Trevor Paterson

A prominent serine-rich region in Vmw175, the major transcriptional regulator protein of herpes simplex virus type 1, is not essential for virus growth in tissue culture

Herpes simplex virus type 1 (HSV-1) encodes five immediate early (IE) genes of which at least three are involved in the transcriptional regulation of later classes of viral genes. Perhaps the most important of these regulatory proteins is Vmw175, a nuclear phosphoprotein of 1298 predicted amino acid residues. In the absence of functional Vmw175 the virus fails to activate early or late genes or to repress IE gene expression. All viruses of the sub-family alphaherpes-virinae encode polypeptides that are closely related to Vmw175. Mutational studies have shown that regions of homology within this family of gene regulators are generally of functional importance. One of the most striking conserved stretches of amino acid sequence is a run of serine residues followed by a highly acidic region in the amino-terminal fifth of the polypeptide. We have constructed an HSV-1 virus which lacks this serine-rich run within Vmw175. Surprisingly, the virus was viable in tissue culture cells and expressed apparently normal amounts of viral polypeptides. In plaque assays it was very slightly temperature-sensitive and, depending on the state of the host cells, could generate plaques with a syncytial morphology. The mutant protein was able to bind to DNA in a manner indistinguishable from that of the wild-type polypeptide. We conclude that despite its conservation in all of the alphaherpes-virinae so far sequenced, the serine-rich homology is not important for virus growth in tissue culture.

Scientific names are ambiguous as identifiers for biological taxa: their context and definition are required for accurate data integration

Biologists use scientific names to label the organisms described in their data; however, these names are not unique identifiers for taxonomic entities. Alternative taxonomic classifications may apply the same name, associated with alternative definition or circumscription. Consequently, labelling data with scientific names alone does not unambiguously distinguish between taxon concepts. Accurate integration and comparison of biological data is required on taxon concepts, as defined in alternative taxonomic classifications. We have derived an abstract, inclusive model for the diverse representations of taxonomic concepts used by taxonomists and in taxonomic databases. This model has been implemented as a proposed standard XML schema for the exchange and comparison of taxonomic concepts between data providers and users. The representation and exchange of taxon definitions conformant with this schema will facilitate the development of taxonomic name/concept resolution services, allowing the meaningful integration and comparison of biological datasets, with greater accuracy than on the basis of name alone.

A LINE-like transposable element in Drosophila, the I factor, encodes a protein with properties similar to those of retroviral nucleocapsids.

I factors are members of the LINE‐like family of transposable elements and move by reverse transcription of an RNA intermediate. Complete I factors contain two open reading frames. The amino acid sequence encoded by the first of these, ORF1, includes the motif CX2CX4HX4C that is characteristic of the nucleocapsid domain of retroviral gag polypeptides followed by a copy of the slightly different sequences CX2CX4HX6C and CX2CX9HX6C. The function of this protein is unknown. We have expressed this protein in Escherichia coli and Spodoptera frugiperda cells and have shown that it binds both DNA and RNA but without any evidence for sequence specificity. The properties of deletion derivatives of the protein indicate that more than one region is responsible for DNA binding and that the CCHC motif is not essential for this. The ORF1 protein expressed in either E.coli or Spodoptera cells forms high molecular weight structures that require the region of the protein including the CCHC motif for their formation. This protein can also accelerate the annealing of complementary single‐stranded oligonucleotides. These results suggest that this protein may associate with the RNA transposition intermediates of the I factor to form particles that enter the nucleus during transposition and that it may stimulate both the priming of reverse transcription and integration. This may be generally true for the product of the first open reading frame of LINE‐like elements.

Mutational dissection of the HSV-1 immediate-early protein Vmw175 involved in transcriptional transactivation and repression

Vmw175 is one of five immediate-early (IE) proteins encoded by herpes simplex virus type-1 (HSV-1). It is required for the transcription of later classes of genes and for the accompanying repression of IE expression. Vmw175 has been shown to be a transactivator of transcription and also to autoregulate its own synthesis. We have made a large number of small, in-frame, insertion and deletion mutants of a plasmid-borne copy of the gene encoding Vmw175. Study of the activity of the resultant mutant polypeptides in transient transfection assays has defined the regions of the protein important for the repression of its own promoter, and for the transactivation of an HSV early promoter in synergy with another HSV IE protein, Vmw110. Large stretches of the protein are relatively unimportant for either function, while the regions most sensitive to disruption correlate to sequences conserved between Vmw175 and VZV 140K, the corresponding transactivating protein of Varicella-Zoster virus. The region from amino acids 275 to 490 is particularly important for both repression and transactivation, whereas that from around 840 to 1100 seems to be more important for transactivation than repression. The nuclear localization signal has been mapped to within residues 682-774.

The effect of DnaA protein levels and the rate of initiation at oriC on transcription originating in the ftsQ and ftsA genes: In vivo experiments

SummaryThe DnaA protein of Escherichia coli, essential for initiation at oriC, binds at a defined sequence which occurs at the chromosomal origin, near plasmid replication origins and in the promoters of the dnaA and mioC genes. This sequence also occurs at many other sites on the E. coli chromosome including three sites within the essential cell division genes ftsQ and A. Using an fts-lac fusion phage, λJFL100, we show here that fts gene expression responds both to reduced and increased intracellular levels of DnaA protein in a manner consistent with the hypothesis that DnaA protein regulates fts gene expression. Experiments using dnaC and dnaB-ts strains, however, suggest that DnaA control of fts transcription may be indirect, at least in part, with fts responding to the rate of initiation at oriC as well as to changes in DnaA protein level per se. It differs in this respect from dnaA gene expression which is unaffected when initiation of replication is inhibited by DnaB or DnaC inactivation. Strains integratively suppressed with pKN500 behave anomalously; neither fts nor dnaA transcription is significantly increased when DnaA is inactivated in these strains.

