Planta 9 by Springer-Verlag 1978
Planta 138, 127-132(1978)
Partial Purification and Characterization of the Soluble DNA Polymerase (Polymerase-~) from Seedlings of Pisum sativum L. Christine Stevens* and John A. Bryant** Department of Botany, UniversityCollege, P.O. Box 78, Cardiff CF1 1XL, U.K.
Soluble D N A polymerase has been extracted from pea seedlings and partially purified by chromatography on columns of DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAEcellulose as a single peak, but is fractionated into three peaks, SI, SIa and SII by DEAE-Sephadex chromatography. SIa and SII may be derived from SI by freeze-thaw treatment or by treatment with (NH4)2 SO~. The ion and pH requirements and the sensitivity to N-ethyl maleimide of the pea seedling soluble D N A polymerase are similar to those of the D N A polymerase-c~ from vertebrates. Abstract.
Key words: D N A polymerase - Pisum.
Introduction
Although D N A polymerases have been extensively studied in vertebrates, and in lower eukaryotes, including algae, little work has been carried out on the D N A polymerases of higher plants. The limited evidence which is available indicates that higher plants contain one D N A polymerase which is tightly bound to the chromatin (Dunham and Cherry, 1973; Srivastava and Grace, 1974; Robinson and Bryant, 1975) and one which is freely soluble (Stout and Arens, 1970; Srivastava and Grace, 1974; Robinson and Bryant, 1975; Mory etal., 1975; Castroviejo etal., 1975; Gardner and Kado, 1976), although it has been suggested that in beet (Beta vulgaris) both forms of the enzyme are bound to the chromatin Present address."TechnologyPolicy Unit, Universityof Aston, Gosta Green, Birmingham B4 7ET, U.K. ** To whom reprint requests should be addressed *
(Tymonko and Dunham, 1977). In plants in which both enzymes have been studied together, the activity of the soluble enzyme shows a better correlation with D N A synthesis than does the activity of the chromatin-bound enzyme (Srivastava and Grace, 1974; Robinson and Bryant, 1975). This situation is similar to that observed in vertebrates (see review by Keir et al., 1977). The molecular weight of the soluble enzyme has been estimated variously as 1.05x l0 s, 1.10x l0 s and 2.1 x l0 s (Mory et al., 1975; Castroviejo etal., 1975; Gardner and Kado, 1976). These estimates suggest a similarity to D N A polymerase c~, the soluble D N A polymerase of vertebrates. There have been no estimates of the molecular weight of the chromatin-bound enzyme of higher plants, although it has been claimed to have a sedimentation coefficient of 3.4 S, as in vertebrates (Srivastava and Grace, 1975; Tymonko and Dunham, 1977). In contradiction to this, it has also been claimed that higher plants do not possess a low molecular weight D N A polymerase (Chang, 1976; Gardner and Kado, 1976; McLennan and Keir, 1977). The chromatin-bound and soluble polymerases of higher plants differ from each other in pH and ion requirements. In general terms, the soluble polymerase has properties similar to those of D N A polymerase ~ (Robinson and Bryant, 1975; Chang, 1976; McLennan and Keir, 1977) whilst the chromatin-bound enzyme has some properties in common with D N A polymerase ~, the chromatinbound enzyme of vertebrates (Robinson and Bryant, 1975; Stevens 1976; Tymonko and Dunham, 1977). In this laboratory we are characterizing the DNA polymerases of the garden pea (Pisum sativum), and in this paper we report the partial purification and properties of the soluble D N A polymerase. Preliminary reports of part of this work have been published previously in abstract form (Stevens et al., 1975; Stevens and Bryant, 1976).
0032-0935/78/0138/0127/$01.20
128
C. Stevens and J.A. Bryant: DNA Polymerase-c~ in Pea Seedlings 800
Materials and Methods 2.0
Plan ts
Pea seeds (Pisum sativum L., c.v. "Feltham First") were surfacesterilized in sodium hypochlorite (2% available chlorine), washed in running water, and planted in moist vermiculite in seed trays. The trays were kept at 25~ in darkness. Shoot apices were harvested after five days.
