scholarly journals Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients

2020 ◽  
Vol 21 (10) ◽  
pp. 3555
Author(s):  
Abdul Qawee Mahyoob Rani ◽  
Tetsushi Yamamoto ◽  
Tatsuya Kawaguchi ◽  
Kazuhiro Maeta ◽  
Hiroyuki Awano ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Alternative Polyadenylation ◽  
Coding Region ◽  
Terminal Exon ◽  
Potential Implication ◽  
Human Genes ◽  
Cardiac Muscles ◽  
Rapid Amplification ◽  
Dmd Gene ◽  
Ecg Abnormalities

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.

scholarly journals Murine erythropoietin gene: cloning, expression, and human gene homology.

1986 ◽  
Vol 6 (3) ◽  
pp. 849-858 ◽  
Author(s):  
C B Shoemaker ◽  
L D Mitsock
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Cdna Probe ◽  
Amino Acid Sequences ◽  
Nucleotide Sequence Analysis ◽  
Coding Region ◽  
Transcription Control ◽  
Erythroid Progenitor ◽  
Human Genes ◽  
Cell Expression

The gene for murine erythropoietin (EPO) was isolated from a mouse genomic library with a human EPO cDNA probe. Nucleotide sequence analysis permitted the identification of the murine EPO coding sequence and the prediction of the encoded amino acid sequence based on sequence conservation between the mouse and human EPO genes. Both the coding DNA and the amino acid sequences were 80% conserved between the two species. Transformation of COS-1 cells with a mammalian cell expression vector containing the murine EPO coding region resulted in secretion of murine EPO with biological activity on both murine and human erythroid progenitor cells. The transcription start site for the murine EPO gene in kidneys was determined. This permitted tentative identification of the transcription control region. The region included 140 base pairs upstream of the cap site which was over 90% conserved between the murine and human genes. Surprisingly, the first intron and much of the 5'- and 3'-untranslated sequences were also substantially conserved between the genes of the two species.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

scholarly journals Induced Pluripotent Stem Cells for Duchenne Muscular Dystrophy Modeling and Therapy

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 253 ◽  
Author(s):  
Lubos Danisovic ◽  
Martina Culenova ◽  
Maria Csobonyeiova
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Reprogramming ◽  
Progressive Degeneration ◽  
Cardiac Muscles ◽  
Dmd Gene ◽  
Induced Pluripotent

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder, caused by mutation of the DMD gene which encodes the protein dystrophin. This dystrophin defect leads to the progressive degeneration of skeletal and cardiac muscles. Currently, there is no effective therapy for this disorder. However, the technology of cell reprogramming, with subsequent controlled differentiation to skeletal muscle cells or cardiomyocytes, may provide a unique tool for the study, modeling, and treatment of Duchenne muscular dystrophy. In the present review, we describe current methods of induced pluripotent stem cell generation and discuss their implications for the study, modeling, and development of cell-based therapies for Duchenne muscular dystrophy.

Duchenne muscular dystrophy: genome editing gives new hope for treatment

2018 ◽  
Vol 94 (1111) ◽  
pp. 296-304 ◽  
Author(s):  
Vassili Crispi ◽  
Antonios Matsakas
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Genetic Diseases ◽  
Wasting Disease ◽  
Cardiac Muscles ◽  
Dmd Gene ◽  
Current Lines ◽  
Dystrophin Protein ◽  
Potential Therapeutics

Duchenne muscular dystrophy (DMD) is a progressive wasting disease of skeletal and cardiac muscles, representing one of the most common recessive fatal inherited genetic diseases with 1:3500–1:5000 in yearly incidence. It is caused by mutations in the DMD gene that encodes the membrane-associated dystrophin protein. Over the years, many have been the approaches to management of DMD, but despite all efforts, no effective treatment has yet been discovered. Hope for the development of potential therapeutics has followed the recent advances in genome editing and gene therapy. This review gives an overview to DMD and summarises current lines of evidence with regard to treatment and disease management alongside the appropriate considerations.

scholarly journals Multiplex analysis of mitochondrial DNA pathogenic and polymorphic sequence variants

2010 ◽  
Vol 391 (10) ◽  
Author(s):  
Jason C. Poole ◽  
Vincent Procaccio ◽  
Martin C. Brandon ◽  
Greg Merrick ◽  
Douglas C. Wallace
Keyword(s):  
Mitochondrial Dna ◽  
Pcr Amplification ◽  
Integrated System ◽  
Sequence Variants ◽  
Coding Region ◽  
Single Nucleotide Variants ◽  
Standard Curve ◽  
Mtdna Mutations ◽  
Pathogenic Mutations ◽  
Optimal Extension

Abstract The mitochondrial DNA (mtDNA) encompasses two classes of functionally important sequence variants: recent pathogenic mutations and ancient adaptive polymorphisms. To rapidly and cheaply evaluate both classes of single nucleotide variants (SNVs), we have developed an integrated system in which mtDNA SNVs are analyzed by multiplex primer extension using the SNaPshot system. A multiplex PCR amplification strategy was used to amplify the entire mtDNA, a computer program identifies optimal extension primers, and a complete global haplotyping system is also proposed. This system genotypes SNVs on multiplexed mtDNA PCR products or directly from enriched mtDNA samples and can quantify heteroplasmic variants down to 0.8% using a standard curve. With this system, we have developed assays for testing the common pathogenic mutations in four multiplex panels: two genotype the 13 most common pathogenic mtDNA mutations and two genotype the 10 most common Leber Hereditary Optic Neuropathy mutations along with haplogroups J and T. We use a hierarchal system of 140 SNVs to delineate the major global mtDNA haplogroups based on a global phylogenetic tree of coding region polymorphisms. This system should permit rapid and inexpensive genotyping of pathogenic and lineage-specific mtDNA SNVs by clinical and research laboratories.

