RESUMEN
BACKGROUND: Congenital limb malformations (CLMs) are common and present to a variety of specialties, notably plastic and orthopaedic surgeons, and clinical geneticists. The authors aimed to characterise causative mutations in an unselected cohort of patients with CLMs requiring reconstructive surgery. METHODS: 202 patients presenting with CLM were recruited. The authors obtained G-banded karyotypes and screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for point mutations using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. Multiplex ligation dependent probe amplification (MLPA) kits were developed and used to measure copy number in GLI3, HOXD13, ROR2, SALL1, SALL4, TBX5 and the ZRS of SHH. RESULTS: Within the cohort, causative genetic alterations were identified in 23 patients (11%): mutations in GLI3 (n = 5), HOXD13 (n = 5), the ZRS of SHH (n = 4), and chromosome abnormalities (n = 4) were the most common lesions found. Clinical features that predicted the discovery of a genetic cause included a bilateral malformation, positive family history, and having increasing numbers of limbs affected (all p<0.01). Additionally, specific patterns of malformation predicted mutations in specific genes. CONCLUSIONS: Based on higher mutation prevalence the authors propose that GLI3, HOXD13 and the ZRS of SHH should be prioritised for introduction into molecular genetic testing programmes for CLM. The authors have developed simple criteria that can refine the selection of patients by surgeons for referral to clinical geneticists. The cohort also represents an excellent resource to test for mutations in novel candidate genes.
Asunto(s)
Deformidades Congénitas de las Extremidades/diagnóstico , Deformidades Congénitas de las Extremidades/genética , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Pruebas Genéticas/métodos , Humanos , Cariotipificación , Deformidades Congénitas de las Extremidades/cirugía , Procedimientos de Cirugía PlásticaRESUMEN
We report an activating fibroblast growth factor receptor 3 (FGFR3) mutation (R248C) occurring in a verrucous epidermal naevus, and not found in other tissues, in a girl with mild facial dysmorphism. We demonstrate the presence of the mutation in keratinocytes cultured from the naevus and we speculate that a low level of the mutation in other tissues may account for her facial dysmorphism. The possibility that the mutation is present in other tissues implies a possible risk to her future offspring.
Asunto(s)
Asimetría Facial/genética , Mosaicismo , Nevo Pigmentado/genética , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Niño , Craneosinostosis/genética , Femenino , HumanosRESUMEN
In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modified Ds transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.
Asunto(s)
Clonación Molecular/métodos , ADN Bacteriano/genética , ADN de Plantas/genética , Genoma de Planta , Solanum lycopersicum/genética , Marcadores Genéticos , Vectores Genéticos , Plásmidos , Polimorfismo Genético , Recombinación Genética , Mapeo RestrictivoRESUMEN
The abscisic acid (ABA) biosynthetic pathway involves the formation of a 9-cis-epoxycarotenoid precursor. Oxidative cleavage then results in the formation of xanthoxin, which is subsequently converted to ABA. A number of steps in the pathway may control ABA synthesis, but particular attention has been given to the enzyme involved in the oxidative cleavage reaction, i.e. 9-cis-epoxycarotenoid dioxygenase (NCED). Cloning of a gene encoding this enzyme in maize was first reported in 1997. Mapping and DNA sequencing studies indicated that a wilty tomato mutant was due to a deletion in the gene encoding an enzyme with a very similar amino acid sequence to this maize NCED. The potential use of this gene in altering ABA content will be discussed together with other genes encoding ABA biosynthetic enzymes.
