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Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana.

Magnotta SM, Gogarten JP - BMC Plant Biol. (2002)

Bottom Line: Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated.Etiolation resulted in a slight increase in transcript levels.All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Hartford, West Hartford, CT, USA. smagnotta@mail.hartford.edu

ABSTRACT

Background: Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene.

Results: Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene.

Conclusions: Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

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Vacuolar type H+-ATPase subunit A 3' untranslated region nucleotide sequence. Nucleotide sequence is depicted in the 5' to 3' direction. The diamond (♦) indicates the coding region stop (TAA). Arrows (↓) indicate sites of Poly (A) tail attachment for the four different transcripts amplified. A total of eight cDNA clones were sequenced from the 3' end, in each case the poly (A) tail was added at an adenine residue in the DNA sequence. Bold underlined motifs correspond to putative polyadenylation signals.
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Figure 3: Vacuolar type H+-ATPase subunit A 3' untranslated region nucleotide sequence. Nucleotide sequence is depicted in the 5' to 3' direction. The diamond (♦) indicates the coding region stop (TAA). Arrows (↓) indicate sites of Poly (A) tail attachment for the four different transcripts amplified. A total of eight cDNA clones were sequenced from the 3' end, in each case the poly (A) tail was added at an adenine residue in the DNA sequence. Bold underlined motifs correspond to putative polyadenylation signals.

Mentions: Subunit A of the vacuolar type H+-ATPase in A. thaliana was shown to exhibit multi site polyadenylation. Sequencing the 3' ends of seven independent clones isolated from the PRL-2 cDNA library and one EST obtained through the Arabidopsis EST database revealed four different length transcripts produced from the single subunit A gene based on different poly (A) tail attachment sites (Fig. 3). Multiple polyadenylation sites are fairly common in plant mRNAs but this phenomenon is rare in animal genes [71,72]. Since the A. thaliana V-ATPase subunit A gene produced at least four different transcripts (all producing the same protein) and is one of two flowering plants observed to date not to have a multigene family for this subunit, experiments were designed to determine if muiti site polyadenylation might be a mechanism for gene regulation in plants.


Multi site polyadenylation and transcriptional response to stress of a vacuolar type H+-ATPase subunit A gene in Arabidopsis thaliana.

Magnotta SM, Gogarten JP - BMC Plant Biol. (2002)

Vacuolar type H+-ATPase subunit A 3' untranslated region nucleotide sequence. Nucleotide sequence is depicted in the 5' to 3' direction. The diamond (♦) indicates the coding region stop (TAA). Arrows (↓) indicate sites of Poly (A) tail attachment for the four different transcripts amplified. A total of eight cDNA clones were sequenced from the 3' end, in each case the poly (A) tail was added at an adenine residue in the DNA sequence. Bold underlined motifs correspond to putative polyadenylation signals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC103671&req=5

Figure 3: Vacuolar type H+-ATPase subunit A 3' untranslated region nucleotide sequence. Nucleotide sequence is depicted in the 5' to 3' direction. The diamond (♦) indicates the coding region stop (TAA). Arrows (↓) indicate sites of Poly (A) tail attachment for the four different transcripts amplified. A total of eight cDNA clones were sequenced from the 3' end, in each case the poly (A) tail was added at an adenine residue in the DNA sequence. Bold underlined motifs correspond to putative polyadenylation signals.
Mentions: Subunit A of the vacuolar type H+-ATPase in A. thaliana was shown to exhibit multi site polyadenylation. Sequencing the 3' ends of seven independent clones isolated from the PRL-2 cDNA library and one EST obtained through the Arabidopsis EST database revealed four different length transcripts produced from the single subunit A gene based on different poly (A) tail attachment sites (Fig. 3). Multiple polyadenylation sites are fairly common in plant mRNAs but this phenomenon is rare in animal genes [71,72]. Since the A. thaliana V-ATPase subunit A gene produced at least four different transcripts (all producing the same protein) and is one of two flowering plants observed to date not to have a multigene family for this subunit, experiments were designed to determine if muiti site polyadenylation might be a mechanism for gene regulation in plants.

Bottom Line: Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated.Etiolation resulted in a slight increase in transcript levels.All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, University of Hartford, West Hartford, CT, USA. smagnotta@mail.hartford.edu

ABSTRACT

Background: Vacuolar type H+-ATPases play a critical role in the maintenance of vacuolar homeostasis in plant cells. V-ATPases are also involved in plants' defense against environmental stress. This research examined the expression and regulation of the catalytic subunit of the vacuolar type H+-ATPase in Arabidopsis thaliana and the effect of environmental stress on multiple transcripts generated by this gene.

Results: Evidence suggests that subunit A of the vacuolar type H+-ATPase is encoded by a single gene in Arabidopsis thaliana. Genome blot analysis showed no indication of a second subunit A gene being present. The single gene identified was shown by whole RNA blot analysis to be transcribed in all organs of the plant. Subunit A was shown by sequencing the 3' end of multiple cDNA clones to exhibit multi site polyadenylation. Four different poly (A) tail attachment sites were revealed. Experiments were performed to determine the response of transcript levels for subunit A to environmental stress. A PCR based strategy was devised to amplify the four different transcripts from the subunit A gene.

Conclusions: Amplification of cDNA generated from seedlings exposed to cold, salt stress, and etiolation showed that transcript levels for subunit A of the vacuolar type H+-ATPase in Arabidopsis were responsive to stress conditions. Cold and salt stress resulted in a 2-4 fold increase in all four subunit A transcripts evaluated. Etiolation resulted in a slight increase in transcript levels. All four transcripts appeared to behave identically with respect to stress conditions tested with no significant differential regulation.

Show MeSH