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A Francis Stewart

Publications and source records attributed to A Francis Stewart.

At least 19 recordsLinked to original sources
PubMed2026

Motor coordination and behavioural deficits in a mouse model of KMT2B-related dystonia.

INTRODUCTION: Pathogenic variants in KMT2B cause early-onset dystonia, but a mouse model that has undergone comprehensive, dystonia-oriented phenotyping is lacking. METHODS: We conducted detailed phenotyping on heterozygous Kmt2b constitutive knockout mice and wild-type littermates, assessing growth, neurobehavioural traits, motor coordination, sensorimotor gating, social behaviour and metabolic parameters, combined with striatal RNA sequencing. RESULTS: Kmt2b knockout mice of both sexes were viable but significantly smaller and lighter than littermate controls. Knockouts were hyperlocomotive in the open field and showed approximately two-fold larger acoustic startle responses; unexpectedly, prepulse inhibition was enhanced rather than reduced at all prepulse intensities. On the balance beam, knockouts crossed more slowly and paused more frequently; female knockouts also paused more on the ladder rung task. Frame-by-frame video analysis revealed a claw-like hindpaw posture characterized by abnormal inward flexion of the digits. Knockout mice spent less time investigating a novel conspecific, while social recognition memory remained intact. Striatal RNA sequencing confirmed reduction of Kmt2b transcript to approximately half of control levels and identified 177 differentially expressed genes, including Maob, encoding monoamine oxidase B; gene set enrichment analysis implicated neurodevelopmental, glial and mitochondrial processes. Nociception, vision, body-weight-adjusted grip strength, and clinical chemistry and haematological measures were largely unaffected. CONCLUSION: Heterozygous Kmt2b knockout mice show hyperlocomotion, altered sensorimotor gating, impaired motor coordination with dystonic-like paw posturing and reduced sociability, alongside a striatal transcriptomic signature implicating neurodevelopmental processes. The model mirrors aspects of human KMT2B-related dystonia and provides a platform for mechanistic study; environmental or pharmacological challenge may be needed to unmask overt dystonic features.

Dystonia↗
PubMed2006

Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus.

Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.

Bacterial Proteins↗
PubMed2006

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation.

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

DNA-Binding Proteins↗
PubMed2006

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development.

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.

Alleles↗
PubMed2006

Recombineering reagents for improved inducible expression and selection marker re-use in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism for cell biology. However, its genetic toolbox is less developed than that of Saccharomyces cerevisiae. In the first part of this study we describe an improved inducible expression vector based on tetracycline regulation of the CaMV35S promoter, which is also capable of chromosomal integration and therefore works in minimal and in rich media. We found that anhydrotetracycline is a superior ligand for induction. Maximum expression levels were observed after 12 h in minimal media (EMM) and after 9 h in rich media (YES), which is faster than the nmt1 promoter system. The system was combined with a convenient recombineering-based subcloning strategy for ease of cloning. In the second part we present four template plasmids, pSVEM-bsd, pSVEM-nat, pSVEM-kan and pSVEM-hph, which harbour four recyclable disruption cassettes based on the Cre recombinase lox71/66 strategy for use in PCR targeting methods. Cre-mediated excision leaves a non-functional mutant lox site in the genome, allowing the reiterative usage of these cassettes for multiple targetings. These cassettes are also configured with dual eukaryotic/prokaryotic promoters so that they can be used for recombineering in E. coli. Amongst other purposes, this permits the rapid and convenient creation of targeting constructs with much longer homology arms for difficult and complex targetings in the Sz. pombe genome.

Escherichia coli↗
PubMed2006

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Acyl Coenzyme A↗
PubMed2006

Engineering embryonic stem cells with recombinase systems.

