Publications by authors named "J Piñas"

18 Publications

  • Page 1 of 1

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

PLoS One 2017 30;12(3):e0174236. Epub 2017 Mar 30.

Institute of Biology Leiden, Faculty of Science, Leiden University, Leiden, The Netherlands.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0174236PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373528PMC
August 2017

Genome interrogation for novel salinity tolerant Arabidopsis mutants.

Plant Cell Environ 2016 12 7;39(12):2650-2662. Epub 2016 Oct 7.

Institute of Biology Leiden, Faculty of Science, Leiden University, Sylviusweg 72, 2333 BE, Leiden, The Netherlands.

Soil salinity is becoming an increasingly large problem in agriculture. In this study, we have investigated whether a capacity to withstand salinity can be induced in the salinity sensitive plant species Arabidopsis thaliana, and whether it can be maintained in subsequent generations. To this end, we have used zinc finger artificial transcription factor (ZF-ATFs) mediated genome interrogation. Already within a relatively small collection Arabidopsis lines expressing ZF-ATFs, we found 41 lines that were tolerant to 100 mM NaCl. Furthermore, ZF-ATF encoding gene constructs rescued from the most strongly salinity tolerant lines were indeed found to act as dominant and heritable agents for salinity tolerance. Altogether, our data provide evidence that a silent capacity to withstand normally lethal levels of salinity exists in Arabidopsis and can be evoked relatively easily by in trans acting transcription factors like ZF-ATFs.
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http://dx.doi.org/10.1111/pce.12805DOI Listing
December 2016

Zinc finger artificial transcription factor-based nearest inactive analogue/nearest active analogue strategy used for the identification of plant genes controlling homologous recombination.

Plant Biotechnol J 2013 Dec 5;11(9):1069-79. Epub 2013 Aug 5.

Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands.

In previous work, we selected a particular transcription factor, designated VP16-HRU, from a pool of zinc finger artificial transcription factors (ZF-ATFs) used for genome interrogation. When expressed in Arabidopsis thaliana under control of the ribosomal protein S5A promoter, the RPS5A::VP16-HRU construct led to a 200- to 300-fold increase in the frequency of somatic intrachromosomal homologous recombination (iHR). Because the expression of each ZF-ATF leads to a large number of transcriptional changes, we designed a strategy employing a collection of structurally similar ZF-ATFs to filter out the transcriptional changes relevant to the phenotype by deep sequencing. In that manner, 30 transcripts were found to be consistently induced in plants with enhanced homologous recombination (HR). For 25 of the cognate genes, their effect on the HR process was assessed using cDNA/gDNA expression constructs. For three genes, ectopic expression indeed led to enhanced iHR frequencies, albeit much lower than the frequency observed when a HR-inducing ZF-ATF was present. Altogether, our data demonstrate that despite the large number of transcriptional changes brought about by individual ZF-ATFs, causal changes can be identified. In our case, the picture emerged that a natural regulatory switch for iHR does not exist but that ZF-ATFs-like VP16-HRU act as an ectopic master switch, orchestrating the timely expression of a set of plant genes that each by themselves only have modest effects, but when acting together support an extremely high iHR frequency.
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http://dx.doi.org/10.1111/pbi.12101DOI Listing
December 2013

ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

Plant Biotechnol J 2013 May 28;11(4):510-5. Epub 2012 Dec 28.

Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, Leiden, The Netherlands.

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10⁻³ per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10⁻³ per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5' and the 3' end of the gene. One plant line contained a PPO gene repaired only at the 5' end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.
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http://dx.doi.org/10.1111/pbi.12040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3719044PMC
May 2013

Frequent hSNF5/INI1 germline mutations in patients with rhabdoid tumor.

Clin Cancer Res 2011 Jan;17(1):31-8

CHU Nantes, Service d'Hémato-Oncologie Pédiatrique, Nantes, France.

Purpose: Germline hSNF5/INI1 mutations are responsible for hereditary cases of rhabdoid tumors (RT) that constitute the rhabdoid predisposition syndrome (RPS). Our study provides the first precise overview of the prevalence of RPS within a large cohort of RT.

Experimental Design: hSNF5/INI1 coding exons were investigated by sequencing and by multiplex ligation-dependent probe amplification.

Results: Seventy-four constitutional DNAs from 115 apparently sporadic RT were analyzed from 1999 to 2009. Germline mutations were found in 26 patients (35%). Data from 9 individuals from 5 RPS families (siblings) were also studied. The median age at diagnosis was much lower (6 months) in patients with germline mutation (P < 0.01) than in patients without (18 months). Nevertheless, 7 of 35 patients with germline mutation (20%) developed the disease after 2 years of age. The mutation could be detected in only 1 parent whereas germline blood DNA was wild type in the 20 other parent pairs, therefore indicating the very high proportion of germ-cell mosaicism or of de novo mutations in RPS. The former hypothesis could be clearly documented in 1 case in which prenatal diagnosis was positive in a new pregnancy. Finally, the 2 years' overall survival was 7% in mutated and 29% in wild-type patients, mainly due to the worse outcome of RT in younger patients.

