No description available.

Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...

Recent studies have suggested a role of two polymorphisms of the dopamine D4 receptor gene (DRD4 exon III and –521C/T) in the modulation of personality traits such as ‘novelty seeking’ or ‘extraversion’, which are supposed to be modulated by individual differences in dopaminergic function. However, several replication studies have not provided positive findings. The present study was performed to further investigate whether DRD4 exon III and –521C/T are associated with individual differences in personality. One hundred and fifteen healthy German volunteers completed the NEO-Five-Factor Inventory (NEO-FFI) and were genotyped for the two DRD4 polymorphisms. We found no association between DRD4exon III and –521C/T, respectively, and estimated novelty seeking, NEO-FFI extraversion or other personality factors. Our findings are in line with several earlier studies which have failed to replicate the initial association results. Hence, our data do not provide evidence for a role of DRD4 exon III and the –521C/T polymorphism in the modulation of novelty seeking and extraversion. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Proper eye development depends on expression and mutual regulation of many genes. Anterior segment dysgenesis (ASD) are a highly heterogeneous group of diseases exhibiting all types of Mendelian inheritance, which manifest as combination of congenital abnormalities of the cornea, iris, anterior chamber angle or lens. Screening of genes associated with ASD does not often lead to the identification of the underlying genetic cause implying that there are still novel variants or genes to be discovered. Molecular genetic analysis in 12 probands with ASD using Sanger and whole-exome sequencing were performed. Functional analysis by Exon trapping assay was provided in variants predicted to effect pre-mRNA splicing. Four PAX6 mutations evaluated as pathogenic or likely pathogenic in a heterozygous state were found in four probands c.183C˃G; p.(Tyr61*), c.1032+1G>A, c.1183+1G>T and c.622C>T; p.(Arg208Trp). One proband was found to be a compound heterozygote for c.244A>G; p.(Met82Val) and c.541delG; p.(Glu181Lysfs*26) mutations in FOXE3. In 7 probands, no potentially pathogenic variants were identified. Exon trapping assay confirmed that mutations c.1032+1G>A and c.1183+1G>T have an effect on pre-mRNA splicing of the PAX6 gene. Detailed molecular-genetic analysis in patients with ASD may contribute to...

No description available.

Malondialdehyde (MDA) is a mutagenic and carcinogenic product of lipid peroxidation which has also been found at elevated levels in smokers. MDA reacts with nucleic acid bases to form pyrimidopurinone DNA adducts, of which 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimidol[1,2-α]purin-10(3H)-one (M1dG) is the most abundant and has been linked to smoking. Mutations in the TP53 tumour suppressor gene are associated with half of all cancers. This research applied a multidisciplinary approach to investigate the toxic effects of MDA on the human lung fibroblasts MRC5, which have an intact p53 response, and their SV40 transformed counterpart, MRC5 SV2, which have a sequestered p53 response. Both cell lines were treated with MDA (0-1000 µM) for 24 and 48 h and subjected to a variety of analyses to examine cell proliferation, cell viability, cellular and nuclear morphology, apoptosis, p53 protein expression, DNA topography and M1dG adduct detection. For the first time, mutation sequencing of the 5’ untranslated region (UTR) of the TP53 gene in response to MDA treatment was carried out. The main findings were that both cell lines showed reduced proliferation and viability with increasing concentrations of MDA, the cell surface and nuclear morphology were altered, and levels of apoptosis and p53 protein expression appeared to increase. A LC MS-MS method for detection of M1dG adducts was developed and adducts were detected in CT-DNA treated with MDA in a dose-dependent manner. DNA appeared to become more fragmented with increasing MDA concentration, and the number of mutations in the 5’ UTR region of the TP53 gene also increased. The majority of mutations observed were insertions, compared to lung cancer mutation data where the majority were G to T transversions. This was unexpected, suggesting that tobacco smoke compounds have a different role in mutagenesis than endogenous lipid peroxidation. Thus, MDA has been found to have a clear effect on human lung fibroblasts at both the cellular and DNA level.

