Expression Of Minigene Research Articles

Expression, purification, and solubility optimization of peptidyl-tRNA hydrolase 1 from Bacillus cereus

Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in Escherichia coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis–Menten parameters reported.

Small Molecule Binding, Docking, and Characterization of the Interaction between Pth1 and Peptidyl-tRNA

Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.

Novel Roles for the E3 Ubiquitin Ligase Atrophin-interacting Protein 4 and Signal Transduction Adaptor Molecule 1 in G Protein-coupled Receptor Signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5′ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5′ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5′ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

The GATA1-HS2 Enhancer Allows Persistent and Position-Independent Expression of a β-globin Transgene

Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.

Characterization of a new splicing mutation in the steroid 21-hydroxylase gene

Novel mutation in CYP21A2 gene causing the steroid 21-hydroxylase deficiency - C to G substitution in 7-position ofintron 2 acceptor splice site (c.290-7C>G) was identified. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in the cultured mammalian cells. The mutation impairs the usage of intron 2 acceptor splice site resulting in intron retention.

CYP17A1 Intron Mutation Causing Cryptic Splicing in 17α-Hydroxylase Deficiency

17α-hydroxylase/17, 20-lyase deficiency (17OHD) is an autosomal recessive disease causing congenital adrenal hyperplasia and a rare cause of hypertension with hypokalemia. The CYP17A1 gene mutation leads to 17OHD and its clinical features. We described an 18 y/o female with clinical features of 17α-hydroxylase/17, 20-lyase deficiency and characterized the functional consequences of an intronic CYP17A1 mutation. The coding regions and flanking intronic bases of the CYP17A1 gene were amplified by PCR and sequenced. The patient is a compound heterozygote for the previously described p.R358X and IVS1 +2T>C mutations. A first intron splice donor site mutation was re-created in minigene and full-length expression vectors. Pre-mRNA splicing of the variant CYP17A1 intron was studied in transfected cells and in a transformed lymphoblastoid cell line. When the full-length CYP17A1 gene and minigene containing the intronic mutation was expressed in transfected cells, the majority (>90%) of mRNA transcripts were incorrectly spliced. Only the p.R358X transcript was detected in the EBV-transformed lymphoblastoid cell line. The IVS1 +2T>C mutation abolished most 17α-hydroxylase/17, 20-lyase enzyme activity by aberrant mRNA splicing to an intronic pseudo-exon, causing a frame shift and early termination.

Two novel mutations in the thyroglobulin gene as cause of congenital hypothyroidism: Identification a cryptic donor splice site in the exon 19

Thyroglobulin (TG) is a homodimeric glycoprotein synthesized by the thyroid gland. To date, 52 mutations of the TG gene have been identified in humans. The purpose of the present study was to identify and characterize new mutations in the TG gene. We report a French patient with congenital hypothyroidism, mild enlarged thyroid gland and low levels of serum TG. Sequencing of DNA, genotyping, expression of chimeric minigenes as well as bioinformatics analysis were performed. DNA sequencing identified the presence of compound heterozygous mutations in the TG gene: the paternal mutation consists of a c.3788–3789insT or c.3788dupT, whereas the maternal mutation consists of g.IVS19+3_+4delAT. Minigene analysis of the g.IVS19+3_+4delAT mutant showed that the exon 19 is skipped during pre-mRNA splicing or partially included by use of cryptic 5′ splice site located to 100 nucleotides downstream of the wild type exon–intron junction. The c.3788–3789insT mutation results in a putative truncated protein of 1245 amino acids, whereas g.IVS19+3_4delAT mutation originates two putative truncated proteins of 1330 and 1349 amino acids. In conclusion, we show that the g.IVS19+3_+4delAT mutation promotes the activation of a cryptic donor splice site in the exon 19 of the TG gene. These results open up new perspectives in the knowledge of the mechanism of splicing for the TG pre-mRNA.

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

Endothelial cell talin1 is essential for embryonic angiogenesis

Using Tln1fl/fl;CreER mice, we show that tamoxifen-induced inactivation of the talin1 gene throughout the embryo produces an angiogenesis phenotype that is restricted to newly forming blood vessels. The phenotype has a rapid onset in early embryos, resulting in vessel defects by 48h and death of the embryo within 72h. Very similar vascular defects were obtained using a Tie2-Cre endothelial cell-specific Tln1 knockout, a phenotype that was rescued by expression of a Tln1 mini-gene in endothelial cells. We show that endothelial cells, unlike most other cell types, do not express talin2, which can compensate for loss of talin1, and demonstrate for the first time that endothelial cells in vivo lacking talin1 are unable to undergo the cell spreading and flattening required to form vessels.

