Biomedical subjects

Michael T McManus

Publications and source records attributed to Michael T McManus.

At least 19 recordsLinked to original sources

PubMed2026

Multiplexed perturbation enables scalable pooled screens.

CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.

Humans↗

PubMed2006

Dicer function is essential for lung epithelium morphogenesis.

DICER is a key enzyme that processes microRNA and small interfering RNA precursors into their short mature forms, enabling them to regulate gene expression. Only a single Dicer gene exists in the mouse genome, and it is broadly expressed in developing tissues. Dicer-null mutants die before gastrulation. Therefore, to study Dicer function in the later event of lung formation, we inactivated it in the mouse lung epithelium using a Dicer conditional allele and the Sonic Hedgehogcre (Shhcre) allele. Branching arrests in these mutant lungs, although epithelial growth continues in distal domains that are expanded compared with normal samples. These defects result in a few large epithelial pouches in the mutant lung instead of numerous fine branches present in a normal lung. Significantly, the initial phenotypes are apparent before an increase in epithelial cell death is observed, leading us to propose that Dicer plays a specific role in regulating lung epithelial morphogenesis independent of its requirement in cell survival. In addition, we found that the expression of Fgf10, a key gene involved in lung development, is up-regulated and expanded in the mesenchyme of Dicer mutant lungs. Previous studies support the hypothesis that precise localization of FGF10 in discrete sites of the lung mesenchyme serves as a chemoattractant for the outgrowth of epithelial branches. The aberrant Fgf10 expression may contribute to the Dicer morphological defects. However, the mechanism by which DICER functions in the epithelium to influence Fgf10 expression in the mesenchyme remains unknown.

Animals↗

PubMed2006

Expression of 1-aminocyclopropane-1-carboxylate (ACC) oxidase genes during the development of vegetative tissues in white clover (Trifolium repens L.) is regulated by ontological cues.

Four 5' flanking sequences, comprising the 5'-UTR and upstream promoter region, have been isolated and cloned from the 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene family of white clover (Trifolium repens L.), and designated TR-ACO1p, TR-ACO2p, TR-ACO3p and TR-ACO4p. Southern analysis confirmed that these sequences correspond to four distinct genes. The four corresponding genomic sequences have also been isolated and each shown to be comprised of four exons interspersed by three introns. The expression pattern, in vivo, directed by all four 5' flanking sequences during leaf development has been examined using GUS fusions and transformation into white clover. Here, the TR-ACO1 5' flanking sequence directs highest expression in the apical tissues, axillary buds, and leaf petiolules in younger tissues and then declines in the ageing tissues, while the TR-ACO2 5' flanking sequence directs expression in both younger, mature green and in ontologically ageing tissue. The TR-ACO3 and TR-ACO4 5' flanking sequences direct more expression in the ontological older tissues, including the axillary buds and leaf petiolules. The TR-ACO1 5' flanking sequence directed expression in the ground meristem and newly emerged leaf tissue at the apical bud of the stolon, but all four 5' flanking sequences directed expression in the ground meristem tissue of axillary buds, vascular tissue, pith and cortex of the internode and node, and the cortex and vascular tissue of the leaf petiolule, with the primacy of each promoter determined by the ontological age of the tissues. These data suggest that in vegetative tissue development of white clover, the primary cues for the transcriptional regulation of the ACO gene family are ontological in nature.

5' Untranslated Regions↗

PubMed2005

The microRNA miR-196 acts upstream of Hoxb8 and Shh in limb development.

MicroRNAs (miRNAs) are an abundant class of gene regulatory molecules (reviewed in refs 1, 2). Although computational work indicates that miRNAs repress more than a third of human genes, their roles in vertebrate development are only now beginning to be determined. Here we show that miR-196 acts upstream of Hoxb8 and Sonic hedgehog (Shh) in vivo in the context of limb development, thereby identifying a previously observed but uncharacterized inhibitory activity that operates specifically in the hindlimb. Our data indicate that miR-196 functions in a fail-safe mechanism to assure the fidelity of expression domains that are primarily regulated at the transcriptional level, supporting the idea that many vertebrate miRNAs may function as a secondary level of gene regulation.

Animals↗

PubMed2005

Slowing down the Ras lane: miRNAs as tumor suppressors?

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression by promoting the degradation of transcribed messages or by inhibiting translation. Although bioinformatic approaches suggest that miRNAs may regulate the expression of a large fraction of the genome, the determination of miRNA gene targets and biological functions has been comparatively limited. Emerging studies suggest that many miRNAs may participate in human disease, including oncogenesis; but for the most part, the observations have been correlative. A recent study by Johnson and colleagues indicates that the let-7 miRNA negatively regulates the oncogenic family of Ras guanosine triphosphatases in both Caenorhabditis elegans and human tumor cell lines, suggesting that let-7 may act as a tumor suppressor. This work raises several important questions: Can other miRNAs act as tumor suppressors or oncogenes? Is miRNA deregulation a critical aspect of tumor development and maintenance? A number of recent studies have begun to address some of these functional questions, providing the field with a greater understanding of the role of miRNAs in cancer.