[ES] Los viroides, genomas mínimos de RNA circular no codificante, monocatenarios y muy estructurados, parasitan factores celulares de las plantas para replicarse autónomamente, establecer infecciones sistémicas y usualmente causar enfermedades. Los de la familia Avsunviroidae se replican y acumulan en cloroplastos por un mecanismo de círculo rodante simétrico. Una RNA polimerasa cloroplástica produce concatémeros lineales de polaridad complementaria que son reducidos a monómeros por las ribozimas de cabeza de martillo (HHR) del concatémero. Producen extremos 5'-hidroxilo y 2',3'-fosfodiéster cíclico, que la isoforma cloroplástica de la tRNA ligasa convierte en enlaces 5',3'-fosfodiéster intramoleculares, generando viroides circulares de polaridad complementaria que pueden entrar en otra ronda de transcripción, simétrica a ésta. En esta Tesis se han analizado las secuencias y estructuras viroidales esenciales para su circularización, usando como modelo el viroide latente de berenjena (ELVd), que induce infecciones asintomáticas en berenjena. Expresamos en Escherichia coli precursores del ELVd(+) lineales flanqueados por dos copias de su HHR. Su procesamiento genera monómeros con los extremos adecuados para la ligación por la tRNA ligasa de la berenjena, que es coexpresada. Mutaciones puntuales y deleciones en el sitio nativo de ligación sugieren que solo el dominio HHR es esencial para la circularización. La conservación de la secuencia y estructura de la HHR con las del sustrato natural del enzima (los tRNAs) nos hacen proponer que la HHR del ELVd secuestra la ligasa mimetizando las características generales del bucle anticodón de los tRNAs. Este sistema de expresión permite también producir RNAs recombinantes, insertándolos en una posición particular del RNA del ELVd. Las quimeras son procesadas por las HHRs flanqueantes y sus extremos ligados por la tRNA ligasa. El andamiaje viroidal circular, compacto y posiblemente asociado a la ligasa, permite aumentar la vida media del RNA de interés y su acumulación en la bacteria. En esta Tesis adaptamos el sistema para producir RNAs de doble cadena (dsRNAs) que desencadenen interferencia por RNA (RNAi), un mecanismo de defensa y regulación génica eucariota basado en la complementariedad de bases entre RNAs. dsRNAs complementarios a genes endógenos reducen los niveles de sus transcritos y generan fenotipos de pérdida de función. Los insectos pueden tomar dsRNAs del ambiente, internalizarlos en sus células y distribuirlos sistémicamente, haciendo al RNAi una estrategia prometedora para el control de plagas. Para producir dsRNAs, separamos las repeticiones invertidas del gen diana que genera la horquilla con el cDNA de un intrón autocatalítico del grupo I de Tetrahymena thermophila, aumentando la estabilidad de los plásmidos de expresión. El intrón es eliminado tras la transcripción, resultando en una molécula viroidal de la que protruye el dsRNA de interés. Flanquear las repeticiones invertidas con una copia adicional permutada del intrón permite separar el ELVd del producto final, un dsRNA circular cerrado en ambos lados por pequeños bucles. Ambas moléculas poseen actividad reguladora: las quimeras viroide-dsRNA con homología al gen de la unión septada suave 1 del gusano de la raíz del maíz (Diabrotica virgifera virgifera) exhiben actividad insecticida oral contra las larvas similar a la de horquillas sintetizadas in vitro, y los dsRNAs circulares sin andamiaje viroidal homólogos al gen de la ATPasa vacuolar (subunidad A) y la proteína ribosomal S13 silencian eficientemente estos genes en adultos de la mosca del Mediterráneo (Ceratitis capitata); este caso es de especial relevancia al ser la primera demostración del RNAi para el control de esta plaga. En conclusión, a pesar de su limitada relevancia agrícola, el ELVd es útil para investigar la biología molecular de la familia Avsunviroidae y una poderosa herramienta biotecnológica en combinación con el sistema de expresión en E. coli. / [CA] Els viroides, genomes mínims d'RNA circular no codificant, monocatenaris i molt estructurats, parasiten factors cel·lulars de