Biomedical subjects

Christine Wells

Publications and source records attributed to Christine Wells.

7 recordsLinked to original sources

PubMed2006

Complex Loci in human and mouse genomes.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

Animals↗

PubMed2006

Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases.

BACKGROUND: Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au. RESULTS: By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. CONCLUSION: These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.

Alternative Splicing↗

PubMed2005

Pilot studies on the parallel production of soluble mouse proteins in a bacterial expression system.

We investigated the parallel production in medium throughput of mouse proteins, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. The methods were scaled up to allow the simultaneous processing of tens or hundreds of protein samples. Scale-up was achieved in two stages. In an initial study, 30 targets were processed manually but with common protocols for all targets. In the second study, these protocols were applied to 96 target proteins that were processed in an automated manner. The success rates at each stage of the study were similar for both the manual and automated approaches. Overall, 15 of the selected 126 target mouse genes (12%) yielded soluble protein products in a bacterial expression system. This success rate compares favourably with other protein screening projects, particularly for eukaryotic proteins, and could be further improved by modifications at the cloning step.

Animals↗

PubMed2004

Towards a mutant map of the mouse--new models of neurological, behavioural, deafness, bone, renal and blood disorders.

With the completion of the first draft of the human genome sequence, the next major challenge is assigning function to genes. One approach is genome-wide random chemical mutagenesis, followed by screening for mutant phenotypes of interest and subsequent mapping and identification of the mutated genes in question. We (a consortium made up of GlaxoSmithKline, the MRC Mammalian Genetics Unit and Mouse Genome Centre, Harwell, Imperial College, London, and the Royal London Hospital) have used ENU mutagenesis in the mouse for the rapid generation of novel mutant phenotypes for use as animal models of human disease and for gene function assignment (Nolan et al., 2000). As of 2003, 35,000 mice have been produced to date in a genome-wide screen for dominant mutations and screened using a variety of screening protocols. Nearly 200 mutants have been confirmed as heritable and added to the mouse mutant catalogue and, overall, we can extrapolate that we have recovered over 700 mutants from the screening programme. For further information on the project and details of the data, see http://www.mgu.har.mrc.ac.uk/mutabase.

Animals↗

PubMed2003

Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Animals↗

PubMed2003

A comprehensive transcript map of the mouse Gnas imprinted complex.

The recent publication of the FANTOM mouse transcriptome has provided a unique opportunity to study the diversity of transcripts arising from a single gene locus. We have focused on the Gnas complex, as imprinting loci themselves provide unique insights into transcriptional regulation. Thirteen full-length cDNAs from the FANTOM2 set were mapped to the Gnas locus. These represented one previously described transcript and 12 putative new transcripts. Of these, eight were found to be differentially expressed from either the maternal or paternal allele. Two clones extended Nespas in the 3' direction, providing evidence of antisense transcription spanning a 30-kb genomic region from a single allele. The transcripts were summarized into six transcriptional units, Nespas, Nesp, Gnasxl, F7, exon 1A, and Gnas. The resolution of the Gnas transcript map by the FANTOM2 clones revealed a pattern of alternate splicing. In addition to the transcripts described previously as splicing onto exon 2 of Gnas, each new sense transcript had an alternate short 3'UTR independent of Gnas. Both spliced and unspliced variants of the new imprinted sense transcripts were found. Whereas the functional significance of these alternate transcripts is not known, the availability of the FANTOM clones has provided remarkable insights into the repertoire of transcripts in the Gnas complex locus.

Animals↗

PubMed2002

Generation of diversity in the innate immune system: macrophage heterogeneity arises from gene-autonomous transcriptional probability of individual inducible genes.

Microbial products such as LPS stimulate macrophages to produce a wide diversity of inducible gene products needed for immediate host defense and priming of an appropriate acquired immune response. In this study, we have examined LPS-inducible gene expression in subclones of a mouse macrophage cell line, RAW264, using cDNA microarrays. Even archetypal target genes such as TNF-alpha were not induced in all subclones, and there was no absolute correlation between expression of pairs of genes. Nevertheless, the array analysis revealed clusters of genes that were more likely to be coexpressed. RAW264 cells stably transfected with luciferase reporter genes driven by LPS-responsive promoters revealed the same kind of clonal heterogeneity. The results indicate that each LPS-inducible gene has its own inherent probability of activation in response to LPS.

Animals↗