High level expression of introduced chimaeric genes in regenerated transformed plants

Jones, Jonathan D. G.; Dunsmuir, Pamela; Bedbrook, John

doi:10.1002/j.1460-2075.1985.tb03949.x

Cited by 458 publications

(261 citation statements)

References 20 publications

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“…Genomic DNA was isolated following the procedure described by Raeder & Broda (1985). Total RNA was isolated according to the protocol described by Jones et al (1985). Southern and Northern blotting was performed using Hybond-N+ membranes (Amersham), according to the manufacturer's recommendations.…”

Section: Methodsmentioning

confidence: 99%

Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride

Esquivel-Naranjo¹,

Herrera-Estrella²

2007

Microbiology

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Section: Methodsmentioning

confidence: 99%

Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride

Esquivel-Naranjo¹,

Herrera-Estrella²

2007

Microbiology

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“…Total RNA was extracted from leaf and pericarp ti., -sues according to the method of Jones et al (1985), except the extraction buffer was replaced with 50 mM Tris-HC1 (pH 8.0) containing 4% p-aminosalicylic acid. RNA samples (20 pg each) were fractionated through a 1.2% agarose-formaldehyde gel and transferred to a Zetabind membrane.…”

Section: Rna Extraction and Blot Analysismentioning

confidence: 99%

Cloning of Tomato (Lycopersicon esculentum Mill.) Arginine Decarboxylase Gene and Its Expression during Fruit Ripening

1993

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Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants. The genes encoding ADC have previously been cloned from oat and Fscherichia coli. Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained. This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein. Using this fragment as a probe, we isolated severa1 partial ADC cDNA clones from a tomato pericarp cDNA library. The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene. The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD. The predicted amino acid sequence exhibits 47 and 38% identity with oat and E. coli ADCs, respectively. Cel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues. During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage. No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage. The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/ or posttranslational regulation of ADC gene expression during tomato fruit ripening.

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“…We determined the degree of variability of chimeric gene expression due to position effects (8) and/or cosuppression effects (14,16,17). To this end, we measured E8-Tag mRNA concentration by S1 nuclease analysis in six plants independently transformed with the same E8-Tag gene.…”

Section: Analysis Of Independently Transformed Plantsmentioning

confidence: 99%

Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum)

Deikman¹,

Kline²,

Fischer³

1992

Plant Physiol.

120

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Tomato (Lycopersicon esculentum) fruit ripening is initiated by an increase in ethylene hormone concentration. E8 gene transcription is fruit-specific and is activated at the onset of ripening and in unripe fruit treated with exogenous ethylene. To understand how E8 gene transcription is controlled during ripening, we analyzed the effect of deletions of flanking DNA sequences on E8 gene expression in transgenic tomato fruit. We found that a minimum of three 5' and one 3' regions influence E8 gene expression during fruit ripening. DNA sequences that confer responsiveness to exogenous ethylene in unripe fruit are distinct from DNA sequences that are sufficient for expression during fruit ripening.Ethylene is one of five classical plant hormones that control plant growth and development. Ethylene affects shoot and root growth and differentiation, promotes leaf abscission, induces fruit ripening and flower senescence, and is an important part of the plant's response to wounding and pathogen attack (13). Experiments have shown that ethylene exerts its effect on plant growth and development, at least in part, by controlling the transcription of specific genes (1,11,18). In tomato (Lycopersicon esculentum) fruits, ripening is controlled by an increase in ethylene hormone production (6,9,19,23).The E8 predicted polypeptide is a member of a family of dioxygenases found in plants and microorganisms (15) MATERIALS AND METHODS Plant Material and TransformationTomato (Lycopersicon esculentum cv Ailsa Craig) plants were grown under standard greenhouse conditions. Fruit maturity stage was determined as described (10). To treat with ethylene, unripe fruit were placed in a 25-L chamber and exposed for 6 h to 4.5 L/min of ethylene (10 ,L/L) in humidified air. Construction of Chimeric Genes and Plant TransformationThe E8-Tag construct (4) was used to generate a nested set of 5' deletions as described by Henikoff (7). Deletion end points were determined by DNA sequencing. For plant transformation, genes bearing 5' deletions were subcloned into the EcoRI site of shuttle vector pMLJ1 using EcoRI/HindIII adapters (New England Biolabs). The 3' deletion was constructed using a unique HgaI restriction endonuclease site. This deletion removed 337 bp of 3'-flanking DNA and left 185 bp of DNA 3' of the poly(A) addition site. The E8-Tag plasmid was digested with HgaI, and DNA polymerase was used to fill in the single-stranded ends of the HgaI restriction endonuclease site. EcoRI linkers (New England Biolabs) were ligated to the fragments, which were then digested with EcoRI. The resulting 4.2-kb E8-Tag fragment was purified by agarose gel electrophoresis and ligated into the EcoRI site of pMLJ1. Plant transformation was carried out as described (4)

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High level expression of introduced chimaeric genes in regenerated transformed plants

Cited by 458 publications

References 20 publications

Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride

Enhanced responsiveness and sensitivity to blue light by blr-2 overexpression in Trichoderma atroviride

Cloning of Tomato (Lycopersicon esculentum Mill.) Arginine Decarboxylase Gene and Its Expression during Fruit Ripening

Organization of Ripening and Ethylene Regulatory Regions in a Fruit-Specific Promoter from Tomato (Lycopersicon esculentum)

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