RESUMEN
BACKGROUND: Traumatic brain injury (TBI) is a risk factor for dementia and is common, especially among Veterans. It is unknown whether TBI exposure moderates the effect of other common medical/psychiatric comorbidities that are also risk factors for dementia. If treatable or preventable risk factors have a different impact on TBI-exposed Veterans, then this may have important public health implications for dementia prevention. OBJECTIVES: Determine prevalence of common medical/psychiatric comorbidities and associated risk of dementia in Veterans with versus without TBI. DESIGN: Observational cohort. SETTING: Nationwide Veterans Health Administrative data 2001-2019. PARTICIPANTS: After excluding baseline dementia, Veterans age ≥55 years with TBI (N=95,139) were age/sex/race-matched 1:2 with Veterans without TBI (N=190,278). MEASUREMENTS: We compared prevalence of hypertension, coronary artery disease (CAD), diabetes, cerebrovascular disease (CVD), epilepsy, depression, and post-traumatic stress disorder (PTSD) among Veterans with and without TBI. We calculated risk of incident dementia associated with each comorbidity using multivariable hazard ratios (HR) with Fine-Grey competing risk of death adjusted for baseline demographics. We estimated population attributable fraction (PAF) of dementia due to each comorbidity among Veterans with versus without TBI. RESULTS: Prevalence of all comorbidities were significantly more prevalent (5.7% to 21.5% higher) among Veterans compared to those without TBI. All comorbidities were associated with increased risk of dementia in both groups. There were significant interactions between comorbidities and TBI in which HRs were slightly lower among Veterans with TBI (adjusted HRs 1.08-1.37) compared to those without TBI (adjusted HRs 1.12-2.13). Nevertheless, PAFs for dementia due to depression, hypertension, CAD, CVD, and epilepsy were slightly higher in Veterans with TBI due to their high prevalence in this group. CONCLUSIONS: Targeting depression, hypertension, CAD, CVD, and epilepsy may be especially important for dementia risk reduction among Veterans with TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo , Demencia , Hipertensión , Veteranos , Humanos , Persona de Mediana Edad , Estudios de Cohortes , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/epidemiología , Factores de Riesgo , Hipertensión/complicaciones , Hipertensión/epidemiología , Prevalencia , Demencia/complicacionesRESUMEN
OBJECTIVE: Marijuana (MJ) use is common. Research shows risks for psychiatric illnesses, including major depressive disorder (MDD) and cognitive deficits with MJ use, particularly early-onset use. We investigated cognitive function, functional connectivity, and genetic risk with MDD alone and combined with MJ use, and differences between early-vs. late-onset/non-MJ use in youth. METHOD: A total of 74 youth in four groups were studied: healthy control, MDD, frequent MJ use and current/past MDD plus frequent MJ use. Psychiatric symptoms, cognitive performance and demographics were measured. Default mode network (DMN) brain connectivity was determined. Risk alleles in six genes of interest were evaluated. RESULTS: DMN differences among groups in reward-processing and motor control regions were found; the effects of MJ use and MDD were distinct. Early-onset MJ use was associated with lower IQ and hyperconnectivity within areas of the DMN. Early-onset MJ use was associated with the BDNF risk allele. CONCLUSIONS: Cognitive deficits linked with early-onset MJ use were present within several years after MJ use began and may result from, predispose to, or share a common cause with early-onset MJ use. The DMN was affected by MDD, MJ and their combination, as well as by early-onset MJ use. BDNF carrier state may predispose to early-onset MJ use.