scholarly journals NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing

2015 ◽  
Vol 90 (4) ◽  
pp. 1718-1728 ◽  
Author(s):  
Olufemi O. Fasina ◽  
Yanming Dong ◽  
David J. Pintel
Keyword(s):  
Rna Processing ◽  
Alternative Polyadenylation ◽  
Genome Replication ◽  
Coding Region ◽  
Content Type ◽  
Mrna Transcripts ◽  
Viral Genome Replication ◽  
Minute Virus ◽  
Polyadenylation Sites ◽  
The Right

ABSTRACTMinute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genusBocaparvovirusof theParvovirinaewhich we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3′-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site.IMPORTANCETheParvovirinaeare small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genusBocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.

NHE3 in an ancestral vertebrate: primary sequence, distribution, localization, and function in gills

2005 ◽  
Vol 289 (5) ◽  
pp. R1520-R1534 ◽  
Author(s):  
Keith P. Choe ◽  
Akira Kato ◽  
Shigehisa Hirose ◽  
Consuelo Plata ◽  
Aleksandra Sindić ◽  
...  
Keyword(s):  
Complete Sequence ◽  
Coding Region ◽  
Degenerate Primers ◽  
Acid Protein ◽  
Rapid Amplification ◽  
And Function ◽  
Apical Membranes ◽  
Carboxyl Tail ◽  
Ancestral Vertebrate ◽  
Amino Acid Protein

In mammals, the Na+/H+ exchanger 3 (NHE3) is expressed with Na+/K+-ATPase in renal proximal tubules, where it secretes H+ and absorbs Na+ to maintain blood pH and volume. In elasmobranchs (sharks, skates, and stingrays), the gills are the dominant site of pH and osmoregulation. This study was conducted to determine whether epithelial NHE homologs exist in elasmobranchs and, if so, to localize their expression in gills and determine whether their expression is altered by environmental salinity or hypercapnia. Degenerate primers and RT-PCR were used to deduce partial sequences of mammalian NHE2 and NHE3 homologs from the gills of the euryhaline Atlantic stingray ( Dasyatis sabina). Real-time PCR was then used to demonstrate that mRNA expression of the NHE3 homolog increased when stingrays were transferred to low salinities but not during hypercapnia. Expression of the NHE2 homolog did not change with either treatment. Rapid amplification of cDNA was then used to deduce the complete sequence of a putative NHE3. The 2,744-base pair cDNA includes a coding region for a 2,511-amino acid protein that is 70% identical to human NHE3 (SLC9A3). Antisera generated against the carboxyl tail of the putative stingray NHE3 labeled the apical membranes of Na+/K+-ATPase-rich epithelial cells, and acclimation to freshwater caused a redistribution of labeling in the gills. This study provides the first NHE3 cloned from an elasmobranch and is the first to demonstrate an increase in gill NHE3 expression during acclimation to low salinities, suggesting that NHE3 can absorb Na+ from ion-poor environments.

Simple PCR Amplification of the Entire Glucocerebrosidase Gene (GBA) Coding Region for Diagnostic Sequence Analysis

DNA Sequence ◽  
1998 ◽  
Vol 8 (6) ◽  
pp. 349-356 ◽  
Author(s):  
Ulrich Finckh ◽  
Pavel Seeman ◽  
Oldrik Cardinal von Widdern ◽  
Arndt Rolfs
Keyword(s):  
Sequence Analysis ◽  
Pcr Amplification ◽  
Coding Region ◽  
Glucocerebrosidase Gene

scholarly journals Fate of genetically modified maize DNA in the oral cavity and rumen of sheep

2003 ◽  
Vol 89 (2) ◽  
pp. 159-166 ◽  
Author(s):  
Paula S. Duggan ◽  
Philip A. Chambers ◽  
John Heritage ◽  
J. Michael Forbes
Keyword(s):  
Biological Activity ◽  
Oral Cavity ◽  
Rumen Fluid ◽  
Genetically Modified ◽  
Pcr Amplification ◽  
Oral Bacteria ◽  
Target Sequence ◽  
Genetically Modified Maize ◽  
Coding Region ◽  
Maize Line

The polymerase chain reaction (PCR) technique was used to investigate the fate of a transgene in the rumen of sheep fed silage and maize grains from an insect-resistant maize line. A 1914-bp DNA fragment containing the entire coding region of the syntheticcryIA(b) gene was still amplifiable from rumen fluid sampled 5 h after feeding maize grains. The same target sequence, however, could not be amplified from rumen fluid sampled from sheep fed silage prepared from the genetically modified maize line. PCR amplification of a shorter (211-bp), yet still highly specific, target sequence was possible with rumen fluid sampled up to 3 and 24 h after feeding silage and maize grains, respectively. These findings indicate that intact transgenes from silage are unlikely to survive significantly in the rumen since a DNA sequence 211-bp long is very unlikely to transmit genetic information. By contrast, DNA in maize grains persists for a significant time and may, therefore, provide a source of transforming DNA in the rumen. In addition, we have examined the biological activity of plasmid DNA that had previously been exposed to the ovine oral cavity. Plasmid extracted from saliva sampled after incubation for 8 min was still capable of transforming competentEscherichia colito kanamycin resistance, implying that DNA released from the diet within the mouth may retain sufficient biological activity for the transformation of competent oral bacteria.

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