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Lentiviral vectors open exciting perspectives for the genetic treatment of a wide array of inherited and acquired diseases, owing to their ability to govern the efficient delivery, integration, and long-term expression of transgenes into nondividing cells both in vitro and in vivo. The genomic complexity of HIV, where a whole set of genes encode virulence factors essential for pathogenesis but not required for gene transfer, allowed a major step toward clinical acceptability through the creation of multiply attenuated packaging systems. Until now, however, vector particles could only be produced by transient transfection because no high-output, stable packaging cell line was available that produced the latest generation of HIV-based vectors. Here we describe such a line, based on the doxycycline-repressible expression of HIV-1 Rev/Gag/Pol and of the vesicular stomatitis virus G envelope (VSV G) in 293 human embryonic kidney cells. Upon induction, the LVG clones can produce 1 to 20 HeLa-transducing units per cell per day for about a week, a yield that compares favorably with that of transiently transfected 293T cells. These virions exhibit functional properties similar to those of viruses produced transiently, in particular the ability to transduce nonmitotic targets. This system will facilitate the further development of lentiviral vectors for gene therapy.
Mol Ther 2000 Aug
PMID:A stable system for the high-titer production of multiply attenuated lentiviral vectors. 1094 45

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds. Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants. This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.
Plant Mol Biol 2001 Feb
PMID:Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana. 1129 73

Two closely related, tandemly arranged, low-temperature- and salt-induced Arabidopsis genes, corresponding to the previously isolated cDNAs RCI2A and RCI2B, were isolated and characterized. The RCI2A transcript accumulated primarily in response to low temperature or high salinity, and to a lesser extent in response to ABA treatment or water deficit stress. The RCI2B transcript was present at much lower levels than RCI2A, and could only be detected by reverse transcription-PCR amplification. The predicted 6 kDa RCI2 proteins are highly hydrophobic and contain two putative membrane-spanning regions. The polypeptides exhibit extensive similarity to deduced low-temperature- and/or salt-induced proteins from barley, wheat grass and strawberry, and to predicted proteins from bacteria, fungi, nematodes and yeast. Interestingly, we found that a deletion of the RCI2 homologous gene, SNA1 (YRD276c), in yeast causes a salt-sensitive phenotype. This effect is specific for sodium, since no growth defect was observed for the sna1 mutant on 1.7 M sorbitol, 1 M KCl or 0.6 M LiCl. Finally, we found that the Arabidopsis RCI2A cDNA can complement the sna1 mutant when expressed in yeast, indicating that the plant and yeast proteins have similar functions during high salt stress.
Plant Mol Biol 2001 Feb
PMID:The low-temperature- and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1. 1129 79

We have previously shown that mRNA and protein encoded by the Pvlea-18 gene from Phaseolus vulgaris L., a member of a new family of late embryogenesis-abundant (LEA) proteins, accumulate in dark-grown bean seedlings not only in response to water deficit but also during optimal irrigation. In this work, we studied Pvlea-18 gene transcriptional regulation by using transgenic Arabidopsis thaliana plants containing a chimeric gene consisting of the Pvlea-18 promoter region and the 3'-nos terminator fused to the GUS gene-coding region. We demonstrate that the chimeric gene is active during Arabidopsis normal development under well-irrigated conditions, and that it is further induced in response to ABA and dehydration treatments. Replacing the 3'-nos terminator with the Pvlea-18 3' region led to an additional increase in expression during development and in response to dehydration, but not in response to exogenous ABA. These results reveal an enhancer effect of the Pvlea-18 3' region, which showed to be higher specifically under dehydration. The small decrease in Pvlea-18 promoter expression observed when transgenic plants treated with fluridone (an ABA biosynthesis inhibitor) were subjected to dehydration suggests that the Pvlea-18 gene dehydration response is predominantly ABA-independent. Finally, we present evidence indicating that Pvlea-18 gene expression is negatively regulated during etiolated growth, particularly in roots, in contrast to the expression pattern observed during normal development.
Plant Mol Biol 2001 Mar
PMID:Downstream DNA sequences are required to modulate Pvlea-18 gene expression in response to dehydration. 1141 10

Of the growing list of promising genes for plant improvement, some of the most versatile appear to be those involved in sugar alcohol metabolism. Mannitol, one of the best characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of mannitol as both a metabolite and an osmoprotectant in celery (Apium graveolens) are well documented. However, there is growing evidence that 'metabolites' can also have key roles in other environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may have significant roles in plant-pathogen interactions. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) is a prime modulator of mannitol accumulation in plants. Because the complex regulation of MTD is central to the balanced integration of mannitol metabolism in celery, its study is crucial in clarifying the physiological role(s) of mannitol metabolism in environmental and metabolic responses. In this study we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data show that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis. These include up-regulation by salicylic acid, hexokinase-mediated sugar down-regulation, and down-regulation by salt, osmotic stress and ABA. In contrast, the massive up-regulation of Mtd expression in the vascular tissues of salt-stressed Arabidopsis roots suggests a possible role for MTD in mannitol translocation and unloading and its interrelation with sugar metabolism.
Plant Mol Biol 2001 Nov
PMID:Analysis of celery (Apium graveolens) mannitol dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses. 1172 47