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Factor Neurotrófico Derivado del Encéfalo/genética , Trastornos del Conocimiento/inducido químicamente , Trastorno Depresivo Mayor/fisiopatología , Abuso de Marihuana/fisiopatología , Adolescente , Edad de Inicio , Mapeo Encefálico/métodos , Trastornos del Conocimiento/genética , Trastorno Depresivo Mayor/genética , Imagen de Difusión por Resonancia Magnética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Abuso de Marihuana/genética , Abuso de Marihuana/psicología , Adulto JovenRESUMEN
A faster rate of nuclear DNA evolution has recently been found for plants occupying warmer low latitudes relative to those in cooler high latitudes. That earlier study by our research group compared substitution rates within the variable internal transcribed spacer (ITS) region of the ribosomal gene complex amongst 45 congeneric species pairs, each member of which differed in their latitudinal distributions. To determine whether this rate differential might also occur within highly conserved DNA, we sequenced the 18S ribosomal gene in the same 45 pairs of plants. We found that the rate of evolution in 18S was 51% faster in the tropical plant species relative to their temperate sisters and that the substitution rate in 18S correlated positively with that in the more variable ITS. This result, with a gene coding for ribosomal structure, suggests that climatic influences on evolution extend to functionally important regions of the genome.
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ADN de Plantas/genética , Evolución Molecular , Plantas/genética , Clima TropicalRESUMEN
A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.
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Mapeo Cromosómico , Ligamiento Genético , Marcadores Genéticos , Lolium/genética , Repeticiones de Minisatélite/genética , Cruzamientos Genéticos , Genoma de Planta , Linaje , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de SecuenciaRESUMEN
Magnesium (Mg(2+)) is the most abundant divalent cation in plant cells and plays a critical role in many physiological processes. We describe the identification of a 10-member Arabidopsis gene family (AtMGT) encoding putative Mg(2+) transport proteins. Most members of the AtMGT family are expressed in a range of Arabidopsis tissues. One member of this family, AtMGT1, functionally complemented a bacterial mutant lacking Mg(2+) transport capability. A second member, AtMGT10, complemented a yeast mutant defective in Mg(2+) uptake and increased the cellular Mg(2+) content of starved cells threefold during a 60-min uptake period. (63)Ni tracer studies in bacteria showed that AtMGT1 has highest affinity for Mg(2+) but may also be capable of transporting several other divalent cations, including Ni(2+), Co(2+), Fe(2+), Mn(2+), and Cu(2+). However, the concentrations required for transport of these other cations are beyond normal physiological ranges. Both AtMGT1 and AtMGT10 are highly sensitive to Al(3+) inhibition, providing potential molecular targets for Al(3+) toxicity in plants. Using green fluorescence protein as a reporter, we localized AtMGT1 protein to the plasma membrane in Arabidopsis plants. We suggest that the AtMGT gene family encodes a Mg(2+) transport system in higher plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Magnesio/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Aluminio/metabolismo , Aluminio/farmacología , Secuencia de Aminoácidos , Animales , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , División Celular/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Humanos , Magnesio/farmacología , Proteínas de Transporte de Membrana/genética , Ratones , Datos de Secuencia Molecular , Familia de Multigenes/genética , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.
Asunto(s)
Silenciador del Gen , Enfermedades de las Plantas/virología , Hojas de la Planta/virología , Potyvirus/genética , Procesamiento Postranscripcional del ARN , Plantas Modificadas Genéticamente , ARN Viral/metabolismo , Solanaceae , NicotianaRESUMEN
Field studies were conducted in North Carolina to determine the responses of mosquitoes found in salt marsh and inland creek flood plain areas to 1-octen-3-ol (octenol), carbon dioxide (CO2), and light in various combinations with Centers for Disease Control (CDC) light traps. Over 56,000 adult mosquito specimens of 12 species in 4 genera were collected in the salt marsh. They exhibited a general response pattern of octenol + CO2 + light > CO2 + light = octenol + CO2 > octenol + light > octenol alone. Significantly, more Aedes sollicitans, Ae. taeniorhynchus, Anopheles bradleyi, and Culex salinarius were attracted to octenol + CO2 + light than to CO2 + light. Over 19,000 specimens of 24 species in 7 genera were collected in the inland creek flood plain. Although the response patterns to the attractants were similar to those in the salt marsh area, there was no significant difference between octenol + CO2 + light and CO2 + light. Aedes vexans, An. crucians, and An. punctipennis were attracted nearly equally to these two attractant combinations. These studies demonstrate that responses to combinations of these attractants are species specific. However, different combinations of attractants can significantly increase the collection of targeted species important in arbovirus transmission. The use of these combinations would be very beneficial in mosquito-borne virus surveillance studies. The use of octenol by itself or in conjunction with light was found the least useful for collecting mosquitoes in both habitats.
