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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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Whole genome blot analysis of vacuolar type H+-ATPase subunit A gene in A. thaliana. Each lane represents 5 μg genomic DNA digested with the indicated restriction enzyme. Restriction enzymes used were; H, HindIII, B, BglII, D, DraI, K, KpnI, P, PvuII, E, EcoRI, Ps, PstI, Ev, EcoRv, Bc, BclI, and Ha, HaeIII. Hybridization was to a single band in each lane except for HaeIII due to the presence of a recognition site in the region corresponding to the probe. Positive hybridization control was 20 pg of unlabeled probe DNA generated via PCR. HindIII digested lambda DNA (2 μg) was used as negative control. Numbers to the left of the blot indicate the approximate size of DNA fragments (in kilobases) as determined by HindIII digestion of Lambda DNA.
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Figure 1: Whole genome blot analysis of vacuolar type H+-ATPase subunit A gene in A. thaliana. Each lane represents 5 μg genomic DNA digested with the indicated restriction enzyme. Restriction enzymes used were; H, HindIII, B, BglII, D, DraI, K, KpnI, P, PvuII, E, EcoRI, Ps, PstI, Ev, EcoRv, Bc, BclI, and Ha, HaeIII. Hybridization was to a single band in each lane except for HaeIII due to the presence of a recognition site in the region corresponding to the probe. Positive hybridization control was 20 pg of unlabeled probe DNA generated via PCR. HindIII digested lambda DNA (2 μg) was used as negative control. Numbers to the left of the blot indicate the approximate size of DNA fragments (in kilobases) as determined by HindIII digestion of Lambda DNA.

Mentions: Genome blot analysis was utilized to determine the number of genes in the A. thaliana genome corresponding to subunit A of the vacuolar type ATPase. Genomic DNA was isolated as described, subjected to restriction enzyme digestion, and immobilized on a positively charged nylon membrane. Ten single restriction enzyme digestions were performed. Hybridization was conducted at moderate stringency with a 300 base pair homologous A. thaliana cDNA probe corresponding to subunit A of the vacuolar type ATPase. Results indicated hybridization to only a single band for nine out of the ten enzymes (Fig. 1). The HaeIII digest showed faint hybridization to a second band that was later shown to be the result of a HaeIII site located in the region of the probe (results not shown).


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)

Whole genome blot analysis of vacuolar type H+-ATPase subunit A gene in A. thaliana. Each lane represents 5 μg genomic DNA digested with the indicated restriction enzyme. Restriction enzymes used were; H, HindIII, B, BglII, D, DraI, K, KpnI, P, PvuII, E, EcoRI, Ps, PstI, Ev, EcoRv, Bc, BclI, and Ha, HaeIII. Hybridization was to a single band in each lane except for HaeIII due to the presence of a recognition site in the region corresponding to the probe. Positive hybridization control was 20 pg of unlabeled probe DNA generated via PCR. HindIII digested lambda DNA (2 μg) was used as negative control. Numbers to the left of the blot indicate the approximate size of DNA fragments (in kilobases) as determined by HindIII digestion of Lambda DNA.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Whole genome blot analysis of vacuolar type H+-ATPase subunit A gene in A. thaliana. Each lane represents 5 μg genomic DNA digested with the indicated restriction enzyme. Restriction enzymes used were; H, HindIII, B, BglII, D, DraI, K, KpnI, P, PvuII, E, EcoRI, Ps, PstI, Ev, EcoRv, Bc, BclI, and Ha, HaeIII. Hybridization was to a single band in each lane except for HaeIII due to the presence of a recognition site in the region corresponding to the probe. Positive hybridization control was 20 pg of unlabeled probe DNA generated via PCR. HindIII digested lambda DNA (2 μg) was used as negative control. Numbers to the left of the blot indicate the approximate size of DNA fragments (in kilobases) as determined by HindIII digestion of Lambda DNA.
Mentions: Genome blot analysis was utilized to determine the number of genes in the A. thaliana genome corresponding to subunit A of the vacuolar type ATPase. Genomic DNA was isolated as described, subjected to restriction enzyme digestion, and immobilized on a positively charged nylon membrane. Ten single restriction enzyme digestions were performed. Hybridization was conducted at moderate stringency with a 300 base pair homologous A. thaliana cDNA probe corresponding to subunit A of the vacuolar type ATPase. Results indicated hybridization to only a single band for nine out of the ten enzymes (Fig. 1). The HaeIII digest showed faint hybridization to a second band that was later shown to be the result of a HaeIII site located in the region of the probe (results not shown).

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