The combined use of site-specific recombination and gene targeting or trapping in embryonic stem cells (ESCs) has resulted in the emergence of technologies that enable the induction of mouse mutations in a prespecified temporal and spatially restricted manner. Their large-scale implementation by several international mouse mutagenesis programs will lead to the assembly of a library of ES cell lines harboring conditional mutations in every single gene of the mouse genome. In anticipation of this unprecedented resource, this chapter will focus on site-specific recombination strategies and issues pertinent to ESCs and mice. The upcoming ESC resource and the increasing sophistication of site-specific recombination technologies will greatly assist the functional annotation of the human genome and the animal modeling of human disease.

Animals↗
PubMed2006

A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Animals↗
PubMed2006

An improved recombineering approach by adding RecA to lambda Red recombination.

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.

Animals↗
PubMed2005

Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene.

Transgenic mouse lines were generated that express the Cre recombinase under the control of the distal promoter of the mouse Lck gene. Cre recombination in four of these lines of transgenic mice was characterized at the single cell level using ROSA26-regulated loxP-Stop-loxP-betageo and loxP-Stop-loxP-YFP reporter mouse lines. Two of the lines showed T cell-restricted Cre recombination, whereas the other two also expressed Cre in B cells, NK cells, and monocytes. Cre recombination began at a late stage of T cell development (at or after up-regulation of the TCR during positive selection) in the two T cell-restricted lines. Lines of mice that express the Cre recombinase at late stages of thymocyte development are of value for determining the impact of mutations on T cell function in the absence of complicating effects on early thymocyte selection.

Animals↗
PubMed2005

Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex.

The replicative DNA helicases can unwind DNA in the absence of polymerase activity in vitro. In contrast, replicative unwinding is coupled with DNA synthesis in vivo. The temperature-sensitive yeast polymerase alpha/primase mutants cdc17-1, pri2-1 and pri1-m4, which fail to execute the early step of DNA replication, have been used to investigate the interaction between replicative unwinding and DNA synthesis in vivo. We report that some of the plasmid molecules in these mutant strains became extensively negatively supercoiled when DNA synthesis is prevented. In contrast, additional negative supercoiling was not detected during formation of DNA initiation complex or hydroxyurea replication fork arrest. Together, these results indicate that the extensive negative supercoiling of DNA is a result of replicative unwinding, which is not followed by DNA synthesis. The limited number of unwound plasmid molecules and synthetic lethality of polymerase alpha or primase with checkpoint mutants suggest a checkpoint regulation of the replicative unwinding. In concordance with this suggestion, we found that the Tof1/Csm3/Mrc1 checkpoint complex interacts directly with the MCM helicase during both replication fork progression and when the replication fork is stalled.

Cell Cycle↗
PubMed2005

Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering.

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.

DNA, Recombinant↗
PubMed2005

The best control for the specificity of RNAi.

RNA interference (RNAi) is revolutionizing functional genomics. However, there are several reasons to be concerned about the specificity and off-target effects of this technique. A recent paper by Kittler et al. describes a straightforward way to validate RNAi specificity, which exploits the increasing availability of bacterial artificial chromosome (BAC) clone resources. Genetic rescue of the RNAi phenotype by BAC transgenesis is the best control yet described for specificity, and has further implications for reverse genetics.

Animals↗
PubMed2005

Current issues in mouse genome engineering.

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.

Animals↗
PubMed2004

A reliable lacZ expression reporter cassette for multipurpose, knockout-first alleles.

Alteration of the mouse genome through homologous recombination in embryonic stem (ES) cells is the most accurate and versatile way to dissect gene function in a vertebrate model. Most often, a selectable marker is used to create a knockout allele by replacing an essential part of the gene. However, knockout strategies are limited because the mutation is present constitutively. Conditional approaches based on the Cre-loxP site-specific recombination (SSR) system address this limitation; however, it requires that all parts of the targeted gene remain in ES cells. Here we report success with a "knockout-first" strategy that ablates gene function by insertion of RNA processing signals without deletion of any of the target gene. Incorporation of site-specific recombination target sites creates a multipurpose allele for both knockout and conditional applications.

Animals↗
PubMed2004

The European dimension for the mouse genome mutagenesis program.

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Animals↗