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Luz , Control de Mosquitos/métodos , Octanoles , Aedes , Animales , Anopheles , Culex , Culicidae , Ambiente , Femenino , North CarolinaRESUMEN
Proteasomes are multicatalytic proteinase complexes which play a central role in intracellular protein degradation. They catalyse key events in cell cycle regulation and in the activation of the transcription factor NFkappaB. Proteasome inhibitors have been useful for the characterization of proteasome catalytic components and in the elucidation of proteasome functions in animal cells. Potent small peptide inhibitors of proteasomes also represent a novel approach to the treatment of inflammatory diseases (which involve activation of NFkappaB) and cancer. Such compounds have recently been shown to be effective in a variety of animal models, and at least one is currently in use in clinical trials.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Complejos Multienzimáticos/antagonistas & inhibidores , Inhibidores de Proteasas/uso terapéutico , Animales , Ensayos Clínicos como Asunto , Cisteína Endopeptidasas , Interpretación Estadística de Datos , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Estructura Molecular , Péptido Hidrolasas/metabolismo , Probabilidad , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa ProteasomalRESUMEN
Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and has a high level of activity against insects that infest a variety of crops. Dairy and poultry feeding studies were conducted to determine the magnitude of spinosad residues in animal products that would result from the consumption of typical feed commodities containing residues of spinosad. Dairy cows were dosed for 28 days with spinosad at rates equivalent to 0, 1, 3, and 10 microg/g in the diet. Chicken hens were dosed for 42 days with spinosad at rates equivalent to 0, 0.1, 0.3, 1, and 5 microg/g in the diet. Milk, eggs, and tissue samples were analyzed by high-performance liquid chromatography and/or immunoassay methods. Spinosad residues occurred in all of the sample types but were lowest in eggs, skim milk, and lean meat and were highest in the fat.
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Antibacterianos/análisis , Huevos/análisis , Macrólidos/análisis , Carne/análisis , Leche/química , Residuos de Plaguicidas/análisis , Animales , Combinación de MedicamentosRESUMEN
Metrosideros bartlettii (Myrtaceae) is a distinctive and extremely rare tree, endemic to New Zealand, first discovered in 1975. Prior to this study, a total of 19 adult individuals of the species had been reported; these are located in three small forest remnants in the far north of the North Island of New Zealand. Here we describe a total of 31 adult M. bartlettii at the three sites, including 12 individuals newly discovered by us. We analyse the genetic diversity of the species, using microsatellites to examine the chloroplast genome and amplified fragment length polymorphisms (AFLPs) to monitor nuclear variation. The results clearly demonstrate that M. bartlettii is a unique species, distinct from its two closest relatives M. robusta and M. excelsa. Analysis of genetic diversity within the 31 remaining individuals of M. bartlettii showed an average heterozygosity (< H >) of 0.18 and a proportion of polymorphic genes (< P >) of 0.44. Population structure, as shown by 286 AFLP loci, varied between the three geographical sites; the site with fewest individuals, containing two trees, showed some separation from the populations at the other two locations. These two latter sites, by contrast, had highly overlapping AFLP population diversity profiles. The implications of these results for conservation of the species are discussed.
Asunto(s)
Conservación de los Recursos Naturales , Magnoliopsida/genética , Árboles/genética , ADN de Cloroplastos , Amplificación de Genes , Variación Genética , Heterocigoto , Repeticiones de Microsatélite , Nueva Zelanda , Polimorfismo GenéticoRESUMEN
Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.
Asunto(s)
Frutas/genética , Proteínas de Homeodominio/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Frutas/crecimiento & desarrollo , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes Homeobox , Hibridación in Situ , Datos de Secuencia Molecular , Fenotipo , Plantas Modificadas Genéticamente , Polen/genética , Polen/crecimiento & desarrollo , ARN Mensajero/genética , ARN de Planta/genética , ARN de Planta/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Análisis de Secuencia de ADN , Distribución Tisular , Factores de Transcripción/genéticaRESUMEN
The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.