Conclusions: Our results show a high proportion of germline mutations in patients with RT that can be found at any age and up to 60% in the youngest patients. Genetic counseling is recommended given the low but actual risk of familial recurrence.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-1795DOI Listing
January 2011

ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation.

Plant Biotechnol J 2009 Oct;7(8):821-35

Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden University, 2333 BE Leiden, The Netherlands.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium-mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from approximately 3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.
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http://dx.doi.org/10.1111/j.1467-7652.2009.00446.xDOI Listing
October 2009

Metastasis of amelanotic melanoma of unknown origin in the parotid gland.

Br J Oral Maxillofac Surg 2009 Oct 22;47(7):569-71. Epub 2009 Jan 22.

Hospital General Vall d'Hebrón, Barcelona, Spain.

We report a rare case of metastasis of an amelanotic melanoma of the parotid gland. To our knowledge this is the first report of a metastasis of an amelanotic melanoma in a parotid lymph node. Superficial parotidectomy with no other complementary treatment controlled the tumour. Progress has been excellent and the patient was well at six years' follow-up.
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http://dx.doi.org/10.1016/j.bjoms.2008.10.011DOI Listing
October 2009

Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis.

Plant J 2006 Nov;48(3):475-83

Clusius Laboratory, Department of Molecular and Developmental Genetics, Institute of Biology Leiden, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by fusion of DNA sequences encoding three-finger Cys(2)His(2) ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF sequences, reconstituted ZF-ATFs were re-introduced in the target line. In this manner, a ZF-ATF was identified that led to a 200-1000-fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR-inducing ZF-ATF exhibited increased resistance to the DNA-damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.
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http://dx.doi.org/10.1111/j.1365-313X.2006.02877.xDOI Listing
November 2006

Selection of Arabidopsis mutants overexpressing genes driven by the promoter of an auxin-inducible glutathione S-transferase gene.

Plant Mol Biol 1999 Mar;39(5):979-90

Institute of Molecular Plant Sciences and RUL-TNO Centre for Phytotechnology, Leiden University, The Netherlands.

Transgenic arabidopsis plants were isolated that contained a T-DNA construct in which the promoter of an auxin-inducible glutathione S-transferase (GST) gene from tobacco was fused to the kanamycin resistance (nptII) as well as to the beta-glucuronidase (gusA) reporter gene. Subsequently, seeds were treated with EMS to obtain mutants in which both reporter gene fusions were up-regulated. Northern analysis showed that the mRNA level of a related, endogenous auxin-inducible GST gene of Arabidopsis was increased in some of these mutants as well. Two of the gup (GST up-regulated) mutants were characterized in more detail and roughly mapped. Both had epinastic cotyledons and leaves, a phenotype that turned out to be linked to the gup mutation.
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http://dx.doi.org/10.1023/a:1006129426712DOI Listing
March 1999

Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation.

Plant Physiol 1999 Mar;119(3):1047-55

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands.

We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC32086PMC
http://dx.doi.org/10.1104/pp.119.3.1047DOI Listing
March 1999

[Nitric oxide synthase activity in nasal mucosa].

Acta Otorrinolaringol Esp 1998 Oct;49(7):503-7

Departamento de ORL, Hospital General Universitario Valle de Hebrón, Barcelona.

Nitric oxide (NO) plays an important regulatory role in airway function and seems to be implicated in the pathophysiology of several airway diseases. We studied the presence of NO synthase activity in human nasal mucosa and nasal polyp tissues obtained from patients undergoing septoplasty or polypectomy, respectively. NO synthase activity was quantified in tissue homogenates using citrulline release assay and was located in tissue sections using NADPH-diaphorase histochemistry. The results indicated that nasal polyps contain higher levels of total NO synthase activity than nasal mucosa tissue. In addition, nasal polyps contained mainly inducible NO synthase activity whereas all NO synthase activity detected in the nasal mucosa was in constitutive form. In both cases, NO synthase activity was localized in epithelial cells. In view of these results, we conclude that NO may be an important inflammatory mediator in the respiratory system and that the epithelium may be a source of NO production.
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October 1998

[Omeprazole: a new treatment for paranasal sinus polyps in Widal syndrome. Preliminary study].

Acta Otorrinolaringol Esp 1998 May;49(4):297-9

Servicio de ORL, Hospital General Universitario Vall d'Hebron, Barcelona.

A preliminary report is made of the potential therapeutic effect of omeprazol in reducing nasosinusal polyps. This study is based on the empirical observation of nasal airflow improvement in patients suffering from nasosinusal polyposis after administering omeprazol. Different phases of the study suggested that patients with Widal's syndrome benefited the most. Based on the results of this study, we have undertaken a randomized, parallel, double-blind, placebo-controlled clinical trial.
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May 1998

[Recurrent peripheral facial paralysis. Our case load from 1995].

