERM proteins mediate the outside-in, rather than inside-out, signalling of adhesion receptors.

Amino Acid Sequence↗

Functional analysis of primary visual cortex (V1) in humans.

Human area V1 offers an excellent opportunity to study, using functional MRI, a range of properties in a specific cortical visual area, whose borders are defined objectively and convergently by retinotopic criteria. The retinotopy in V1 (also known as primary visual cortex, striate cortex, or Brodmann's area 17) was defined in each subject by using both stationary and phase-encoded polar coordinate stimuli. Data from V1 and neighboring retinotopic areas were displayed on flattened cortical maps. In additional tests we revealed the paired cortical representations of the monocular "blind spot." We also activated area V1 preferentially (relative to other extrastriate areas) by presenting radial gratings alternating between 6% and 100% contrast. Finally, we showed evidence for orientation selectivity in V1 by measuring transient functional MRI increases produced at the change in response to gratings of differing orientations. By systematically varying the orientations presented, we were able to measure the bandwidth of the orientation "transients" (45 degrees).

Brain Mapping↗

In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer.

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.

Animals↗

Wannier functions analysis of the nonlinear Schrödinger equation with a periodic potential.

In the present paper we use the Wannier function basis to construct lattice approximations of the nonlinear Schrödinger equation with a periodic potential. We show that the nonlinear Schrödinger equation with a periodic potential is equivalent to a vector lattice with long-range interactions. For the case-example of the cosine potential we study the validity of the so-called tight-binding approximation, i.e., the approximation when nearest neighbor interactions are dominant. The results are relevant to the Bose-Einstein condensate theory as well as to other physical systems, such as, for example, electromagnetic wave propagation in nonlinear photonic crystals.

Journal Article↗