John A. Bryant

Partial purification and characterization of the soluble DNA polymerase (polymerase-α) from seedlings of Pisum sativum L.

Soluble DNA polymerase has been extracted from pea seedlings and partially purified by chromatography on columns of DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAE-cellulose 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)2SO4. The ion and pH requirements and the sensitivity to N-ethyl maleimide of the pea seedling soluble DNA polymerase are similar to those of the DNA polymerase-α from vertebrates.

Development of nuclease activity in cotyledons of Pisum sativum L.

SummaryThe RNA content of pea cotyledons shows little change during the first five days of germination at 22°C. From day five onwards there is a rapid net degradation of RNA, which continues until day thirteen. The DNA content of the cotyledons increases slightly during the first nine days of germination, after which there is a net decrease. Acid and alkaline ribonuclease activities increase markedly between day one and day five, and then decline between day five and day nine. There is a second increase in the activities of both enzymes from day nine onwards. Soluble deoxyribonuclease activity exhibits a single peak, seven days after the onset of germination. The first increase in acid ribonuclease activity is only partially inhibited by cycloheximide at concentrations which severely inhibit protein synthesis.

Molecular weights of the major DNA polymerases in a higher plant, Pisum sativum L. (PEA).

The molecular weights of the soluble (alpha-like) and chromatin-bound (beta-like) DNA polymerases of pea have been determined. The bulk of the soluble activity consists of molecular species of ca 101,500 and ca 140,000 molecular weights. Smaller (49,000) and larger (182,000 and 234,000) species are also observed in some preparations. The chromatin-bound DNA polymerase exhibits a molecular weight of ca 50,000, although a larger (ca 88,000) species is also detected under conditions which favour aggregate formation.

Chromatin-bound DNA polymerase from higher plants: A DNA polymerase-?-like enzyme

Chromatin-bound DNA polymerase has been extracted from pea (Pisum sativum L.) seedlings, and partially purified by solubilization from chromatin followed by chromatography on columns of either DEAE-cellulose or DEAE-Sephadex. The enzyme elutes from DEAE-cellulose as a single peak, but is fractionated into two peaks, CI and CII, by DEAE-Sephadex chromatography. If the enzyme is stored at-15°C for several days prior to chromatography, a third peak, CIII, derived from CII, is obtained. The polymerase is devoid of nuclease activity, and is relatively insensitive to N-ethyl-maleimide. These features, taken with the ion requirements and with data obtained from other plant species, lead to the suggestion that the chromatin-bound DNA polymerase of higher plants is similar to the DNA polymerase-β from vertebrates.

DNA polymerase activity and DNA synthesis in roots of pea (Pisum sativum) seedlings

Abstract Soluble DNA polymerase (DNA polymerase-α) and chromatin-bound DNA polymerase (DNA polymerase-β) have been assayed in serial sections cut from the roots of 5-day-old pea seedlings. The activity of DNA polymerase-α is high in regions of the root which exhibit high rates of DNA replication, and declines during cell differentiation and maturation. The activity of DNA polyrnerase-β is low in cells which show high rates of DNA replication, and increases during differentiation and maturation.

Correlation between deoxyribonuclease activity and DNA replication in the embryonic axes of germinating peas (Pisum sativum L.)

Crude chromatin preparations from pea seedlings contain calcium-dependent deoxyribonuclease activity, at least part of which is endonucleolytic. During germination, there is a dramatic increase in chromatin-bound deoxyribonuclease activity in the embryonic axis immediately prior to the onset of DNA replication. Evidence has been obtained for the presence of an inhibitor of deoxyribonuclease activity in chromatin preparations from embryonic axes not undergoing DNA replication.

Iso-enzymes of acid ribonuclease in cotyledons of Pisum sativum L.

SummaryAcid ribonuclease from cotyledons of Pisum sativum is very stable, with a temperature optimum of 65° C. It has a molecular weight of 17,500 and there is evidence that a fragment, with a molecular weight of 3,100, retains enzyme activity. Acid ribonuclease from the cotyledons of five-day old seedlings may be fractionated into two iso-enzymes, I and II, by CM-cellulose chromatography. The increase in activity of iso-enzyme I is not inhibited by cycloheximide, whereas the increase in iso-enzyme II activity is strongly inhibited by cycloheximide. Cotyledons of 9-day old seedlings contain only iso-enzyme I, whilst cotyledons of 15-day old seedlings contain three iso-enzymes, I, IIa and III.

Enzymology of nuclear dna replication in plants

DNA replication is a complicated, multi‐step process and must therefore involve an array of different enzyme activities. These enzymes may be loosely associated together to form a replication complex. A number of different enzymes which are assumed to be involved in DNA replication have been identified. These include endodeoxyribonuclease, topoisomerase, “primase”, DNA polymerase, ribonuclease‐H, DNA ligase, and DNa methylase. At least two of these enzymes, DNA polymerase and DNA ligase, exist as multiple forms. The best characterized of the enzymes is DNA polymerase, where it has been possible to assign a role in DNA replication to one specific form, polymerase‐α. With DNA polymerase‐α, there is also some information on regulatory mechanisms, and on subunit composition. By contrast, others of the enzymes listed above, despite partial characterization, have not yet been identified as having a specific function in replication. Further progress will be made by a number of approaches, including further chara...

Partial purification and properties of a chromatin-bound deoxyribonuclease from the embryo axes of germinating pea

Abstract A deoxyribonuclease associated with chromatin extracts of the embryo axes of germinating pea seeds has been partially purified. The enzyme rapidly catalyses the hydrolysis of denatured DNA and has low activity on native DNA substrates. The enzyme can be partially purified from chromatin by solubilization with ammonium sulphate followed by anion-exchange chromatography and gel filtration. The purified DNase is inhibited by 8 mM Ca 2+ and has a M r of ca 18 000. The enzyme hydrolyses the synthetic duplex alternating copolymer poly(dA-dT): poly(dA-dT) at a 10-fold higher rate than the duplex copolymer poly(dG-dC): poly(dG-dC). This, and the limited extent of hydrolysis of native DNA, suggests that the sites of action of the DNase in native DNA are in regions exhibiting ‘structural breathing’, i.e. transient single-strandedness or local single-stranded regions caused by the presence of mismatched bases.

Changes in the activity of poly(adenosine diphosphate-ribose) polymerase during germination of pea

Abstract Poly(adenosine diphosphate-ribose) polymerase activity has been detected in nuclei from pea seedlings. The reaction is a specific polymerization of the adenosine diphosphate-ribose moiety of NAD, giving an average chain length of 2.4 residues. The enzyme shows marked changes in activity during germination: it exhibits a clear peak between 24 and 36 hr of germination, after which the activity declines to the level observed in ungerminated seeds.