scholarly journals Combining Protein Expression and Molecular Data Improves Mutation Characterization of Dystrophinopathies

2021 ◽  
Vol 12 ◽  
Author(s):  
Gisela Gaina ◽  
Rolf H. A. M. Vossen ◽  
Emilia Manole ◽  
Doina Anca Plesca ◽  
Elena Ionica
Keyword(s):  
Protein Expression ◽  
Copy Number Variants ◽  
Point Mutations ◽  
Becker Muscular Dystrophy ◽  
Molecular Data ◽  
Inherited Disorders ◽  
Reading Frame ◽  
Hrm Analysis ◽  
Dystrophin Protein

Duchenne and Becker muscular dystrophy are X-linked recessive inherited disorders characterized by progressive weakness due to skeletal muscle degeneration. Different mutations in the DMD gene, which encodes for dystrophin protein, are responsible for these disorders. The aim of our study was to investigate the relationship between type, size, and location of the mutation that occurs in the DMD gene and their effect on dystrophin protein expression in a cohort of 40 male dystrophinopathy patients and nine females, possible carriers. We evaluated the expression of dystrophin by immunofluorescence and immunoblotting. The mutational spectrum of the DMD gene was established by MLPA for large copy number variants, followed by HRM analysis for point mutations and sequencing of samples with an abnormal melting profile. MLPA revealed 30 deletions (75%) and three duplications (7.5%). HRM analysis accounted for seven-point mutations (17.5%). We also report four novel small mutations (c. 8507G>T, c.3021delG, c.9563_9563+1insAGCATGTTTATGATACAGCA, c.7661-60T>A) in DMD gene. Our work shows that the DNA translational open reading frame and the location of the mutation both influence the expression of dystrophin and disease severity phenotype. The proposed algorithm used in this study demonstrates its accuracy for the characterization of dystrophinopathy patients.

scholarly journals Characterization of NOL7 Gene Point Mutations, Promoter Methylation, and Protein Expression in Cervical Cancer

2012 ◽  
Vol 31 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Colleen L. Doçi ◽  
Tanmayi P. Mankame ◽  
Alexander Langerman ◽  
Kelly R. Ostler ◽  
Rajani Kanteti ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Protein Expression ◽  
Promoter Methylation ◽  
Point Mutations

scholarly journals Structural Perturbations of Exon Skipping Edits within the Dystrophin D20:24 Region

2020 ◽  
Author(s):  
Xin Niu ◽  
Nick Menhart
Keyword(s):  
Protein Expression ◽  
Genetic Defect ◽  
Exon Skipping ◽  
Dystrophin Gene ◽  
Protein Domain ◽  
Reading Frame ◽  
Structural Perturbations ◽  
Protein Domain Structure ◽  
And Function ◽  
Dystrophin Protein

AbstractExon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from inclusion in the mature mRNA. Of course, this also results in loss of the corresponding region from the cognate protein, which is one possible therapeutic aim. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame in the dystrophin gene, creating a frameshift and abolishing protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is the restoration of the reading frame by skipping an exon adjacent to the patient’s original defect. However, the therapeutically expressed protein is of course edited, and missing both the region of the underlying genetic defect, as well as the therapeutically skipped exon. While restoring some protein expression is good, how removing some region from the middle of a protein effects its structure and function is unclear. Complicating this in the case of DMD is the fact that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are known, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. What has been unclear is whether and how exon editing can be done to minimize these perturbations. In this study we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein, and identify for the first time, exon edits that appear to maintain structural stability similar to wildtype protein. We also identify factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the underlying exon structure interacts with the protein domain structure.

scholarly journals Characterization of a Baculovirus Alkaline Nuclease

2000 ◽  
Vol 74 (14) ◽  
pp. 6401-6407 ◽  
Author(s):  
Lulin Li ◽  
George F. Rohrmann
Keyword(s):  
Essential Gene ◽  
Point Mutations ◽  
Reading Frame ◽  
Baculovirus Gene ◽  
Salt Requirement ◽  
Low Levels ◽  
Alkaline Nuclease ◽  
Maximal Expression ◽  
Rule Out

ABSTRACT All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in theHerpesviridae. In this report we describe the characterization of the alkaline nuclease (AN) homolog of theAutographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg2+ over Mn2+, had optimal activity at 35°C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.

scholarly journals Characterization of the Genetic Diversity Present in a Diverse Sesame Landrace Collection Based on Phenotypic Traits and EST-SSR Markers Coupled With an HRM Analysis

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 656
Author(s):  
Evangelia Stavridou ◽  
Georgios Lagiotis ◽  
Parthena Kalaitzidou ◽  
Ioannis Grigoriadis ◽  
Irini Bosmali ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Geographical Origin ◽  
Expressed Sequence Tag ◽  
Phenotypic Diversity ◽  
Molecular Data ◽  
Phenotypic Traits ◽  
Dissimilarity Matrix ◽  
Heterotic Groups ◽  
Hrm Analysis ◽  
Sesame Genotypes

