RESUMEN
Cystic fibrosis (CF) occurrence in Arab populations is not common and still remains underidentified. Furthermore, the lack of disease awareness and diagnosis facilities have mislead the identification of cystic fibrosis for decades. The knowledge about cystic fibrosis (CF) in Egypt is very limited, and a few reports have drawn attention to the existence of CF or CFTR-related disorders (CFTR-RDs) in the Egyptian population. Therefore a comprehensive genetic analysis of the CFTR gene was realized in patients of North Egypt. DNA samples of 56 Egyptian patients were screened for the CFTR gene mutations. The 27 exons and their flanking regions of the CFTR gene were amplified by PCR, using the published primer pairs, and were studied by automated direct DNA sequencing to detect disease-causing mutations. Moreover, large duplication/deletion was analysed by MLPA technique. CFTR screening revealed the identification of thirteen mutations including four novel ones: c.92G>A (p.Arg31His), c.2782G>C (p.Ala928Pro), c.3718-24G>A, c.4207A>G (p.Arg1403Gly) and nine previously reported mutations: c.454A>T (p.Met152Leu), c.902A>G (p.Tyr301Cys), c.1418delG, c.2620-15C>G, c.2997_3000delAATT, c.3154T>G (p.Phe1052Val), c.3872A>G (p.Gln1291Arg), c.3877G>A (p.Val1293Ile), c.4242+10T>C. Furthermore, eight polymorphisms were found: c.743+40A>G, c.869+11C>T, c.1408A>G, c.1584G>A, c.2562T>G, c.3870A>G, c.4272C>T, c.4389G>A. These mutations and polymorphisms were not previously described in the Egyptian population except for the c.1408A>G polymorphism. Here we demonstrate the importance of the newly discovered mutations in Egyptian patients and the presence of CF, whereas the p.Phe508del mutation is not detected. The identification of CFTR mutations will become increasingly important in undocumented populations. The current findings will help us expand the mutational spectrum of CF and establish the first panel of the CFTR gene mutations in the Egyptian population and design an appropriate strategy for future genetic diagnosis of CF.
Asunto(s)
Población Negra/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/diagnóstico , Adolescente , Adulto , Secuencia de Bases , Niño , Fibrosis Quística/genética , Egipto , Exones , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Polimorfismo Genético , Adulto JovenRESUMEN
Spinophilin/neurabin 2 has been isolated independently by two laboratories as a protein interacting with protein phosphatase 1 (PP1) and F-actin. Gene analysis and biochemical approaches have contributed to define a number of distinct modular domains in spinophilin that govern protein-protein interactions such as two F-actin-, three potential Src homology 3 (SH3)-, a receptor- and a PP1-binding domains, a PSD95/DLG/zo-1 (PDZ) and three coiled-coil domains, and a potential leucine/isoleucine zipper (LIZ) motif. More than 30 partner proteins of spinophilin have been discovered, including cytoskeletal and cell adhesion molecules, enzymes, guanine nucleotide exchange factors (GEF) and regulator of G-protein signalling protein, membrane receptors, ion channels and others proteins like the tumour suppressor ARF. The physiological relevance of some of these interactions remains to be demonstrated. However, spinophilin structure suggests that the protein is a multifunctional protein scaffold that regulates both membrane and cytoskeletal functions. Spinophilin plays important functions in the nervous system where it is implicated in spine morphology and density regulation, synaptic plasticity and neuronal migration. Spinophilin regulates also seven-transmembrane receptor signalling and may provide a link between some of these receptors and intracellular mitogenic signalling events dependent on p70(S6) kinase and Rac G protein-GEF. Strikingly a role for spinophilin in cell growth was demonstrated and this effect was enhanced by its interaction with ARF. Here we review the current knowledge of the protein partners of spinophilin and present the available data that are contributing to the appreciation of spinophilin functions.
Asunto(s)
Proteínas de Microfilamentos/fisiología , Proteínas del Tejido Nervioso/fisiología , Transducción de Señal , Animales , Membrana Celular/metabolismo , Sistema Nervioso Central/metabolismo , Citoesqueleto/metabolismo , Humanos , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Modelos Biológicos , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genéticaRESUMEN
The impact of the hobo transposable element in the global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by the injection of hobo elements into the Hikone strain, which lacked them previously. Extensive surveys of transgenic lines followed for 250 generations have identified 13 inversions with hobo inserts at most breakpoints. One of these inversions is pericentric on chromosome 2. It has been maintained in the line where it was discovered and in several sublines at frequencies from 0.19 to 0.45, generating stable chromosomal polymorphisms, similar to cosmopolitan paracentric inversions in natural populations. Individuals homozygous for this inversion were viable and fertile, allowing the creation of a new homozygous strain.
