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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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Subunit A 3' end transcript control amplification. Whole RNA was extracted from seedlings and cDNA was generated as described in materials and methods. Polymerase Chain Reaction amplifications were performed with the appropriate primer pairs. Amplification products were fractionated by agarose gel electrophoresis, transferred to solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. All four transcripts were successfully amplified. Bands produced from the PCR reaction were all of the appropriate, expected size. In addition, the transcript-4 probe successfully detected all four amplification products.
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Figure 5: Subunit A 3' end transcript control amplification. Whole RNA was extracted from seedlings and cDNA was generated as described in materials and methods. Polymerase Chain Reaction amplifications were performed with the appropriate primer pairs. Amplification products were fractionated by agarose gel electrophoresis, transferred to solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. All four transcripts were successfully amplified. Bands produced from the PCR reaction were all of the appropriate, expected size. In addition, the transcript-4 probe successfully detected all four amplification products.

Mentions: Having established that overall subunit A transcript levels were responsive to environmental stress conditions an assay was designed to examine the response of the four individual lengthed transcripts to the same stress conditions. The goal was to determine if any of the four were differentially expressed in response to stress. A PCR based approach was taken to evaluate transcript levels for the four different subunit A mRNAs. Primer pairs were designed (Table 1) that would only amplify one of the four transcripts. This was accomplished by anchoring the downstream primer in the poly (A) tail. The upstream primer was located in the coding region of the subunit A cDNA. Anchoring the downstream primer in the poly (A) tail prevented that oligonucleotide from priming at any other site since 10 out of 18 nucleotides were T's at the 5' end. The primer pairs were designed such that the four amplification products would differ in size by approximately 100 base pairs for ease of visualization on agarose gels and blots (Fig. 5 and Fig. 6). Primer pair T-11 and T-12 corresponds to transcript-1 and produce an amplification product of 579 base pairs. Primer pair T-21 and T-22 corresponds to transcript-2 and amplify a band of 455 base pairs. Primers T-31 and T-32 correspond to transcript-3 and amplify a 362 base pair product. Lastly, primer pair T-41 and T-42 corresponds to transcript-4 and produce a 238 base pair amplification product. The transcript-4 amplification product, which is the smallest and nested within the other three, acted as probe to detect all four transcripts. This nesting was accomplished to avoid differences in signal during hybridization based on different probe lengths and labeling efficiency.


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)

Subunit A 3' end transcript control amplification. Whole RNA was extracted from seedlings and cDNA was generated as described in materials and methods. Polymerase Chain Reaction amplifications were performed with the appropriate primer pairs. Amplification products were fractionated by agarose gel electrophoresis, transferred to solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. All four transcripts were successfully amplified. Bands produced from the PCR reaction were all of the appropriate, expected size. In addition, the transcript-4 probe successfully detected all four amplification products.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC103671&req=5

Figure 5: Subunit A 3' end transcript control amplification. Whole RNA was extracted from seedlings and cDNA was generated as described in materials and methods. Polymerase Chain Reaction amplifications were performed with the appropriate primer pairs. Amplification products were fractionated by agarose gel electrophoresis, transferred to solid support and hybridized with a digoxigenin labeled probe corresponding to the transcript-4 amplification product. All four transcripts were successfully amplified. Bands produced from the PCR reaction were all of the appropriate, expected size. In addition, the transcript-4 probe successfully detected all four amplification products.
Mentions: Having established that overall subunit A transcript levels were responsive to environmental stress conditions an assay was designed to examine the response of the four individual lengthed transcripts to the same stress conditions. The goal was to determine if any of the four were differentially expressed in response to stress. A PCR based approach was taken to evaluate transcript levels for the four different subunit A mRNAs. Primer pairs were designed (Table 1) that would only amplify one of the four transcripts. This was accomplished by anchoring the downstream primer in the poly (A) tail. The upstream primer was located in the coding region of the subunit A cDNA. Anchoring the downstream primer in the poly (A) tail prevented that oligonucleotide from priming at any other site since 10 out of 18 nucleotides were T's at the 5' end. The primer pairs were designed such that the four amplification products would differ in size by approximately 100 base pairs for ease of visualization on agarose gels and blots (Fig. 5 and Fig. 6). Primer pair T-11 and T-12 corresponds to transcript-1 and produce an amplification product of 579 base pairs. Primer pair T-21 and T-22 corresponds to transcript-2 and amplify a band of 455 base pairs. Primers T-31 and T-32 correspond to transcript-3 and amplify a 362 base pair product. Lastly, primer pair T-41 and T-42 corresponds to transcript-4 and produce a 238 base pair amplification product. The transcript-4 amplification product, which is the smallest and nested within the other three, acted as probe to detect all four transcripts. This nesting was accomplished to avoid differences in signal during hybridization based on different probe lengths and labeling efficiency.

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