Mutations In Different Domains Research Articles

Computational analysis of missense mutations in dystrophin protein: Insights into domain-specific effects and functional implications

BackgroundDystrophin is a structural protein primarily found in skeletal muscles that connects trans-membrane component of dystrophin-glycoprotein complex to the intracellular cytoskeleton network. N-terminal domain of dystrophin binds to F-actin and its C-terminal domain attaches to dystrophin-associated complex (DAG) in the membrane. It serves as the bridge connecting the extracellular matrix to the actin based cytoskeleton of muscle cells across the plasma membrane. This complex works together to strengthen muscle fibers and shield them from damage as they contract and relax. ObjectiveOur Objective is to investigate the different pathogenic mutations in four distinct domains of dystrophin protein and to decipher how these mutations impact protein structure and function. In this study, we investigated the impact of disease causing missense mutations in isoforms of dystrophin – P.11532–1 (K18N.A165V, D3187G, F3228L); P11532–4 (Y223N, D3179G) and P-11532-11 (Y227N,D3183G).Occurrence of pathogenic mutations in dystrophin might compromise structural stability, interfere with protein-protein interactions or alter cellular signaling pathways collectively. All these can contribute to the progressive muscle degeneration observed in Duchene or Becker muscular dystrophy(DMD or BMD) and X-lined dilated cardiomyopathy(CMD3B) affected individuals. MethodsEvolutionary conservation analysis using multiple sequence alignment followed by pathogenicity prediction using web based server was performed. Homology modeling of mutants was performed bySWISS MODEL a fully automated homology-modeling server, accessible through the Expasy web server. These models were then analyzed using various servers such as Dynamut. Pymol was used for visualization and structural analysis. ResultsAnalyzed mutations cause structural instability by either gaining or losing specific interactions involved in the folding of protein. Loss of pi-pi stacking interactions has the potential to significant impact the overall stability of the protein. It is important to note that the majority of the mutations lead to stability defects rather than functional defects, ultimately contributing to different forms of muscular dystrophy. ConclusiónThe mutations in different domains of Dystrophin protein significantly alters the protein structure. This may in turn impact its ability to interact with other proteins. Understanding the specific impacts of these mutations can pave the way for development of targeted therapies that aims at restoring functionality of dystrophin and ameliorating the debilitating effects of muscular dystrophy.

Genetic analysis of IRF2BPL in a Taiwanese dystonia cohort: The genotype and phenotype correlation.

IRF2BPL mutation has been associated with a rare neurodevelopmental disorder with abnormal movements, including dystonia. However, the role of IRF2BPL in dystonia remains elusive. We aimed to investigate IRF2BPL mutations in a Taiwanese dystonia cohort. A total of 300 unrelated patients with molecularly unassigned isolated (n = 256) or combined dystonia (n = 44) were enrolled between January 2015 and July 2023. The IRF2BPL variants were analyzed based on whole exome sequencing. The in silico prediction of the identified potential pathogenic variant was performed to predict its pathogenicity. We also compared the clinical and genetic features to previous literature reports. We identified one adolescent patient carrying a de novo heterozygous pathogenic variant of IRF2BPL, c.379C>T (p.Gln127Ter), who presented with generalized dystonia, developmental regression, and epilepsy (0.33% of our dystonia cohort). This variant resides within the polyglutamine (poly Q) domain before the first PEST sequence block of the IRF2BPL protein, remarkably truncating the protein structure. Combined with other patients with IRF2BPL mutations in the literature (n = 60), patients with variants in the poly Q domain have a higher rate of nonsense mutations (p < 0.001) and epilepsy (p = 0.008) than patients with variants in other domains. Furthermore, as our index patient, carriers with substitutions before the first PEST sequence block have significantly older age of onset (p < 0.01) and higher non-epilepsy symptoms, including generalized dystonia (p = 0.003), and ataxia (p = 0.003). IRF2BPL mutation is a rare cause of dystonia in our population. Mutations in different domains of IRF2BPL exhibit different phenotypes.