Asunto(s)
Antígenos Virales , Proteínas de la Cápside , Cápside/fisiología , Nicotiana/virología , Plantas Tóxicas , Potexvirus/fisiología , Rotavirus/fisiología , Vectores Genéticos , Ensamble de VirusRESUMEN
Metrosideros subg. Metrosideros (Myrtaceae) comprises approximately 26 species distributed widely across the Pacific basin. They occur on the ancient Gondwanan landmasses of New Zealand and New Caledonia, as well as on the volcanic islands of the remote Pacific, from Melanesia to tropical Polynesia and the Bonin Island. Phylogenetic analysis based on nuclear ribosomal DNA spacer sequences from all named species showed Metrosideros umbellata of New Zealand as basal in the subgenus, with the remaining species falling into three monophyletic clades. One includes the seven New Caledonian species together with three daughters in western Oceania that probably dispersed during the mid/late Tertiary. A second contains six taxa located in east Melanesia and Samoa that may also have arisen from a mid/late Tertiary dispersal, in this instance from New Zealand. The third includes three New Zealand endemics along with all of the taxa in remote Polynesia and accounts for much of the total range of the subgenus. These dispersed taxa in Polynesia either are identical to the New Zealand species Metrosideros excelsa or differ by a single nucleotide change. We suggest that they are all derived from a Pleistocene dispersal out of New Zealand. A relatively recent dispersal is surprising, given that this wind-dispersed genus has occupied New Zealand for much of the Tertiary and that some of the islands in remote Polynesia date to at least the Miocene. We attribute this dramatic range expansion to climate change-specifically changes in wind flow patterns-in the southern hemisphere during worldwide glaciation.
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Evolución Biológica , Núcleo Celular/metabolismo , ADN Ribosómico/genética , Plantas/genética , Tiempo (Meteorología) , Secuencia de Bases , Datos de Secuencia Molecular , Océano PacíficoRESUMEN
To examine the biological role of Al-stress-induced genes, nine genes derived from Arabidopsis, tobacco (Nicotiana tabacum L.), wheat (Triticum aestivum L.), and yeast (Saccharomyces cerevisiae) were expressed in Arabidopsis ecotype Landsberg. Lines containing eight of these genes were phenotypically normal and were tested in root elongation assays for their sensitivity to Al, Cd, Cu, Na, Zn, and to oxidative stresses. An Arabidopsis blue-copper-binding protein gene (AtBCB), a tobacco glutathione S-transferase gene (parB), a tobacco peroxidase gene (NtPox), and a tobacco GDP-dissociation inhibitor gene (NtGDI1) conferred a degree of resistance to Al. Two of these genes, AtBCB and parB, and a peroxidase gene from Arabidopsis (AtPox) also showed increased resistance to oxidative stress induced by diamide, while parB conferred resistance to Cu and Na. Al content of Al-treated root tips was reduced in the four Al-resistant plant lines compared with wild-type Ler-0, as judged by morin staining. All four Al-resistant lines also showed reduced staining of roots with 2',7'-dichloro fluorescein diacetate (H(2)DCFDA), an indicator of oxidative stress. We conclude that Al-induced genes can serve to protect against Al toxicity, and also provide genetic evidence for a link between Al stress and oxidative stress in plants.
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Aluminio/toxicidad , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Genes de Plantas , Arabidopsis/metabolismo , Resistencia a Medicamentos/genética , Expresión Génica/efectos de los fármacos , Genes Fúngicos , Metales/toxicidad , Estrés Oxidativo/genética , Plantas Modificadas Genéticamente , Plantas Tóxicas , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/genética , Nicotiana/genética , Triticum/genéticaRESUMEN
Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
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Ácidos Borónicos , Cisteína Endopeptidasas/metabolismo , Inhibidores Enzimáticos/metabolismo , Complejos Multienzimáticos/metabolismo , Animales , Células Cultivadas , Quimotripsina/metabolismo , Complejo de la Endopetidasa ProteasomalRESUMEN
We describe the isolation and characterization of 13 cDNA clones that are differentially expressed in male cones of Pinus radiata (D. Don). The transcripts of the 13 genes are expressed at different times between meiosis and microspore mitosis, timing that corresponds to a burst in tapetal activity in the developing anthers. In situ hybridization showed that four of the genes are expressed in the tapetum, while a fifth is expressed in tetrads during a brief developmental window. Six of the seven cDNAs identified in database searches have striking similarity to genes expressed in angiosperm anthers. Seven cDNAs are homologs of defense and pathogen response genes. The cDNAs identified are predicted to encode a chalcone-synthase-like protein, a thaumatin-like protein, a serine hydrolase thought to be a putative regulator of programmed cell death, two lipid-transfer proteins, and two homologs of the anther-specific A9 genes from Brassica napus and Arabidopsis. Overall, our results support the hypothesis that many of the reproductive processes in the angiosperms and gymnosperms were inherited from a common ancestor.