Animals↗

PubMed2005

TAZ, a transcriptional modulator of mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator-activated receptor gamma (PPARgamma), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARgamma-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation.

Acyltransferases↗

PubMed2005

The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb.

The RNaseIII-containing enzyme Dicer is believed to be required for the processing of most, if not all, microRNAs (miRNAs) and for processing long dsRNA into small interfering RNAs. Because the complete loss of Dicer in both zebrafish and mice results in early embryonic lethality, it has been impossible to determine what role, if any, Dicer has in patterning later tissues in the developing vertebrate embryo. To bypass the early requirement of Dicer in development, we have created a conditional allele of this gene in mice. Using transgenes to drive Cre expression in discrete regions of the limb mesoderm, we find that removal of Dicer results in the loss of processed miRNAs. Phenotypically, developmental delays, in part due to massive cell death as well as disregulation of specific gene expression, lead to the formation of a much smaller limb. Thus, Dicer is required for the formation of normal mouse limbs. Strikingly, however, we did not detect defects in basic patterning or in tissue-specific differentiation of Dicer-deficient limb buds.

Alleles↗

PubMed2005

Molecular and biochemical characterisation of a serine acetyltransferase of onion, Allium cepa (L.).

We have previously cloned a cDNA, designated SAT1, corresponding to a gene coding for a serine acetyltransferase (SAT) from onion (Allium cepa L.). The SAT1 locus was mapped to chromosome 7 of onion using a single-stranded conformation polymorphism (SSCP) in the 3' UTR of the gene. Northern analysis has demonstrated that expression of the SAT1 gene is induced in leaf tissue in response to low S-supply. Phylogenetic analysis has placed SAT1 in a strongly supported group (100% bootstrap) that comprises sequences that have been characterised biochemically, including Allium tuberosum, Spinacea oleracea, Glycine max, Citrullus vulgaris, and SAT5 (AT5g56760) of Arabidopsis thaliana. This group can be divided further with the SAT1 of A. cepa sequence grouping strongly with the A. tuberosum sequence. Translation of SAT1 from onion generates a protein of 289 amino acids with a calculated molecular mass of 30,573 Da and pI of 6.52. The conserved G277 and H282 residues that have been identified as critical for L-cysteine inhibition are observed at G272 and H277. SAT1 has been cloned into the pGEX plasmid, expressed in E. coli and SAT activity of the recombinant enzyme has been measured as acetyl-CoA hydrolysis detected at 232 nm. A Km of 0.72 mM was determined for l-serine as substrate, a Km of 92 microM was calculated with acetyl-CoA as substrate, and an inhibition curve for L-cysteine generated an IC50 value of 3.1 microM. Antibodies raised against the recombinant SAT1 protein recognised a protein of ca. 33 kDa in whole leaf onion extracts. These properties of the SAT1 enzyme from onion are compared with other SAT enzymes characterised from closely related species.

Acetyltransferases↗

PubMed2005

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: influence on protein stability and activity.

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV35S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn189) associated with the point of glycan attachment (Asn189) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35S:prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent Km) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (Ea) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases.

Amino Acid Sequence↗

PubMed2005

MicroRNAs and endocrine biology.

microRNAs (miRNAs) are highly conserved, non-coding RNAs that powerfully regulate gene expression at the post-transcriptional level. These fascinating molecules play essential roles in many biological processes in mammals, including insulin secretion, B-cell development, and adipocyte differentiation. This review provides a general background regarding current knowledge about miRNA biogenesis and the potential contributions of these RNAs to endocrine function.

Adipocytes↗

PubMed2004

MicroRNA-responsive 'sensor' transgenes uncover Hox-like and other developmentally regulated patterns of vertebrate microRNA expression.

MicroRNAs (miRNAs) are a class of short ( approximately 22-nt) noncoding RNA molecules that downregulate expression of their mRNA targets. Since their discovery as regulators of developmental timing in Caenorhabditis elegans, hundreds of miRNAs have been identified in both animals and plants. Here, we report a technique for visualizing detailed miRNA expression patterns in mouse embryos. We elucidate the tissue-specific expression of several miRNAs during embryogenesis, including two encoded by genes embedded in homeobox (Hox) clusters, miR-10a and miR-196a. These two miRNAs are expressed in patterns that are markedly reminiscent of those of Hox genes. Furthermore, miR-196a negatively regulates Hoxb8, indicating that its restricted expression pattern probably reflects a role in the patterning function of the Hox complex.

Animals↗

PubMed2004

Small RNAs and immunity.

Small RNAs of 21-23 nucleotides are powerful regulators of gene expression and play essential roles in biological processes that include development, maintenance of genome stability, and viral adaptive defense mechanisms. Such small RNAs are simple in design yet rich in biology and have captivated the attention of biologists in many fields. This review discusses the potential roles of small RNAs in immune biology and speculates on their potential participation in lymphogenesis and antiviral mechanisms.