PATH-67. SHH/GLI3 SIGNALOPATHIES AND NEURO-ONCO-GENETIC COUNSELING : A TUNISIAN PEDIGREE

Abstract Here, we present a 3-generation pedigree of a Tunisian non-consanguineous family with six offspring affected by heterogeneous forms of the GLI3-related PHS spectrum. Our aim was to assess the neuro-onco-genetic risks and provide counseling for appropriate clinical follow-up. A non-consanguineous couple was referred to our genetic counseling due to the termination of their first pregnancy, which was diagnosed with multiple congenital anomalies. An autopsy examination, including pathological and radiographic analysis, was conducted. Furthermore, a medical genetic assessment considering a family history spanning over three generations as well as cytogenetic analysis were carried out. The index case was diagnosed with GLI3-related PHS based on facial dysmorphic features, auricular malformations, and unilateral postaxial toe syndactyly, along with brain macro-calcifications. The index case's father and his brother exhibited facial dysmorphic features. Furthermore, two cousins from one of their paternal uncles presented another form, associated respectively with unilateral renal agenesis and unilateral hand agenesis. The remaining three cases, who belong to the first generation, and passed away during infancy, exhibited the same condition, associated to congenital heart defect in one of them. The heterogeneity of GLI3-related overlapping conditions (SHH/GLI3 signalopathies) result from the location of GLI3 mutations in different domains of the gene, as well as the resulting functional balance between activating and repressive forms of GLI3 during development. The report of a late diagnosis of GLI3-related PHS following the discovery of hypothalamic hamartomas as well as the description of a new nonsense germline mutation in a child with Greig syndrome and medulloblastoma, support that neuro-oncological investigation of GLI3 families could be beneficial.

Novel MKRN3 Missense Mutations Associated With Central Precocious Puberty Reveal Distinct Effects on Ubiquitination.

Loss-of-function mutations in the maternally imprinted genes, MKRN3 and DLK1, are associated with central precocious puberty (CPP). Mutations in MKRN3 are the most common known genetic etiology of CPP. This work aimed to screen patients with CPP for MKRN3 and DLK1 mutations and analyze the effects of identified mutations on protein function in vitro. Participants included 84 unrelated children with CPP (79 girls, 5 boys) and, when available, their first-degree relatives. Five academic medical institutions participated. Sanger sequencing of MKRN3 and DLK1 5' upstream flanking and coding regions was performed on DNA extracted from peripheral blood leukocytes. Western blot analysis was performed to assess protein ubiquitination profiles. Eight heterozygous MKRN3 mutations were identified in 9 unrelated girls with CPP. Five are novel missense mutations, 2 were previously identified in patients with CPP, and 1 is a frameshift variant not previously associated with CPP. No pathogenic variants were identified in DLK1. Girls with MKRN3 mutations had an earlier age of initial pubertal signs and higher basal serum luteinizing hormone and follicle-stimulating hormone compared to girls with CPP without MRKN3 mutations. Western blot analysis revealed that compared to wild-type MKRN3, mutations within the RING finger domain reduced ubiquitination whereas the mutations outside this domain increased ubiquitination. MKRN3 mutations were present in 10.7% of our CPP cohort, consistent with previous studies. The novel identified mutations in different domains of MKRN3 revealed different patterns of ubiquitination, suggesting distinct molecular mechanisms by which the loss of MRKN3 results in early pubertal onset.

In Silico Analyses of All STAT3 Missense Variants Leading to Explore Divergent AD-HIES Clinical Phenotypes.