Acta Otorrinolaringol Esp 1998 May;49(4):280-2

Servicio de ORL, Hospital General Universitario Vall d'Hebron, Universidad Autónoma, Barcelona.

Of 117 cases of peripheral facial palsy seen in our emergency room in 1995, 10 (8.5%) were relapses. Seven were males and 3, females. Mean age at the first appearance was 23.6 years. The mean interval between the first facial palsy and the first recurrence was 10.1 years, and between the first and second recurrence, 4.6 years. Five of the 10 patients (50%) had a second recurrence. Two of the 10 patients (20%) had a family history of facial palsy. Only 1 (10%) was diabetic. Seven of 10 patients achieved a complete functional recovery and 3 had sequelae. Computed tomography was normal in every patient. In our series, a family history of facial palsy and low ENoG results were related with a poorer prognosis. ENoG scan was used as a prognostic and follow-up factor.
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May 1998

[An etiopathogenetic and prognostic study of sudden sensorineural hypoacusis].

An Otorrinolaringol Ibero Am 1990 ;17(6):605-40

Servicio ORL, Residencia Valle Hebron, Barcelona.

From October 1987 till November 1989 the AA. have studied 40 cases with Sensorineural sudden hypoacusis. The control term varied between 3 months and 2 years. The whole group followed an scheduled prospective protocol with the aim of find out the more significative factors regarding the etiology and prognostic of the process. On the other hand, minimize the cost of explorations, spare nuisances to the patients and select the most reliable methods were the purposes of the AA.
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April 1991

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum.

Exp Cell Res 1988 Dec;179(2):332-43

Cell Biology and Genetics Unit, University of Leiden, The Netherlands.

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.
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http://dx.doi.org/10.1016/0014-4827(88)90272-8DOI Listing
December 1988

Postaggregative differentiation induction by cyclic AMP in Dictyostelium: intracellular transduction pathway and requirement for additional stimuli.

Dev Biol 1986 Nov;118(1):52-63

Cyclic AMP induces postaggregative differentiation in aggregation competent cells of Dictyostelium by interacting with cell surface cAMP receptors. We investigated the transduction pathway of this response and additional requirements for the induction of postaggregative differentiation. Optimal induction of postaggregative gene expression requires that vegetative cells are first exposed to 2-4 hr of nanomolar cAMP pulses, and subsequently for 4-6 hr to steady-state cAMP concentrations in the micromolar range. Cyclic AMP pulses, which are endogenously produced before and during aggregation, induce full responsiveness to cAMP as a morphogen. The transduction pathway from the cell surface cAMP receptor to postaggregative gene expression may involve Ca2+ ions as intracellular messengers. A cAMP-induced increase in intracellular cAMP or cGMP levels is not involved in the transduction pathway.
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http://dx.doi.org/10.1016/0012-1606(86)90072-2DOI Listing
November 1986

Analogs of cyclic AMP as chemoattractants and inhibitors of Dictyostelium chemotaxis.

J Bacteriol 1982 Jan;149(1):99-105

Aggregative amoebae of Dictyostelium discoideum, D. mucoroides, D. purpureum, and D. rosarium react chemotactically to cyclic AMP (cAMP). We measured the chemotactic activity of 14 cAMP analogs and found that these four species have a similar sensitivity to chemical modifications of cAMP; this suggests that the cAMP receptor is identical in all of these species. Besides the induction of a chemotactic response, cAMP analogs also may delay or prevent cell aggregation. cAMP analogs like N1-O-cAMP, 2'-H-cAMP, and 5'NH-cAMP are chemotactically nearly as active as cAMP and induced no, or only a short, delay of cell aggregation. Other cAMP derivatives, such as 6-Cl-cPMP and 8-Br-cAMP, are chemotactically active only at high concentrations and delayed cell aggregation for several hours. Still other cAMP analogs, which do not induce a chemotactic reaction in D. mucoroides, D. purpureum, and D. rosarium, either prevented cell aggregation [cAMPS(S), cAMPS(R), and 3'-NH-cAMP[ or had no effect on cell aggregation [cAMPN(CH3)2(S) and cAMPN(CH3)2(R)]. cAMP analog 3'-NH-cAMP prevented cell aggregation by the inhibition of chemotaxis, whereas cell locomotion was not affected. Although we cannot provide a satisfactory explantation for these observations, our data suggest that occupation and activation of the cAMP receptors do not always induced a chemotactic response.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC216596PMC
http://dx.doi.org/10.1128/JB.149.1.99-105.1982DOI Listing
January 1982

[Contribution to the study of the crying cat syndrome].

Authors:
J Piñas

Acta Otorinolaryngol Iber Am 1970 ;21(1):38-44

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September 1970