Asunto(s)
Inversión Cromosómica , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Transposasas/genética , Animales , Animales Modificados Genéticamente , Aberraciones Cromosómicas , Mapeo CromosómicoRESUMEN
The impact of the hobo transposable element in global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by injection of hobo elements into the Hikone strain, which lacked them. In the present extensive survey, the chromosomal distribution of hobo insertion sites in the line 28 was found to be homogeneous and similar for all chromosomal arms, except 3L, when compared with other transgenic lines. However, some original features were observed in this line at the genetic and chromosomal levels. Several hotspots of insertion sites were observed on the X, second and third chromosomes. Five sites with a high frequency of hobo insertions were present on the 3L arm in most individuals tested, suggesting the action of selection for hobo element in some sites. The presence of doublets or triplet was also observed, implying that hobo inserts can show local jumps or insertions in preferred regions. This local transposition occurred independently in 11 specific genomic regions in many individuals and generations. The dynamics of this phenomenon were analysed across generations. These results support the use of the hobo system as an important tool in fundamental and applied Drosophila genetics.
Asunto(s)
Animales Modificados Genéticamente , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Animales , Southern Blotting , Línea Celular , Mapeo Cromosómico , Técnicas Genéticas , Hibridación in SituRESUMEN
This study is an attempt to trace the fate of hobo elements in the genomes of E strains of Drosophila melanogaster that have been transfected with pHFL1, a plasmid containing an autonomous hobo. Such long-term population studies (over 105 generations) could be very useful for better understanding the population and genomic dynamics of transposable elements and their pattern of insertions. Molecular analyses of hobo elements in the transfected lines were performed using Southern blots of XhoI-digested genomic DNAs. The complete element was observed in all six injected lines. In two lines we observed, at generation 100, two deleted elements, which did not correspond to Th1 and Th2. The results obtained by the in situ method show that the number of hybridization sites increases in each line and prove that the hobo element may be amplified in an RM genome. The hobo activity does not seem to be systematically correlated with the number of hobo elements. After generation 85, the evolution of the hobo element's insertion site number depends on the injected line. In all lines, the total number of insertions remains quite small, between 0 and 11. Hobo elements are located on each of the chromosomal arms. We describe 'hotspots'-insertion sites present in all lines and in all generations. On the 3R arm, a short inversion appeared once at generation 85.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Animales Modificados Genéticamente , Femenino , Masculino , Transposasas/genética , Transposasas/metabolismoRESUMEN
Several laboratory surveys have shown that transposable elements (TEs) can cause chromosomal breaks and lead to inversions, as in dysgenic crosses involving P-elements. However, it is not presently clear what causes inversions in natural populations of Drosophila. The only direct molecular studies must be taken as evidence against the involvement of mobile elements. Here, in Drosophila lines transformed with the hobo transposable element, and followed for 100 generations, we show the appearance of five different inversions with hobo inserts at breakpoints. Almost all breakpoints occurred in hobo insertion sites detected in previous generations. Therefore, it can be assumed that such elements are responsible for restructuring genomes in natural populations.
Asunto(s)
Aberraciones Cromosómicas/genética , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Inversión Cromosómica , Hibridación in SituRESUMEN
The transposable element hobo has been introduced into the previously empty Drosophila melanogaster strain Hikone so that its dynamics can be followed and it can be compared with the P element. Five transformed lines were followed over 58 generations. The results were highly dependent on the culture temperature, the spread of hobo element being more efficient at 25 degrees C. The multiplication of hobo sequences resulted in a change in the features of these lines in the hobo system of hybrid dysgenesis. The number of hobo elements remained low (two to seven copies) and the insertions always corresponded to complete sequences. Our findings suggest that, despite their genetic similarities, P and hobo elements differ in many aspects, such as mobility and regulation mechanisms.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Genes de Insecto , Animales , Southern Blotting , Cruzamientos Genéticos , ADN/análisis , Femenino , Regulación de la Expresión Génica , Hibridación Genética , Masculino , PlásmidosRESUMEN
The invasion kinetics of hobo transposable element in the Drosophila melanogaster genome was studied by in situ hybridization on the polytene chromosomes. Six independent lines of Drosophila melanogaster flies that had been previously transformed by microinjection of the pHFL1 plasmid containing a complete hobo element were followed over 50 generations. We observed that hobo elements were scattered on each of the chromosome arms, with more insertion sites on the 3R arm. The total number of insertion sites remains quite small, between four and six, at generation 52. On the 2R arm, a short inversion appeared once at generation 52. Most of the integration sites reported here were already described for several transposons but some of them appear to be hotspots for hobo elements.
Asunto(s)
Cromosomas/ultraestructura , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Southern Blotting , Centrómero/ultraestructura , Mapeo Cromosómico , Embrión no Mamífero , Genoma , Hibridación in SituRESUMEN
Hobo elements are a family of transposable elements found in Drosophila melanogaster and its three sibling species: D. simulans, D. mauritiana and D. sechellia. Studies in D. melanogaster have shown that hobo may be mobilized, and that the genetic effects of such mobilizations included the general features of hybrid dysgenesis: mutations, chromosomal rearrangements and gonadal dysgenis in F1 individuals. At the evolutionary level some hobo-hybridizing sequences have also been found in the other members of the melanogaster subgroup and in many members of the related montium subgroup. Surveys of older collected strains of D. melanogaster suggest that complete hobo elements were absent prior to 50 years ago and that they have recently been introduced into this species by horizontal transfer. In this paper we review our findings and those of others, in order to precisely describe the geographical distribution and the evolutionary history of hobo in the D. melanogaster complex. Studies of the DNA sequences reveal a different level of divergence between the group D. melanogaster, D. simulans and D. mauritiana and the fourth species D. sechellia. The hypothesis of multiple transfers in the recent past into the D. melanogaster complex from a common outside source is discussed.