Autosomal dominant hyper-IgE syndrome (AD-HIES) is linked to dominant negative mutations of the STAT3 protein whose molecular basis for dysfunction is unclear and presenting with a variety of clinical manifestations with only supportive treatment. To establish the relationship between the impact of STAT3 mutations in different domains and the severity of the clinical manifestations, 105 STAT3 mutations were analyzed for their impact on protein stability, flexibility, function, and binding affinity using in Silico approaches. Our results showed that 73% of the studied mutations have an impact on the physicochemical properties of the protein, altering the stability, flexibility and function to varying degrees. In particular, mutations affecting the DNA binding domain (DBD) and the Src Homology 2 (SH2) have a significant impact on the protein structure and disrupt its interaction either with DNA or other STAT3 to form a heterodomain complex, leading to severe clinical phenotypes. Collectively, this study suggests that there is a close relationship between the domain involving the mutation, the degree of variation in the properties of the protein and the degree of loss of function ranging from partial loss to complete loss, explaining the variability of clinical manifestations between mild and severe.

Genetic landscape of FOXC2 mutations in lymphedema-distichiasis syndrome: Different mechanism of pathogenicity for mutations in different domains

Lymphedema-dissociated syndrome (LDS), of which the pathogenesis is not fully understood, afflicts many patients. In this study, we investigated the effect of FOXC2 gene loss-of-function on the development of LDS disease. Two Han Chinese families with LDS were recruited in this study, pathogenic mutations were identified by Sanger sequencing. Reverse-transcription PCR, subcellular localization, dual fluorescein enzymes, and other in vitro experiments were used to study the functional effects of eight FOXC2 mutations. Two pathogenic FOXC2 duplication mutations (c.930_936dup and c.931-937dup) were identified in the two families. Both mutations caused uneven distribution in the nucleus and a chromatin contraction phenotype, weakening the DNA binding activity and transcription activity. We then performed functional analysis on six additional mutations in different domains of FOXC2 that were reported to cause LDS. We found mutations located in the forkhead domain and central region dramatically reduced the transactivation ability, while mutations in activation domain-2 enhanced this ability. All 8 mutations down-regulated the transcription of ANGPT2 and affected the activity of the ERK-RAS pathway, which may cause abnormal formation of lymphatic vessels. Our findings also showed that all 8 mutations decreased the ability of interaction between FOXC2 and the Wnt4 promoter, suggesting mutations in FOXC2 may also affect the Wnt4-Frizzled-RYK signaling pathway, leading the abnormal differentiation of the meibomian glands into hair follicle cells during the embryonic period and causing distichiasis. This study expanded and revealed the potential pathogenesis mechanism.

Mitochondrial Fission is Essential to Maintain Cristae Morphology and Bioenergetics

Mitochondria and peroxisomes are both dynamic signaling organelles that constantly undergo fission. While mitochondrial fission and fusion are known to coordinate cellular metabolism, proliferation, and apoptosis, the physiological relevance of peroxisome dynamics and the implications for cell fate are not fully understood. DRP1 (dynamin‐related protein 1) is an essential GTPase that executes both mitochondrial and peroxisomal fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause developmental defects, we are using patient‐derived fibroblasts and iPSC‐derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. Using super resolution imaging, we find that patient cells, in addition to displaying elongated mitochondrial and peroxisomal morphology, present with aberrant cristae structure. Given the direct link between cristae morphology and oxidative phosphorylation efficiency, we explored the impact of these mutations on cellular energy production. Patient cells display a lower coupling efficiency of the electron transport chain, increased proton leak, and Complex III deficiency. In addition to these metabolic abnormalities, mitochondrial hyperfusion results in hyperpolarized mitochondrial membrane potential. Intriguingly, human fibroblasts are capable of cellular reprogramming into iPSCs and appear to display peroxisome‐mediated mitochondrial adaptations that could help sustain these cell fate transitions. Understanding the mechanism by which DRP1 mutations cause cellular dysfunction will give insight into the role of mitochondrial and peroxisome dynamics in neurodevelopment.

Identification of the Q Gene Playing a Role in Spike Morphology Variation in Wheat Mutants and Its Regulatory Network.