Responses to drought and salinity in barley (Hordeum vulgare L. cv. Tokak) were monitored by microarray hybridization of 1463 DNA elements derived from cDNA libraries of 6 and 10 h drought-stressed plants. Functional identities indicated that many cDNAs in these libraries were associated with drought stress. About 38% of the transcripts were novel and functionally unknown. Hybridization experiments were analyzed for drought- and salinity-regulated sequences, with significant changes defined as a deviation from the control exceeding 2.5-fold. Responses of transcripts showed stress-dependent expression patterns and time courses. Nearly 15% of all transcripts were either up- or down-regulated under drought stress, while NaCl led to a change in 5% of the transcripts (24 h, 150 mM NaCl). Transcripts that showed significant up-regulation under drought stress are exemplified by jasmonate-responsive, metallothionein-like, late-embryogenesis-abundant (LEA) and ABA-responsive proteins. Most drastic down-regulation in a category was observed for photosynthesis-related functions. Up-regulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress. A number of functionally unknown transcripts from cDNA libraries of drought-stressed plants showed up-regulation by drought but down-regulation by salt stress, documenting how precisely transcript profiles report different growth conditions and environments.
Plant Mol Biol
PMID:Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. 1199 34

The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions. We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis. Antisense constructs of cDNAs for senescence-related 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation. Several transgenic lines showed higher PA contents than wild-type plants. The number and weight of seeds also increased. Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment. These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants. This shows a positive correlation between PA content and stress tolerance in plants.
Mol Cells 2002 Apr 30
PMID:Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants. 1201 42

We have isolated and characterized a gene encoding cytosolic glutathione reductase from Brassica campestris (B. campestris). The gene (BcgGR1) is presented as a single copy in the B. campestris genome and is composed of 17 exons and 16 introns in the trancribed region with coding sequence beginning in the 2nd exon and ending in the 17th exon. BcgGR1 is expressed strongly in roots and calli, and moderately in stems and leaves. The transcription is strongly induced by various stress treatments including ozone, paraquat, salt, hydrogen peroxide, chilling or ABA but depressed by heat treatment. The transcript level of BcgGR1 is increased significantly at 2 h after the onset of ozone (300 ppb), paraquat (10 microM) or salt (250 mM NaCI) treatments and reached a maximum level by 10-24 h. However, the maximum induction of BcgGR1 is reached at 2-4 h after the onset of hydrogen peroxide (10 mM), chilling (10 degrees C) or ABA (1 mM) treatments. The rapid reduction of BcgGR1 transcripts after 4 h in ABA treatment is distinguished from hydrogen peroxide and chilling treatments.
Mol Cells 2002 Apr 30
PMID:Genomic cloning and characterization of glutathione reductase gene from Brassica campestris var. Pekinensis. 1201 46

A wound-inducible Arabidopsis plastid omega-3 fatty acid desaturase (fad7) cDNA was obtained. Transgenic tobacco plants were produced by integration of the antisense fad7 DNA fragments under the control of a CaMV 35S promoter into the genome. Two transgenic T1 lines, AsFAD714 and 716, showed a strong expression of the antisensefad7 and reduced amounts of linolenic acid compared with the control plants. The two T1 lines were highly sensitive to dehydration conditions, showing growth retardation on the MS medium in the presence of 250 mM NaCl, and severe wilting under drought conditions. The expression of the transcriptional factor gene abf4 transducing ABA-dependent signal in response to drought stress was strongly induced in the control plants, but far less in the AsFAD716 line. This suggests that the inhibitory effect of the antisense fad7 gene expression on the ABF-mediated stress-responsive gene regulation may reduce drought tolerance in the AsFAD716 line. However, no significant difference in the ABA concentration was found between the control and the AsFAD716 line under normal and drought conditions.
Mol Cells 2002 Apr 30
PMID:Antisense expression of an Arabidopsis omega-3 fatty acid desaturase gene reduces salt/drought tolerance in transgenic tobacco plants. 1201 49

Homeodomain leucine zipper (HDZip) genes encode putative transcription factors that are unique to plants. A function in regulating processes that are specific for plants is postulated, such as responses to environmental cues and developmental signals. This is supported by a growing body of evidence resulting from studies of HDZip genes in a variety of species. In addition to the previously isolated CPHB-1 and -2 genes, this paper reports the isolation of members of five families of Craterostigma plantagineum homeobox leucine zipper genes (CPHB) via a yeast one-hybrid screening approach. Based on the sequence homology and protein interactions the encoded proteins (CPHB-3/4/5/6/7) were classified into HDZip class II and I genes. Homo- and heterodimerization of CPHB proteins within the same structurally related class has been demonstrated and the DNA-binding activity of CPHB proteins to two homeodomain recognition elements (HDE1 and HDE2) has been compared in yeast. All families of CPHB genes were modulated in their expression in response to dehydration in leaves and roots. CPHB-6 and CPHB-7 transcripts accumulated in leaves during early stages of dehydration and decreased after prolonged dehydration. Both transcripts were also induced in ABA-treated callus. CPHB-3/4/5 were down-regulated by dehydration in both leaves and roots. The results support the role of HDZips in regulating programs of gene expression in C. plantagineum that lead to desiccation tolerance.
Plant Mol Biol 2002 Aug
PMID:Characterization of five novel dehydration-responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum. 1208 68


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