les plantes per a replicar-se autònomament, establir infeccions sistèmiques i usualment causar malalties. Els de la família Avsunviroidae es repliquen i acumulen en cloroplasts per un mecanisme de cercle rodant simètric. Una RNA polimerasa cloroplàstica produeix concatèmers lineals de polaritat complementària que són reduïts a monòmers per els ribozims de cap de martell (HHR) del concatèmer. Produeixen extrems 5'-hidroxil i 2',3'-fosfodièster cíclic, que la isoforma cloroplàstica de la tRNA lligasa converteix en enllaços 5',3'-fosfodièster intramoleculars, generant viroides circulars de polaritat complementària que poden entrar en una nova ronda de transcripció, simètrica a la primera. En aquesta Tesi s'han analitzat les seqüències i estructures viroidals essencials per a la seua circularització, emprant com a model el viroide latent d'albergínia (ELVd), que indueix infeccions asimptomàtiques en albergínia. Expressem en Escherichia coli precursors de l'ELVd(+) lineals flanquejats per dos còpies del seu HHR. El seu processament produeix monòmers amb els extrems apropiats per a la lligació mediada per la tRNA ligasa de l'albergínia, que és coexpressada. Mutacions puntuals i delecions en el lloc nadiu de lligació suggereixen que només el domini HHR és essencial per a la circularització. La conservació de la seqüència i estructura del HHR amb les del substrat natural de l'enzim (els tRNAs) ens fan proposar que el HHR de l'ELVd segresta la lligasa mimetitzant les característiques generals del bucle anticodó dels tRNAs. Aquest sistema d'expressió també permet produir RNAs recombinants, inserint-los en una posició particular de l'RNA de l'ELVd. Les quimeres són processades pels HHR flanquejants i els seus extrems lligats per la tRNA lligasa. L'RNA viroïdal circular, compacte i possiblement associat a la lligasa, permet augmentar la vida mitjana de l'RNA d'interés i la seua acumulació en els bacteris. En aquesta Tesi adaptem el sistema per a produir RNAs de doble cadena (dsRNAs) que desencadenen interferència per RNA (RNAi), un mecanisme de defensa i regulació gènica eucariota basat en la complementarietat de bases entre RNAs. dsRNAs complementaris a gens endògens redueixen els nivells dels seus transcrits i generen fenotips de pèrdua de funció. Els insectes poden prendre dsRNAs de l'ambient, internalitzar-los en les seues cèl·lules i distribuir-los sistèmicament, fent a l'RNAi una estratègia prometedora en el control de plagues. Per a produir dsRNAs, separem les repeticions invertides del gen diana que genera la forqueta amb el cDNA d'un intró autocatalític del grup I de Tetrahymena thermophila, augmentant l'estabilitat dels plasmidis d'expressió. L'intró és eliminat després de la transcripció, resultant en una molècula viroïdal de la qual protrueix el dsRNA d'interés. Flanquejar les repeticions invertides amb una còpia addicional permutada de l'intró permet separar l'ELVd del producte final, un dsRNA circular tancat als dos costats per xicotets bucles. Els dos tipus de molècules posseeixen activitat reguladora: les quimeres viroide-dsRNA amb homologia al gen de la unió septada suau 1 del cuc de l'arrel de la dacsa (Diabrotica virgifera virgifera) exhibeixen activitat insecticida oral contra les larves similar a la de forquetes sintetitzades in vitro, i els dsRNAs circulars sense l'RNA viroïdal homòlegs al gen de la ATPasa vacuolar (subunitat A) i la proteïna ribosomal S13 silencien eficientment aquests gens en adults de la mosca del Mediterrani (Ceratitis capitata); aquest cas és d'especial rellevància perquè és la primera demostració de l'RNAi per al control d'aquesta plaga. En conclusió, malgrat la seua limitada rellevància agrícola l'ELVd és útil per a investigar la biologia molecular de la família Avsunviroidae i una poderosa ferramenta biotecnològica en combinació amb el sistema d'expressió en E. coli. / [EN] Viroids, minimal genomes of non-coding circular RNA, single-stranded and highly structured, parasitize plant cellular factors to replicate autonomously, establish systemic infections, and typically cause disease. Those of the family Avsunviroidae replicate