The wheat AP2 family gene Q controls domestication traits, including spike morphology and threshability, which are critical for the widespread cultivation and yield improvement of wheat. Although many studies have investigated the molecular mechanisms of the Q gene, its direct target genes, especially those controlling spike morphology, are not clear, and its regulatory pathways are not well established. In this study, we conducted gene mapping of a wheat speltoid spike mutant and found that a new allele of the Q gene with protein truncation played a role in spike morphology variation in the mutant. Dynamic expression levels of the Q gene throughout the spike development process suggested that the transcript abundances of the mutant were decreased at the W6 and W7 scales compared to those of the WT. We identified several mutation sites on the Q gene and showed that mutations in different domains resulted in distinct phenotypes. In addition, we found that the Q gene produced three transcripts via alternative splicing and that they exhibited differential expression patterns in nodes, internodes, flag leaves, and spikes. Finally, we identified several target genes directly downstream of Q, including TaGRF1-2D and TaMGD-6B, and proposed a possible regulatory network. This study uncovered the target genes of Q, and the results can help to clarify the mechanism of wheat spike morphology and thereby improve wheat grain yield.

CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT Arrhythmogenesis.

Human-induced pluripotent stem cells (hiPSCs) provide a powerful platform to study biophysical and molecular mechanisms underlying the pathophysiology of genetic mutations associated with cardiac arrhythmia. Human iPSCs can be generated by reprograming of dermal fibroblasts of normal or diseased individuals and be differentiated into cardiac myocytes. Obtaining biopsies from patients afflicted with point mutations causing arrhythmia is often a cumbersome process even when patients are available. Recent development of CRISPR/Cas9 gene editing system makes it, however, possible to introduce arrhythmia-associated point mutations at the desired loci of the wild-type hiPSCs in relatively short times. This platform was used by us to compare the Ca2+ signaling phenotypes of cardiomyocytes harboring point mutations in cardiac Ca2+ release channel, type-2 ryanodine receptor (RyR2), since over 200 missense mutations in RYR2 gene appear to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1). We have created cardiac myocytes harboring mutations in different domains of RyR2, to study not only their Ca2+ signaling consequences but also their drug and domain specificity as related to CPVT1 pathology. In this chapter, we describe our procedures to establish CRISPR/Cas9 gene-edited hiPSC-derived cardiomyocytes.

Mimicking human Drp1 disease-causing mutations in yeast Dnm1 reveals altered mitochondrial dynamics

The dynamin-related protein 1 (Drp1) and its homologs in various eukaryotes are essential to maintain mitochondrial morphology and regulate mitochondrial division. Several mutations in different domains of Drp1 have been reported, which result in debilitating conditions. Four such disease-causing mutations of the middle domain of Drp1 were mimicked in the yeast dynamin-related GTPase (Dnm1) and were characterized in this study. Mitochondrial morphology and protein function were observed to be altered to a variable extent in cells expressing the mutated variants of Dnm1. Several aspects related to the protein such as punctate formation, localization to mitochondria, dynamic behavior and structure were analyzed by microscopy, biochemical studies and molecular dynamics simulations. Significant effects on the protein structure and function were observed in cells expressing A430D and G397D mutations. Overall, our data provide insight into the molecular and cellular alterations resulting from middle domain mutations in Dnm1.

Autosomal dominant hyper IgE syndrome from a single centre in Chongqing, China (2009-2018).