Asunto(s)
Evolución Biológica , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Drosophila/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas/ultraestructura , Datos de Secuencia Molecular , Eliminación de Secuencia , Especificidad de la EspecieRESUMEN
Screening for resistance to fenpropimorph was undertaken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessive fen1-1 mutation was characterized by a 1.5-fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. The fen1-1 mutation was linked to MAT locus on chromosome III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopentenyl diphosphate (IPP) isomerase activity was specifically increased 1.5-fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. The fen1-1 mutation permits viability in aerobiosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the corresponding strain bearing the wild type FEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergosterol requirement in yeast.
Asunto(s)
Antifúngicos/toxicidad , Isomerasas de Doble Vínculo Carbono-Carbono , Farmacorresistencia Microbiana , Genes Fúngicos , Saccharomyces cerevisiae/efectos de los fármacos , Esteroles/biosíntesis , Mapeo Cromosómico , Cromosomas Fúngicos , Cartilla de ADN , Farmacorresistencia Microbiana/genética , Ergosterol/biosíntesis , Expresión Génica , Hemiterpenos , Isomerasas/biosíntesis , Isomerasas/metabolismo , Metionina/metabolismo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The purpose of this paper is the genetic visualization by in situ hybridization of 130 sex-linked recessive lethals plus a non-lethal induced by I-R dysgenesis. This collection of lethals involves inducer strains which differ in the position of the I elements on the X chromosomes. The I-R interaction was strong. Our previous results have shown that about 30% of the induced recessive lethals are associated with cytologically visible chromosomal rearrangements. (1) The rearrangements induced by I-R-type hybrid dysgenesis often exhibit homology with the I factor at the level of one or both junction points, depending on the types of chromosome rearrangements. These results suggest that the chromosome rearrangements arise directly from the transposition of I elements. However, the breakpoints of some types of cytologically non-visible deficiencies and of 2 small cytologically visible deficiencies do not present detectable homology with the I factor. (2) The majority of rearrangements do not involve the I elements already present on the paternal X chromosome. (3) The hybridization signal distributions on the X chromosome are not uniform. They present peaks of various heights which may correspond to specific anchoring areas of copies of I in the course of integration. (4) The data presented here agree with the literature with respect to the mean number of copies of I per X chromosome and to the excess of copies of I at locus 1A. Two rearrangement formation mechanisms are envisaged: crossing-over and 'target' exchanges.
Asunto(s)
Aberraciones Cromosómicas , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Genes Letales/genética , Hibridación Genética/genética , Animales , Distribución de Chi-Cuadrado , Deleción Cromosómica , Inversión Cromosómica , Intercambio Genético , Femenino , Genes Recesivos , Ligamiento Genético , Mutación , Hibridación de Ácido Nucleico , Secuencias Reguladoras de Ácidos Nucleicos , Translocación Genética , Cromosoma XRESUMEN
Yeast mutant strains auxotrophic for ergosterol and blocked in mevalonate diphosphate decarboxylase (erg19) and farnesyl diphosphate (FPP) synthetase (erg20) were isolated. The main feature of the mutants blocked in FPP synthetase is their ability to excrete prenyl alcohols, such as geraniol and farnesol. The isolation of the functional ERG20 gene allowed us to show that farnesyl diphosphate synthetase could be a rate limiting enzyme in ergosterol biosynthesis in yeast.
Asunto(s)
Carboxiliasas/metabolismo , Dimetilaliltranstransferasa/metabolismo , Saccharomyces cerevisiae/enzimología , Esteroles/biosíntesis , Monoterpenos Acíclicos , Carboxiliasas/genética , Dimetilaliltranstransferasa/genética , Ergosterol/biosíntesis , Farnesol/metabolismo , Expresión Génica , Genes Fúngicos , Mutación , Saccharomyces cerevisiae/genética , Terpenos/metabolismoRESUMEN
Two yeast mutant strains auxotrophic for ergosterol and blocked in farnesyl diphosphate synthetase (EC 2.5.1.1) were isolated. Genetic analysis has shown that these mutant strains carry additional mutations in the ergosterol pathway besides erg20-1 and erg20-2 which affect FPP synthetase. The novel feature of these mutants is their ability to excrete prenyl alcohols (farnesol and geraniol). As geraniol is toxic for yeast cells, the above leaky mutations in FPP synthetase have to be associated with others in the sterol pathway, in order to slow down geraniol synthesis.