A selection of sesame (Sesamum indicum L.) landraces of different eco-geographical origin and breeding history have been characterized using 28 qualitative morpho-physiological descriptors and seven expressed sequence tag-simple sequence repeat (EST-SSR) markers coupled with a high-resolution melting (HRM) analysis. The most variable qualitative traits that could efficiently discriminate landraces, as revealed by the correlation analyses, were the plant growth type and position of the branches, leaf blade width, stem pubescence, flowering initiation, capsule traits and seed coat texture. The agglomerative hierarchical clustering analysis based on a dissimilarity matrix highlighted three main groups among the sesame landraces. An EST-SSR marker analysis revealed an average polymorphism information content (PIC) value of 0.82, which indicated that the selected markers were highly polymorphic. A principal coordinate analysis and dendrogram reconstruction based on the molecular data classified the sesame genotypes into four major clades. Both the morpho-physiological and molecular analyses showed that landraces from the same geographical origin were not always grouped in the same cluster, forming heterotic groups; however, clustering patterns were observed for the Greek landraces. The selective breeding of such traits could be employed to unlock the bottleneck of local phenotypic diversity and create new cultivars with desirable traits.

scholarly journals Characterization of synthetic riboswitch in cell-free protein expression systems

RNA Biology ◽  
2021 ◽  
pp. 1-12
Author(s):  
Yaroslav Chushak ◽  
Svetlana Harbaugh ◽  
Kathryn Zimlich ◽  
Bryan Alfred ◽  
Jorge Chávez ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Systems ◽  
Free Protein

scholarly journals Characterization of CRISPR Spacer and Protospacer Sequences in Paenibacillus larvae and Its Bacteriophages

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 459
Author(s):  
Casey Stamereilers ◽  
Simon Wong ◽  
Philippos K. Tsourkas
Keyword(s):  
Comparative Studies ◽  
Point Mutations ◽  
Paenibacillus Larvae ◽  
Bacterial Disease ◽  
American Foulbrood ◽  
Antibiotic Resistant ◽  
Future Studies ◽  
Complete Genomes ◽  
Point Of Reference

The bacterium Paenibacillus larvae is the causative agent of American foulbrood, the most devastating bacterial disease of honeybees. Because P. larvae is antibiotic resistant, phages that infect it are currently used as alternative treatments. However, the acquisition by P. larvae of CRISPR spacer sequences from the phages could be an obstacle to treatment efforts. We searched nine complete genomes of P. larvae strains and identified 714 CRISPR spacer sequences, of which 384 are unique. Of the four epidemiologically important P. larvae strains, three of these have fewer than 20 spacers, while one strain has over 150 spacers. Of the 384 unique spacers, 18 are found as protospacers in the genomes of 49 currently sequenced P. larvae phages. One P. larvae strain does not have any protospacers found in phages, while another has eight. Protospacer distribution in the phages is uneven, with two phages having up to four protospacers, while a third of phages have none. Some phages lack protospacers found in closely related phages due to point mutations, indicating a possible escape mechanism. This study serve a point of reference for future studies on the CRISPR-Cas system in P. larvae as well as for comparative studies of other phage–host systems.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Molecular cloning and analysis of l(1)ogre, a locus of Drosophila melanogaster with prominent effects on the postembryonic development of the central nervous system.

Genetics ◽  
1990 ◽  
Vol 126 (4) ◽  
pp. 1033-1044 ◽  
Author(s):  
T Watanabe ◽  
D R Kankel
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Postembryonic Development ◽  
P Element ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
The Central Nervous System ◽  
Highly Hydrophobic ◽  
Conceptual Translation

Abstract Previous genetic studies have shown that wild-type function of the l(1)ogre (lethal (1) optic ganglion reduced) locus is essential for the generation and/or maintenance of the postembryonic neuroblasts including those from which the optic lobe is descended. In the present study molecular isolation and characterization of the l(1)ogre locus was carried out to study the structure and expression of this gene in order to gain information about the nature of l(1)ogre function and its relevance to the development of the central nervous system. About 70 kilobases (kb) of genomic DNA were isolated that spanned the region where l(1)ogre was known to reside. Southern analysis of a l(1)ogre mutation and subsequent P element-mediated DNA transformation mapped the l(1)ogre+ function within a genomic fragment of 12.5 kb. Northern analyses showed that a 2.9-kb message transcribed from this 12.5-kb region represented l(1)ogre. A 2.15-kb portion of a corresponding cDNA clone was sequenced. An open reading frame (ORF) of 1,086 base paris was found, and a protein sequence of 362 amino acids with one highly hydrophobic segment was deduced from conceptual translation of this ORF.

Immunohistochemical Detection of Cancer-Testis Antigen PRAME

2021 ◽  
pp. 106689692110120
Author(s):  
Cecilia Lezcano ◽  
Annette M. Müller ◽  
Denise Frosina ◽  
Enmily Hernandez ◽  
Jerica A. Geronimo ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Data ◽  
Immunohistochemical Detection ◽  
Normal Tissues ◽  
Variable Extent ◽  
Formalin Fixed Paraffin Embedded ◽  
Myxoid Liposarcomas ◽  
Fetal Ovary ◽  
Ct Antigens ◽  
Cancer Testis

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.

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