A Universal Character Model and Ontology of Defined Terms for Taxonomic Description

Taxonomists classify biological specimens into groups (taxa) on the basis of similarities between their observed features (‘characters’). The description of these ‘characters’ is therefore central to taxonomy, but there is currently no agreed model, defined terminology nor methodology for composing these descriptions. This lack of a common conceptual model, together with the individualistic working practices of taxonomists, means that descriptions are not composed consistently, and are not easy to interpret and re-use, nor are datasets comparable. The purpose of the Prometheus II project is to improve the interpretation and comparison of plant descriptions. To this end we propose a new conceptual model for unambiguously representing character descriptions, and have developed a controlled vocabulary as an ontology of defined terms, which will be used to describe specimens according to our character model.

A mutant of herpes simplex virus type 1 immediate early polypeptide Vmw175 binds to the cap site of its own promoter in vitro but fails to autoregulate in vivo

Vmw175, the product of herpes simplex virus type 1 immediate early (IE) gene 3, is essential for viral replication. It is required for the activation of transcription from both early and late gene promoters and also for the repression of IE gene expression. Vmw175 is able to bind specifically to certain DNA sequences, some of which (including that at the cap site of IE gene 3) contain the consensus sequence ATCGTC. The presence of this sequence at the cap site has been correlated with the ability of Vmw175 to autoregulate its own promoter. This report describes the characterization of five viruses with temperature-sensitive (ts) lesions in Vmw175. Four of these mutants express Vmw175 which is ts in its ability to bind to DNA in vitro and to autoregulate IE-3 gene expression in the infected cell. Although Vmw175 produced by the remaining mutant, ts1225, fails to autoregulate IE-3 expression at the non-permissive temperature (NPT) its DNA-binding properties are indistinguishable from those of the wild-type protein. This suggests that the ability of Vmw175 to bind to the IE-3 cap site (as measured in vitro) is insufficient for autoregulation (in vivo). All five newly characterized ts mutants are partially permissive for early gene transcription at the NPT, although Vmw175 expressed by four of them is unable to bind to the IE-3 cap site sequence at elevated temperatures. This suggests that binding to one class of recognition sequences by Vmw175, as measured in vitro, is not absolutely required for the activation of early gene promoters during virus infection. The lesions in these five ts mutants lie in the carboxy-terminal third of the polypeptide; three of the mutations (those in ts1219, ts1221 and ts1225) were identified by DNA sequence analysis and were found to affect amino acid residues that are conserved in the homologous proteins from varicella-zoster virus and pseudorabies virus.

VIPER: a visualisation tool for exploring inheritance inconsistencies in genotyped pedigrees

BackgroundPedigree genotype datasets are used for analysing genetic inheritance and to map genetic markers and traits. Such datasets consist of hundreds of related animals genotyped for thousands of genetic markers and invariably contain multiple errors in both the pedigree structure and in the associated individual genotype data. These errors manifest as apparent inheritance inconsistencies in the pedigree, and invalidate analyses of marker inheritance patterns across the dataset. Cleaning raw datasets of bad data points (incorrect pedigree relationships, unreliable marker assays, suspect samples, bad genotype results etc.) requires expert exploration of the patterns of exposed inconsistencies in the context of the inheritance pedigree. In order to assist this process we are developing VIPER (Visual Pedigree Explorer), a software tool that integrates an inheritance-checking algorithm with a novel space-efficient pedigree visualisation, so that reported inheritance inconsistencies are overlaid on an interactive, navigable representation of the pedigree structure.Methods and resultsThis paper describes an evaluation of how VIPER displays the different scales and types of dataset that occur experimentally, with a description of how VIPERs display interface and functionality meet the challenges presented by such data. We examine a range of possible error types found in real and simulated pedigree genotype datasets, demonstrating how these errors are exposed and explored using the VIPER interface and we evaluate the utility and usability of the interface to the domain expert.Evaluation was performed as a two stage process with the assistance of domain experts (geneticists). The initial evaluation drove the iterative implementation of further features in the software prototype, as required by the users, prior to a final functional evaluation of the pedigree display for exploring the various error types, data scales and structures.ConclusionsThe VIPER display was shown to effectively expose the range of errors found in experimental genotyped pedigrees, allowing users to explore the underlying causes of reported inheritance inconsistencies. This interface will provide the basis for a full data cleaning tool that will allow the user to remove isolated bad data points, and reversibly test the effect of removing suspect genotypes and pedigree relationships.

The Prometheus Description Model: an examination of the taxonomic description-building process and its representation

A model for representing taxonomic descriptive data is presented. The model has been developed in response to the growing requirement for the global exchange of descriptive data. Meaningful exchange of data requires that data be represented in a form that can be consistently parsed and interpreted, requiring a common data model and the constrained and explicitly defined use of descriptive terms. The model presented here is divided into two parts that address both of these issues. A new data model for the representation and storage of taxonomic descriptive data is proposed that builds on and extends the best features of current descriptive data models and formats. An ontology-based model for defining and constraining the use of descriptive terms is also presented. The model is based on an analysis of current taxonomic working practices and the processes involved in generating a description. The model takes a specimen-oriented approach allowing descriptive data to be represented through a range of levels of abstraction from actual measurements of structures on a specimen to

A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.

The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.