Viral and bacterial minigene products are presented by MHC class I molecules with similar efficiencies

MHC class I molecules present short peptides, usually 8–10 amino acids in length, to CD8+ T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein.

The severity of phenotype linked to SUCLG1 mutations could be correlated with residual amount of SUCLG1 protein

BackgroundSuccinate-CoA ligase deficiency is responsible for encephalomyopathy with mitochondrial DNA depletion and mild methylmalonic aciduria. Mutations in SUCLA2, the gene encoding a β subunit of succinate-CoA ligase, have been reported...

BAC TG-EMBED: one-step method for high-level, copy-number-dependent, position-independent transgene expression

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.

Long-term Physiologically Regulated Expression of the Low-density Lipoprotein Receptor In Vivo Using Genomic DNA Mini-gene Constructs

Familial hypercholesterolemia (FH) is a condition caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Expression of LDLR is highly regulated and excess receptor expression is cytotoxic. To incorporate essential gene regulation into a gene therapy vector for FH, we generated vectors in which the expression of therapeutic human LDLR gene, or luciferase reporter gene, is driven by 10 kb of human LDLR genomic DNA encompassing the promoter region including elements essential for physiologically regulated expression. Using luciferase expression and specific LDL binding and internalization assays, we have shown in vitro that the genomic promoter element confers long-term, physiologically regulated gene expression and complementation of receptor deficiency in culture for 240 cell-generations. This was demonstrated in the presence of sterols or statins, modifiers of LDLR promoter activity. In vivo, we demonstrate efficient liver-specific delivery and expression of luciferase following hydrodynamic tail-vein injection and confirm that expression from the LDLR promoter element is sensitive to statin administration. We also demonstrate long-term LDLR expression from the 10-kb promoter element up to 9 months following delivery. The vector system that we describe provides the efficient delivery, long-term expression, and physiological regulation required for a successful gene therapy intervention for FH.

Impaired M3 Muscarinic Acetylcholine Receptor Signal Transduction Through Blockade of Binding of Multiple Proteins to its Third Intracellular Loop

Several motifs found in the third intracellular loop of the M₃ muscarinic receptor are critical for G protein activation and scaffold protein interaction. However, how multiprotein complexes form is not fully understood. A minigene encoding the third intracellular loop of the M₃ muscarinic receptor was constructed to explore whether peptides from this intracellular region could act as inhibitors of the muscarinic multiprotein complex formation and signaling. We found that this construct, when co-expressed with the M₃ receptor, has the ability to act as a competitive antagonist of G protein receptors and receptor-scaffold/accessory proteins. Transient transfection of human embryonic kidney-293 cells with DNA encoding the human M₃ and M₅ receptor subtypes results in a carbachol-dependent increase of inositol phosphate. Co-expression of the M₃ third cytoplasmic loop minigene dramatically reduces both carbachol-mediated G protein activation and inositol phosphate accumulation. Minigene expression also abrogates activation of M₃ and M₅ receptor mitogen-activated protein kinases pathway. Furthermore, minigene expression led to reduced AKT activation. These data, together with results of co-immunoprecipitation of different scaffold and kinase proteins, provide experimental evidence for the role for the third cytoplasmic loop of the human M₃ muscarinic receptor in G-protein activation and multiprotein complex formation.

Molecular analysis of congenital goitres with hypothyroidism caused by defective thyroglobulin synthesis. Identification of a novel c.7006C&gt;T [p.R2317X] mutation and expression of minigenes containing nonsense mutations in exon 7

Thyroglobulin (TG) deficiency is an autosomal-recessive disorder that results in thyroid dyshormonogenesis. A number of distinct mutations have been identified as causing human hypothyroid goitre. The purpose of this study was to identify and characterize new mutations in the TG gene in an attempt to increase the understanding of the genetic mechanism responsible for this disorder. A total of six patients from four nonconsanguineous families with marked impairment of TG synthesis were studied. Single-strand conformation polymorphism (SSCP) analysis, sequencing of DNA, genotyping, expression of chimeric minigenes and bioinformatic analysis were performed. Four different inactivating TG mutations were identified: one novel mutation (c.7006C>T [p.R2317X]) and three previously reported (c.886C>T [p.R277X], c.6701C>A [p.A2215D] and c.6725G>A [p.R2223H]). Consequently, one patient carried a compound heterozygous for p.R2223H/p.R2317X mutations; two brothers showed a homozygous p.A2215D substitution and the remaining three patients, from two families with typical phenotype, had a single p.R277X mutated allele. We also showed functional evidences that premature stop codons inserted at different positions in exon 7, which disrupt exonic splicing enhancer (ESE) sequences, do not interfere with exon definition and processing. In this study, we have identified a novel nonsense mutation p.R2317X in the acetylcholinesterase homology domain of TG. We have also observed that nonsense mutations do not interfere with the pre-mRNA splicing of exon 7. The results are in accordance with previous observations confirming the genetic heterogeneity of TG defects.