Autosomal dominant hyper IgE syndrome (AD-HIES) caused by STAT3 gene mutation is a rare primary immunodeficiency disease. To better understand the disease, we described the clinical characteristics of 20 AD-HIES patients in Chongqing, China and explored the effect of mutations in different domains of STAT3 gene on the function of STAT3 protein by Western blot and confocal microscopy. The mean age at onset was 0.12years. The mean age at diagnosis was 5.31years. The most common presentation was eczema, pneumonia, skin abscesses and chronic mucocutaneous candidiasis. Seven patients suffered from BCG complications. R382W/Q were identified in 12 patients, V637M mutation in three patients. Three patients have died. The phosphorylated STAT3 was expressed more in wild-type(WT) and R382W mutant STAT3 in the cytoplasm of COS7 cells with epidermal growth factor(EGF) stimulation, less in the V637M mutation and T620S mutation. Dynamic observation showed that STAT3 cytoplasmic accumulation and nuclear translocation occurred rapidly after EGF stimulation in WT-STAT3-GFP, the time of accumulation and nuclear translocation was later and the expression was less in R382W-STAT3-GFP compared with WT-STAT3-GFP, followed by V637M and T620S mutation. These results suggested that our patients had earlier onset, diagnostic age and higher rate of BCG complications. However, our patients had higher incidence of mortality though the earlier diagnostic age. We did not find a significant genotype/phenotype correlation, but Src homology 2 domain mutations (V637M and T620S) had a greater effect on STAT3 phosphorylation and nuclear translocation than DNA-binding domain mutation (R382W) in vitro.

Distribution of Disease-Causing Mutations through Different Protein Domains in Patients with Severe Combined Immunodeficiency

Background and Aim: Severe combined immunodeficiency (SCID) has been described as the most severe form of primary immunodeficiency disorders (PID). The disease can be caused by mutations in more than 20 different genes with prevalence of 1 in 50000 to 100000 live births. In the present study, we described the protein domain position of variants in 14 main genes in patients with SCID. We also aimed to investigate the correlation between the variant distribution of protein domains and its pathogenicity and clinical outcome of the variant. Materials and Methods: Molecular genetic analysis including Sanger sequencing, targeted gene panel and whole exome sequencing were performed on 50 patients with SCID. Moreover, protein domains characteristics were extracted from different databases such as Uniprot and PDB and the reported mutations were obtained from HGMD and ENSEMBL databases. Results: Our results showed that the mortality rate had a significant correlation with severity of clinical manifestations in the patients (p-value=0.000). There was also a significant relationship between the protein type and mutation severity (p-value=0.001) and severity of clinical manifestations (p-value=0.025). However, there was no significant relationship between the mortality rate and occurrence of mutations in different domains of proteins (p-value=0.304) and the severity of mutations (p-value= 0.586). Conclusion: In severe genetic diseases such as SCID, mutations in related genes have affected the structure of the protein enough to cause severe symptoms. However, there are differences in the pathogenicity of the mutations based on their location on the protein domains. In order to determine these variations and predict the outcome of mutations, it is necessary to use in silico and laboratory methods along with statistical and data mining tools to track these minor differences.

Mutations in different domains of lamin A change the mechanical properties of the nucleus

Mutations in different domains of lamin A change the mechanical properties of the nucleus

Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin.

Mutations in the RHO gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two RHO mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The in vitro biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.

Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Polymerase γ catalytic subunit (POLG) gene encodes the enzyme responsible for mitochondrial DNA (mtDNA) synthesis. Mutations affecting POLG are the most prevalent cause of mitochondrial disease because of defective mtDNA replication and lead to a wide spectrum of clinical phenotypes characterized by mtDNA deletions or depletion. Enhancing mitochondrial deoxyribonucleoside triphosphate (dNTP) synthesis effectively rescues mtDNA depletion in different models of defective mtDNA maintenance due to dNTP insufficiency. In this study, we studied mtDNA copy number recovery rates following ethidium bromide-forced depletion in quiescent fibroblasts from patients harboring mutations in different domains of POLG. Whereas control cells spontaneously recovered initial mtDNA levels, POLG-deficient cells experienced a more severe depletion and could not repopulate mtDNA. However, activation of deoxyribonucleoside (dN) salvage by supplementation with dNs plus erythro-9-(2-hydroxy-3-nonyl) adenine (inhibitor of deoxyadenosine degradation) led to increased mitochondrial dNTP pools and promoted mtDNA repopulation in all tested POLG-mutant cells independently of their specific genetic defect. The treatment did not compromise POLG fidelity because no increase in multiple deletions or point mutations was detected. Our study suggests that physiologic dNTP concentration limits the mtDNA replication rate. We thus propose that increasing mitochondrial dNTP availability could be of therapeutic interest for POLG deficiency and other conditions in which mtDNA maintenance is challenged.-Blázquez-Bermejo, C., Carreño-Gago, L., Molina-Granada, D., Aguirre, J., Ramón, J., Torres-Torronteras, J., Cabrera-Pérez, R., Martín, M. Á., Domínguez-González, C., de la Cruz, X., Lombès, A., García-Arumí, E., Martí, R., Cámara, Y. Increased dNTP pools rescue mtDNA depletion in human POLG-deficient fibroblasts.