Asunto(s)
Ácido Abscísico/biosíntesis , Plantas Modificadas Genéticamente/metabolismo , Aldehído Oxidasa , Aldehído Oxidorreductasas/genética , Dioxigenasas , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxidorreductasas/genética , Oxigenasas/genética , Proteínas de Plantas , Plantas Modificadas Genéticamente/enzimología , Plantas Modificadas Genéticamente/genéticaRESUMEN
The tomato mutant notabilis has a wilty phenotype as a result of abscisic acid (ABA) deficiency. The wild-type allele of notabilis, LeNCED1, encodes a putative 9-cis-epoxycarotenoid dioxygenase (NCED) with a potential regulatory role in ABA biosynthesis. We have created transgenic tobacco plants in which expression of the LeNCED1 coding region is under tetracycline-inducible control. When leaf explants from these plants were treated with tetracycline, NCED mRNA was induced and bulk leaf ABA content increased by up to 10-fold. Transgenic tomato plants were also produced containing the LeNCED1 coding region under the control of one of two strong constitutive promoters, either the doubly enhanced CaMV 35S promoter or the chimaeric 'Super-Promoter'. Many of these plants were wilty, suggesting co-suppression of endogenous gene activity; however three transformants displayed a common, heritable phenotype that could be due to enhanced ABA biosynthesis, showing increased guttation and seed dormancy. Progeny from two of these transformants were further characterized, and it was shown that they also exhibited reduced stomatal conductance, increased NCED mRNA and elevated seed ABA content. Progeny of one transformant had significantly higher bulk leaf ABA content compared to the wild type. The increased seed dormancy was reversed by addition of the carotenoid biosynthesis inhibitor norflurazon. These data provide strong evidence that NCED is indeed a key regulatory enzyme in ABA biosynthesis in leaves, and demonstrate for the first time that plant ABA content can be increased through manipulating NCED.
Asunto(s)
Ácido Abscísico/biosíntesis , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxigenasas/genética , Solanum lycopersicum/genética , Secuencia de Bases , Cartilla de ADN , Dioxigenasas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Solanum lycopersicum/enzimología , Solanum lycopersicum/metabolismo , Proteínas de Plantas , ARN Mensajero/genética , Tetraciclina/farmacologíaRESUMEN
Two genes encoding enzymes in the abscisic acid (ABA) biosynthesis pathway, zeaxanthin epoxidase (ZEP) and 9-cis-epoxycarotenoid dioxygenase (NCED), have previously been cloned by transposon tagging in Nicotiana plumbaginifolia and maize respectively. We demonstrate that antisense down-regulation of the tomato gene LeZEP1 causes accumulation of zeaxanthin in leaves, suggesting that this gene also encodes ZEP. LeNCED1 is known to encode NCED from characterization of a null mutation (notabilis) in tomato. We have used LeZEP1 and LeNCED1 as probes to study gene expression in leaves and roots of whole plants given drought treatments, during light/dark cycles, and during dehydration of detached leaves. During drought stress, NCED mRNA increased in both leaves and roots, whereas ZEP mRNA increased in roots but not leaves. When detached leaves were dehydrated, NCED mRNA responded rapidly to small reductions in water content. Using a detached leaf system with ABA-deficient mutants and ABA feeding, we investigated the possibility that NCED mRNA is regulated by the end product of the pathway, ABA, but found no evidence that this is the case. We also describe strong diurnal expression patterns for both ZEP and NCED, with the two genes displaying distinctly different patterns. ZEP mRNA oscillated with a phase very similar to light-harvesting complex II (LHCII) mRNA, and oscillations continued in a 48 h dark period. NCED mRNA oscillated with a different phase and remained low during a 48 h dark period. Implications for regulation of water stress-induced ABA biosynthesis are discussed.
Asunto(s)
Ácido Abscísico/biosíntesis , Solanum lycopersicum/metabolismo , Ácido Abscísico/farmacología , Northern Blotting , Ritmo Circadiano , ADN sin Sentido/genética , ADN Complementario , Oscuridad , Dioxigenasas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Luz , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Oxidorreductasas/genética , Oxigenasas/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas , Raíces de Plantas/enzimología , Raíces de Plantas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transformación Genética , Agua/farmacología , Xantófilas , Zeaxantinas , beta Caroteno/análogos & derivados , beta Caroteno/metabolismoRESUMEN
The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.
Asunto(s)
Ácido Abscísico/genética , Mutación , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Zea mays/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Dioxigenasas , Datos de Secuencia Molecular , Oxigenasas/química , Oxigenasas/genética , Homología de Secuencia de AminoácidoRESUMEN
The protean manifestations of the clinical presentation of carcinoma of the lung are well known. In the following case report we describe an unusual presentation of such a carcinoma. We further describe this occurrence in Latin as befits what we believe to be a new presentation.