HnRNP M interacts with PSF and p54 nrb and co-localizes within defined nuclear structures

The abundant heterogeneous nuclear ribonucleoprotein M (hnRNP M) is able to associate with early spliceosomes and to influence splicing patterns of specific pre-mRNAs. Here, by a combination of immunoprecipitation and pull-down assays, we have identified PSF (polypyrimidine tract-binding protein-associated splicing factor) and p54 nrb, two highly related proteins involved in transcription and RNA processing, as new binding partners of hnRNP M. HnRNP M was found to co-localize with PSF within a subset of nuclear paraspeckles and to largely co-fractionate with PSF and p54 nrb in biochemical nuclear matrix preparations. In cells transfected with an alternatively spliced preprotachykinin (PPT) minigene expression of hnRNP M promoted exon skipping while expression of PSF favours exon inclusion. The latter effect was reverted specifically by co-expressing the full length hnRNP M or a deletion mutant capable of interaction with PSF and p54 nrb. Together our data provide new insights and some functional implications on the hnRNP M network of interactions.

Restoration of sarcolemmal nNOS is essential to normalize α‐adrenoceptor control of muscle blood flow in transgenic mdx mice

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle, where it is localized to the sarcolemma via the dystrophin glycoprotein complex (DGC). We previously reported that nNOS‐derived NO blunts α‐adrenergic vasoconstriction in contracting muscle and that this protective effect is absent in boys with muscular dystrophy and in mdx mice in which mutations in the dystrophin gene disrupt the DGC and displace nNOS. We therefore hypothesized that restoration of sarcolemmal nNOS is essential to rescue the normal contraction‐induced attenuation of α‐adrenergic vasoconstriction. In the resting hindlimbs of transgenic mdx mice expressing minidystrophin genes that restore the DGC with (ΔH2‐R15) or without (ΔH2‐R19) restoring nNOS, intraarterial norepinephrine evoked similar decreases in femoral blood flow and vascular conductance. These constrictor responses were markedly attenuated by 81±3% in the contracting hindlimbs of the ΔH2‐R15 transgenics (p&lt;0.05 vs. rest; n=8), but by only 16±4% in the ΔH2‐R19 transgenics (p&gt;0.05 vs. rest; n=6) and by 10±5% in transgene negative littermates (n=3). Minigene expression yielded similar results in mdx4cv mice, which have a different dystrophin mutation and genetic background than mdx mice. These data indicate that recovery of sarcolemmal nNOS is essential to restore normal blood flow regulation in mdx skeletal muscle. Supported by the NIH and MDA.

Functional analysis of three splicing mutations identified in the PMM2 gene: Toward a new therapy for congenital disorder of glycosylation type Ia

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N-glycosylation. The most prevalent form of CDG-type Ia-is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256-1G>C and c.640-9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640-15479C>T). Cell-based splicing assays strongly suggest that all these are disease-causing splicing mutations. The c.256-1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640-9T>G mutation was found responsible for the activation of a cryptic intronic splice-site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine-rich (SR) proteins. Finally, the deep intronic change c.640-15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation-specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen.

Cardiac Expression of a Mini-dystrophin That Normalizes Skeletal Muscle Force Only Partially Restores Heart Function in Aged Mdx Mice

Duchenne muscular dystrophy (DMD) affects both skeletal and cardiac muscle. It is currently unclear whether the strategies developed for skeletal muscle can ameliorate cardiomyopathy. Synthetic mini-/micro-dystrophin genes have yielded impressive skeletal muscle protection in animal models. The 6-kb DeltaH2-R19 minigene is particularly promising because it completely restores skeletal muscle force to wild-type levels. Here, we examined whether expressing this minigene in the heart, but not skeletal muscle, could normalize cardiac function in the mdx model of DMD cardiomyopathy. Transgenic mdx mice were generated to express the DeltaH2-R19 minigene under the control of the alpha-myosin heavy-chain promoter. Heart structure and function were examined in adult and very old mice. The DeltaH2-R19 minigene enhanced cardiomyocyte sarcolemmal strength and prevented myocardial fibrosis. It also restored the dobutamine response and enhanced treadmill performance. Surprisingly, heart-restricted DeltaH2-R19 minigene expression did not completely normalize electrocardiogram and hemodynamic abnormalities. Overall, systolic function and ejection fraction were restored to normal levels but stroke volume and cardiac output remained suboptimal. Our results demonstrate that the skeletal muscle-proven DeltaH2-R19 minigene can correct cardiac histopathology but cannot fully normalize heart function. Novel strategies must be developed to completely restore heart function in DMD.