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Cycadopsida/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Edulcorantes , Aciltransferasas/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Brassica/genética , Cycadopsida/citología , ADN Complementario , Meiosis , Mitosis , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Estructuras de las Plantas , Reproducción , Semillas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Árboles/genéticaRESUMEN
Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n > or = 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.
Asunto(s)
Cloroplastos/genética , Cartilla de ADN , Genes de Plantas , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Bases de Datos Factuales , Colorantes Fluorescentes , Variación Genética , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Plantas Tóxicas , Polimorfismo Genético , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Nicotiana/genéticaRESUMEN
We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.
Asunto(s)
CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/genética , Fosfolípidos/metabolismo , Triticum/genética , Secuencia de Aminoácidos , Secuencia de Bases , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Clonación Molecular , Cartilla de ADN , ADN Complementario , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Hojas de la Planta/enzimología , Plantas Modificadas Genéticamente , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Triticum/enzimologíaRESUMEN
Eleven aluminum stress-induced genes derived from plants (wheat, Arabidopsis and tobacco) were introduced into Saccharomyces cerevisiae to test if expression of these genes confers Al tolerance. Al sensitivity tests showed that expression of two genes, either an Arabidopsis gene for blue copper binding protein (BCB), or a tobacco gene for the GDP dissociation inhibitor (NtGDI1), conferred Al tolerance. Determinations of total content and localization of Al ions in these transformants suggested that the BCB gene product functions in restricting Al uptake, while expression of the NtGDI1 gene promotes release of Al ions after uptake.
Asunto(s)
Aluminio/toxicidad , Proteínas de Arabidopsis , Proteínas Portadoras/genética , Proteínas de Unión al GTP/genética , Inhibidores de Disociación de Guanina Nucleótido , Proteínas de Plantas/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Aluminio/farmacocinética , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Proteínas Portadoras/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al GTP/metabolismo , Galactosa/farmacología , Regulación Fúngica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Glucosa/farmacología , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
Early during fruit ripening in kiwifruit (Actinidia deliciosa var. deliciosa [A. Chev.], C.F. Liang and A.R. Ferguson cv. Hayward), starch is broken down to sucrose and hexose sugars. Concomitantly, sucrose-phosphate synthase (SPS, EC 2.3.1.14) activity measured with saturating substrate increased, suggesting that SPS is induced in response to a higher requirement for sucrose synthesis. A 2584 bp long partial cDNA clone encoding SPS was isolated from ripening kiwifruit. cDNA fragments encoding the 5' end were isolated by PCR, and sequencing revealed at least four closely related (> 96% identity) mRNAs expressed early in kiwifruit ripening. Southern hybridisations in a diploid relative of kiwifruit, Actinidia chinensis (Planch.) var. chinensis, were consistent with the presence of a small gene family. Western analysis indicated a 125 kDa SPS protein present in all tissues of A. chitensis at all stages of development. Steady-state levels of SPS mRNA in A. chinensis increased near fruit maturity as net starch degradation began on the vine, and increased again during ethylene treatment of fruit after harvest. After removal from ethylene SPS transcript levels decreased, only to increase again as fruit moved into the climacteric and starch breakdown was completed. Exposure to low temperatures also caused an increase in SPS transcript level. These results indicate that SPS mRNA increases in kiwifruit in response to the presence of new substrate sourced from starch degradation, in response to ethylene and in response to low temperature.