Asunto(s)
Neoplasias de los Bronquios/patología , Carcinoma de Células Escamosas/patología , Estornudo , Terminología como Asunto , Humanos , Masculino , Persona de Mediana Edad , Estornudo/fisiologíaRESUMEN
The ABA biosynthetic pathway has been studied in detail and the steps impaired in some ABA-deficient mutants are known. However, little is known of the molecular control mechanisms regulating ABA production in planta. A direct route for improving our understanding of these mechanisms is to transposon tag and clone the wild-type counterparts of the ABA mutant alleles. On the basis of the observation that maize transposons move preferentially to linked sites in both homologous and heterologous systems and in doing so disrupt gene function, a targeted transposon mutagenesis strategy is being developed towards cloning ABA biosynthetic genes from tomato. The possibility of using marker genes to identify T-DNA insertion sites in selected parts of the genome has been examined and compared with an inverse PCR/RFLP approach to mapping T-DNAs.
RESUMEN
The metabolism of deuterium-labelled analogues of ABA by normal and flacca mutant tomato plants was investigated. Comparison of the biological activity of ABA, ABA alcohol, ABA aldehyde and their 2-trans isomers was made in both mutant and non-mutant genotypes. While in normal plants ABA alcohol and ABA aldehyde were as effective as ABA in inducing stomatal closure, in the flacca mutants only ABA itself was biologically active. Both ABA alcohol and ABA aldehyde were converted to the inactive compound trans- ABA alcohol instead of ABA when fed to flacca plants. As trans-ABA aldehyde was also readily converted to trans-ABA alcohol by flacca plants, it was not possible to establish whether isomerization precedes reduction or vice versa in the synthesis of trans-ABA alcohol from ABA aldehyde.
RESUMEN
A new abscisic acid (ABA) analogue has been isolated from tomato plants. High levels of the compound are found in flacca mutants compared with normal isogenic controls. The analogue also accumulates in response to water stress. Three alternative structures, consistent with the mass spectrum, have been proposed. The possibility that the compound may be a biosynthetic precursor of ABA is considered.
Asunto(s)
Ácido Abscísico/genética , Plantas/genética , Ácido Abscísico/metabolismo , Ácidos Ciclohexanocarboxílicos , Modelos Químicos , Mutación , Plantas/metabolismoRESUMEN
A series of double mutant homozygotes have been produced from three wilty tomato mutants; flacca, sitiens and notabilis. The phenotypic interaction between the mutant genes has been studied. The severity of phenotype in the double mutants does not correspond to that predicted from the single mutant homozygotes. The results are discussed in relation to the probable involvement of the mutants in abscisic acid metabolism.
RESUMEN
An attempt has been made to construct a genetic bridge between the cultivated tomato, Lycopersicon esculentum and its wild relatives L. peruvianum and L. chilense. A complex interspecific hybrid genotype SB 2 has been assembled which shows strong sexual compatibility with both L. esculentum and L. chilense, and a rather weaker degree of compatibility with one specific race of L. peruvianum. The crossing relations of a hybrid between SB 2 and L. peruvianum have also been investigated. This hybrid retains a high level of sexual compatibility with L. esculentum and only shows a slight increase in its ability to set seed on the L. peruvianum parent.
RESUMEN
Two tomato mutants, Lycopersicon esculentum flacca and lateral suppressor, are assigned to map position 59 of chromosome 7. The tight linkage between these two gene loci was detected as a result of attempts to establish whether they would exhibit phenotypic interaction. The possibility that both mutants result in abnormalities of abscisic acid (ABA) accumulation is considered. ABA analysis supports the suggestion that plants homozygous for flacca have a substantially lower concentration but indicates that lateral suppressor homozygotes do not differ from normal in ABA content. An attempt is made to reconcile the results with those of Tucker (1976, New. Phytol. 77, 561-568) by suggesting that lateral suppressor plants may accumulate high levels of an ABA metabolite which is indistinguishable from ABA using the Commelina epidermal strip bioassay.