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Planta 138, 127-132(1978)
Partial Purification and Characterization of the Soluble DNA Polymerase (Polymerase-~) from Seedlings of Pisum sativum L. Christine Stevens* and John A. Bryant** Department of Botany, UniversityCollege, P.O. Box 78, Cardiff CF1 1XL, U.K.
Soluble D N A polymerase has been extracted from pea seedlings and partially purified by chromatography on columns of DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAEcellulose as a single peak, but is fractionated into three peaks, SI, SIa and SII by DEAE-Sephadex chromatography. SIa and SII may be derived from SI by freeze-thaw treatment or by treatment with (NH4)2 SO~. The ion and pH requirements and the sensitivity to N-ethyl maleimide of the pea seedling soluble D N A polymerase are similar to those of the D N A polymerase-c~ from vertebrates. Abstract.
Key words: D N A polymerase - Pisum.
Introduction
Although D N A polymerases have been extensively studied in vertebrates, and in lower eukaryotes, including algae, little work has been carried out on the D N A polymerases of higher plants. The limited evidence which is available indicates that higher plants contain one D N A polymerase which is tightly bound to the chromatin (Dunham and Cherry, 1973; Srivastava and Grace, 1974; Robinson and Bryant, 1975) and one which is freely soluble (Stout and Arens, 1970; Srivastava and Grace, 1974; Robinson and Bryant, 1975; Mory etal., 1975; Castroviejo etal., 1975; Gardner and Kado, 1976), although it has been suggested that in beet (Beta vulgaris) both forms of the enzyme are bound to the chromatin Present address."TechnologyPolicy Unit, Universityof Aston, Gosta Green, Birmingham B4 7ET, U.K. ** To whom reprint requests should be addressed *
(Tymonko and Dunham, 1977). In plants in which both enzymes have been studied together, the activity of the soluble enzyme shows a better correlation with D N A synthesis than does the activity of the chromatin-bound enzyme (Srivastava and Grace, 1974; Robinson and Bryant, 1975). This situation is similar to that observed in vertebrates (see review by Keir et al., 1977). The molecular weight of the soluble enzyme has been estimated variously as 1.05x l0 s, 1.10x l0 s and 2.1 x l0 s (Mory et al., 1975; Castroviejo etal., 1975; Gardner and Kado, 1976). These estimates suggest a similarity to D N A polymerase c~, the soluble D N A polymerase of vertebrates. There have been no estimates of the molecular weight of the chromatin-bound enzyme of higher plants, although it has been claimed to have a sedimentation coefficient of 3.4 S, as in vertebrates (Srivastava and Grace, 1975; Tymonko and Dunham, 1977). In contradiction to this, it has also been claimed that higher plants do not possess a low molecular weight D N A polymerase (Chang, 1976; Gardner and Kado, 1976; McLennan and Keir, 1977). The chromatin-bound and soluble polymerases of higher plants differ from each other in pH and ion requirements. In general terms, the soluble polymerase has properties similar to those of D N A polymerase ~ (Robinson and Bryant, 1975; Chang, 1976; McLennan and Keir, 1977) whilst the chromatin-bound enzyme has some properties in common with D N A polymerase ~, the chromatinbound enzyme of vertebrates (Robinson and Bryant, 1975; Stevens 1976; Tymonko and Dunham, 1977). In this laboratory we are characterizing the DNA polymerases of the garden pea (Pisum sativum), and in this paper we report the partial purification and properties of the soluble D N A polymerase. Preliminary reports of part of this work have been published previously in abstract form (Stevens et al., 1975; Stevens and Bryant, 1976).
0032-0935/78/0138/0127/$01.20
128
C. Stevens and J.A. Bryant: DNA Polymerase-c~ in Pea Seedlings 800
Materials and Methods 2.0
Plan ts
Pea seeds (Pisum sativum L., c.v. "Feltham First") were surfacesterilized in sodium hypochlorite (2% available chlorine), washed in running water, and planted in moist vermiculite in seed trays. The trays were kept at 25~ in darkness. Shoot apices were harvested after five days.
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o o
1.0 >
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,
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