and accumulate in chloroplasts by a symmetrical rolling circle mechanism. A chloroplast RNA polymerase produces linear concatemers of complementary polarity that are reduced to monomers by the hammerhead ribozymes (HHR) of the concatemer. They produce 5'-hydroxyl and 2',3'-cyclic phosphodiester ends, which the chloroplastic isoform of tRNA ligase converts to intramolecular 5',3'-phosphodiester bonds, generating circular viroids of complementary polarity that can enter another round of transcription, symmetric to the first one. In this Thesis, the viroid sequences and structures essential for its circularization have been analyzed, using as a model the eggplant latent viroid (ELVd), which induces asymptomatic infections in eggplant. We expressed in Escherichia coli linear ELVd(+) precursors flanked by two copies of its HHR. Its processing generates monomers with suitable ends for ligation by the eggplant tRNA ligase, which is co-expressed. Point mutations and deletions at the wild-type ligation site suggest that only the HHR domain is essential for circularization. The conservation of the sequence and structure of the HHR with those of the natural substrate of the enzyme (the tRNAs) lead us to propose that the HHR of the ELVd hijacks the ligase, mimicking the general characteristics of the anticodon loop of the tRNAs. This expression system also allows the production of recombinant RNAs, inserting them into a particular position of the ELVd RNA. Chimeras are processed by flanking HHRs and their ends ligated by the tRNA ligase. The compact, circular viroidal scaffold, possibly associated with the ligase, allows increasing the half-life of the RNA of interest and its accumulation in the bacteria. In this Thesis we adapt the system to produce double-stranded RNAs (dsRNAs) that trigger RNA interference (RNAi), a eukaryotic gene regulation and defense mechanism based on base complementarity between RNAs. dsRNAs complementary to endogenous genes reduce the levels of their transcripts and generate loss-of-function phenotypes. Insects can take dsRNAs from the environment, internalize them into cells, and distribute them systemically, making RNAi a promising pest control strategy. To produce dsRNAs, we separated the inverted repeats of the target gene that generates the hairpin with the cDNA of a group-I autocatalytic intron from Tetrahymena thermophila, increasing the stability of the expression plasmids. The intron is removed after transcription, resulting in a viroidal molecule from which the dsRNA of interest protrudes. Flanking the inverted repeats with an additional copy of the intron in a permuted form allows the ELVd molecule to be separated from the final product, a circular dsRNA molecule capped on both sides by small loops. Both molecules have regulatory activity: the viroid-dsRNA chimeras with homology to the smooth septate junction 1 gene of the corn rootworm (Diabrotica virgifera virgifera) exhibit oral insecticidal activity against larvae similar to that of in vitro synthesized hairpins, and the circular dsRNAs without the viroid scaffold homologous to the vacuolar ATPase (subunit A) and ribosomal protein S13 genes efficiently silence those genes in adult Medfly (Ceratitis capitata); this case is of special relevance as it is the first demonstration of RNAi for the control of this pest. In conclusion, despite its limited agricultural relevance, the ELVd is useful for investigating the molecular biology of the Avsunviroidae family and a powerful biotechnological tool in combination with the E. coli expression system. / This work was supported by the Ministerio de Ciencia e Innovación (Spain; co-financed by the European Regional Development Fund) [BIO2017-83184-R] and [BIO2017‐ 91865‐EXP]; Universitat Politècnica de València [PAID-01-17]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). / Ortolá Navarro, B. (2023). Heterologous expression of circular RNAs in Escherichia coli for analyzing the ligation process of chloroplastic viroids and producing double-stranded RNAs with insecticidal activity [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192635