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Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea./
Author:	Hsieh, Hsu-Liang.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 1995,
Description:	152 p.
Notes:	Source: Dissertations Abstracts International, Volume: 57-11, Section: B.
Contained By:	Dissertations Abstracts International57-11B.
Subject:	Molecular biology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9617254
ISBN:	9798209396697

Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea.
Hsieh, Hsu-Liang.

Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea. - Ann Arbor : ProQuest Dissertations & Theses, 1995 - 152 p.

Source: Dissertations Abstracts International, Volume: 57-11, Section: B.

Thesis (Ph.D.)--The University of Texas at Austin, 1995.

This item must not be sold to any third party vendors.

A cDNA encoding a 47 kD nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Northern blot analysis shows that the NTPase mRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and farred light, as is characteristic of very low fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene. In addition, the cDNA and truncated cDNA have been successfully expressed in E. coli BL21 (DE 3). The resulting recombinant NTPase and C-terminus of NTPase were used for calmodulin binding and phosphorylation studies. Native pea NTPase and recombinant NTPase but not C-terminus of NTPase are recognized by polyclonal antibody pc480. This suggests that the epitopes recognized by pc480 are located in the N-terminal region of NTPase. The recombinant enzyme also has NTPase activity 70 times higher than the nuclear NTPase using ATP as a substrate. Recombinant NTPase has a preference for ATP more than other nucleoside triphosphate substrates. Calmodulin (CaM) binds to recombinant NTPase, but not to the C-terminus of NTPase. Both recombinant NTPase and the C-terminus of NTPase can be phosphorylated by a recombinant human protein kinase CK II. Phosphorylation does not affect CaM binding to recombinant NTPase. However, CaM does inhibit CK II phosphorylation of recombinant NTPase and this CaM inhibition is blocked by 5 mM EGTA.

ISBN: 9798209396697Subjects--Topical Terms:

517296
Molecular biology.

Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea.
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100 1 $a Hsieh, Hsu-Liang. $3 3688898
245 1 0 $a Molecular cloning and characterization of acDNA encoding a chromatin-associated, calmodulin-regulated nucleoside triphosphatase (NTPase) in pea.
260 1 $a Ann Arbor : $b ProQuest Dissertations & Theses, $c 1995
300 $a 152 p.
500 $a Source: Dissertations Abstracts International, Volume: 57-11, Section: B.
500 $a Publisher info.: Dissertation/Thesis.
500 $a Advisor: Roux, Stanley J.
502 $a Thesis (Ph.D.)--The University of Texas at Austin, 1995.
506 $a This item must not be sold to any third party vendors.
506 $a This item must not be added to any third party search indexes.
520 $a A cDNA encoding a 47 kD nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Northern blot analysis shows that the NTPase mRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and farred light, as is characteristic of very low fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene. In addition, the cDNA and truncated cDNA have been successfully expressed in E. coli BL21 (DE 3). The resulting recombinant NTPase and C-terminus of NTPase were used for calmodulin binding and phosphorylation studies. Native pea NTPase and recombinant NTPase but not C-terminus of NTPase are recognized by polyclonal antibody pc480. This suggests that the epitopes recognized by pc480 are located in the N-terminal region of NTPase. The recombinant enzyme also has NTPase activity 70 times higher than the nuclear NTPase using ATP as a substrate. Recombinant NTPase has a preference for ATP more than other nucleoside triphosphate substrates. Calmodulin (CaM) binds to recombinant NTPase, but not to the C-terminus of NTPase. Both recombinant NTPase and the C-terminus of NTPase can be phosphorylated by a recombinant human protein kinase CK II. Phosphorylation does not affect CaM binding to recombinant NTPase. However, CaM does inhibit CK II phosphorylation of recombinant NTPase and this CaM inhibition is blocked by 5 mM EGTA.
590 $a School code: 0227.
650 4 $a Molecular biology. $3 517296
650 4 $a Cellular biology. $3 3172791
650 4 $a Botany. $3 516217
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710 2 $a The University of Texas at Austin. $3 718984
773 0 $t Dissertations Abstracts International $g 57-11B.
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791 $a Ph.D.
792 $a 1995
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856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=9617254