Animals↗

PubMed2003

RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription.

Influenza A virus causes widespread infection in the human respiratory tract, but existing vaccines and drug therapy are of limited value. Here we show that short interfering RNAs (siRNAs) specific for conserved regions of the viral genome can potently inhibit influenza virus production in both cell lines and embryonated chicken eggs. The inhibition depends on the presence of a functional antisense strand in the siRNA duplex, suggesting that viral mRNA is the target of RNA interference. However, siRNA specific for nucleocapsid (NP) or a component of the RNA transcriptase (PA) abolished the accumulation of not only the corresponding mRNA but also virion RNA and its complementary RNA. These siRNAs also broadly inhibited the accumulation of other viral, but not cellular, RNAs. The findings reveal that newly synthesized NP and PA proteins are required for influenza virus transcription and replication and provide a basis for the development of siRNAs as prophylaxis and therapy for influenza infection in humans.

Animals↗

PubMed2003

A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.

RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.

Animals↗

PubMed2003

MicroRNAs and cancer.

The general basis of cancer is the loss of cell identity and inappropriate proliferation of cells. Classically, a universal paradigm in oncogenesis is the accumulation of mutations in the open reading frames of protein-encoding oncogenes and tumor suppressors. The identification of new classes of noncoding RNAs (ncRNA) important for development and cell homeostasis will likely change this current paradigm. Recent data suggests that a special class of ncRNAs called microRNAs might be involved in human disease. This review proposes a role for microRNAs in oncogenesis.

Animals↗

PubMed2003

Identification and characterisation of proteinase inhibitors and their genes from seeds of apple (Malus domestica).

Trypsin and papain proteinase inhibitors have been identified and purified from aqueous extracts of apple seeds (Malus domestica). Superdex G75 gel filtration chromatography identified a higher molecular weight (HMW) papain inhibitory fraction (22-26 kDa) and a lower molecular weight papain and trypsin inhibitory fraction (6-12 kDa). The lower molecular weight fraction was separated into a trypsin inhibitor (designated Trp1) and early (designated Pap1) and late (designated Pap2) eluting papain inhibitors after anion exchange (Hitrap SP) chromatography. For Pap2, two inhibitory peaks (designated Pap2-1 and Pap2-2) were identified after further anion exchange (Resource S) chromatography. Each of these lower molecular weight inhibitors was purified by reverse phase HPLC to homogeneity as determined by SDS-PAGE and by mass spectrometry. The HMW papain inhibitory fraction was purified further by anion-exchange (Hitrap Q followed by Resource Q) column chromatography where a minor inhibitor (HMWPap1) and major inhibitor (HMWPap2) fraction were identified. The relative abundance in seeds of apple and the spectrum of proteinase inhibition has been determined for all of these inhibitors. Reverse-phase HPLC separated HMWPap2 into a minor (HMWPap2-1) and a major (HMWPap2-2) inhibitory fraction, and SDS-PAGE and mass spectrometry confirmed that HMWPap2-2 was purified to homogeneity. Amino acid composition data were obtained from Trp1, Pap1, Pap2-2, and HMWPap2-2, and N-terminal sequence data from Trp1, Pap2-1, Pap2-2, and HMWPap2-2, with two of these sequences (Pap2-2 and HMWPap2-2) perfectly matching predicted protein sequences based on EST sequences from an apple database. The relationship of these inhibitors with those of other species is discussed.

Amino Acid Sequence↗

PubMed2002

Small interfering RNA-mediated gene silencing in T lymphocytes.

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.

Animals↗

PubMed2002

The proteomics of senescence in leaves of white clover, Trifolium repens (L.).

A proteomics approach has been used to study changes in protein abundance during leaf senescence in white clover. Changes in cell ultrastructure were also examined using transmission electron microscopy. The most obvious ultrastructural changes during senescence occurred in chloroplasts, with progressive loss of thylakoid integrity and accumulation of osmiophilic globules in the stroma. Quantitative analysis of 590 leaf protein spots separated by two-dimensional electrophoresis indicated that approximately 40% of the spots showed significant senescence related changes in abundance. Approximately one-third of the protein spots present in mature green leaves were also visible by two-dimensional electrophoresis of an isolated chloroplast fraction, and these spots represented a major proportion of the proteins showing senescence related declines in abundance. Chloroplast proteins that were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting included rubisco large and small subunits, a rubisco activase and the 33 kDa protein of the photosystem II oxygen-evolving complex. These proteins declined in abundance late in senescence, indicating that the photosynthetic apparatus was being degraded. A chloroplast glutamine synthetase showed partial decline in abundance during late senescence but was maintained at levels that may support provision of glutamine for export to other tissues. The results emphasise the importance of proteolysis, chloroplast degradation and remobilisation of nitrogen in leaf senescence.

Cellular Senescence↗