Novel GLI3 variant causing overlapped Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS) phenotype with agenesis of gallbladder and pancreas

BackgroundA proper balance between the activator and the repressor form of GLI3, a zinc-finger transcription factor downstream of hedgehog signaling, is essential for proper development of various organs during development. Mutations in different domains of the GLI3 gene underlie several congenital diseases including Greig cephalopolysyndactyly syndrome (GCPS) and Pallister-Hall syndrome (PHS).Case presentationHere, we describe the case of an overlapped phenotype of these syndromes with agenesis of the gallbladder and the pancreas, bearing a c.2155 C > T novel likely pathogenic variant of GLI3 gene by missense point mutation causing p.P719S at the proteolytic cleavage site.ConclusionsAlthough agenesis of the gallbladder and the pancreas is uncommon in GLI3 morphopathy, a slight difference in the gradient or the balance between activator and repressor in this case may hinder sophisticated spatial and sequential hedgehog signaling that is essential for proper development of gallbladder and pancreas from endodermal buds.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

Aquaporin 1, Aquaporin 3 and Aquaporin 5 expression and EGFR mutation in malignant pleural mesotheliomas: an imunohistochemical and molecular study

ABSTRACTMalignant pleural mesothelioma is a rare and fatal malignancy. This disease is, unfortunately, at its advanced stage when it is diagnosed. Survival time is usually not more than a few months. The aim of this study was to analyse the expression of Aquaporin 1, Aquaporin 3 and Aquaporin 5 in malignant pleural mesotheliomas and to explore the relationship of these levels of expression with epidermal growth factor receptor (EGFR) gene mutation and prognostic parameters. In this study, 60 cases diagnosed as malignant pleural mesothelioma among the pleural biopsy materials in the archives of the Pathology Department of Medical Faculty of Dicle University in 2003–2013 were evaluated. The tissues were stained immunohistochemically with antibodies against Aquaporin 1, Aquaporin 3 and Aquaporin 5, and the existence of EGFR mutation was investigated in the tissues by real-time polymerase chain reaction (PCR). The obtained results showed expression of Aquaporin 1, Aquaporin 3 and Aquaporin 5 in varied amounts in malignant pleural mesotheliomas. However, no significant relation was obtained thus far between the expression levels of these aquaporins and the prognostic parameters. No mutations were detected in the EGFR gene exons 18–21 by using real-time PCR. It could be suggested that although Aquaporin 1, Aquaporin 3 and Aquaporin 5 are expressed in malignant pleural mesothelioma, they do not have any effect on the prognostic parameters. Mutations in different domains of EGFR gene, other than exons 18–21, should be sought to develop new targeted treatments.

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

Targeted mutagenesis of Drosophila RNaseZ gene by homologous recombination.

For the first time we used a homologous recombination method to obtain complete and precise deletion of Drosophila dRNaseZ gene. In the founder line of flies in which the RNaseZ sequence was replaced by attP site, the full-length sequence of the gene was reintegrated, and its functionality was shown. This approach will allow us to generate further gene mutations in different domains of dRNaseZ protein and discover a broad spectrum and uncover functions outside of tRNA processing.