The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Partial purification and characterization of four distinct activities

Abstract

Four extramitochondrial DNA polymerases from the marine photosynthetic diatom Cylindrotheca fusiformis were isolated and purified more than 1200-fold by chromatography on DNA-cellulose and DEAE-Sephadex. The enzymes were equally susceptible to inhibition by the thiol-blocking agents N-ethylmaleimide and p-chloromercuribenzoate, the zinc chelator o-phenathroline, and the nucleic acid interchelators ethidium bromide and acriflavin; they displayed similar pH optima, preferred activated DNA, and had strict dependence on high K+ for maximum activity. They were differentiated on the basis of their kinetic parameters, template-primer utilization and salt requirements. The four activities varied with growth stage of C. fusiformis. Activities of polymerases A and D doubled in exponential-phase cells as compared with those in stationary-phase cells, and the increase in polymerase B and chloroplast activity C was 20-40%. The relationship of the diatom polymerases to the complements in other organisms is discussed.

Identification and Expression Pattern of DNA Polymerase α Gene in a Marine Diatom, Skeletonema costatum

“…DNA polymerase activity has been found in spinach, pea, and unicellular algae such as Chlamydomonas, Chlorella, and Euglena (Schönherr and Keir, 1972;Ross and Harris, 1978;Ward and Weissbach, 1986). In a diatom, Cylindrotheca fusiformis Reimann & Lewin, DNA polymerase activity is inhibited by N-ethylmaleimide, and the peak activity coincides with the DNA synthesis phase in the cell cycle (Sullivan and Volcani, 1973;Okita and Volcani, 1977). These properties are similar to the characteristics of DNA polymerase ␣ and ␦ (Burgers, 1989).…”

Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp. strain N1

“…Cylindrotheca N1 ctDNA was purified by first isolating chloroplasts by the procedures described by Okita and Volcani [16]. The isolated chloroplasts were then resuspended in a chloroplast lysis buffer consisting of 0.5~o SDS, 0.7~o sarkosyl, 0.1 M EDTA, 0.1 M NaC1 and 50 mM Tris-C1, pH 8.3, containing 500 #g/ml each of proteinase K and RNase A.…”

Compartmental Analysis Suggests Macropinocytosis at the Onset of Diatom Valve Formation

“…The presence of Si inside cytoplasm and organelles (e.g. mitochondria, microsomes, chloroplasts) as well as its role in controlling DNA replication indicates that Si is present constitutively [5,42]. SIT protein expression is quite constant in the course of girdle band and valve formation, in spite of the fact that mRNA levels increase during girdle band formation [36].…”

“…The K m value of silicic acid transformation inside the SDV to solid silica of the valve (k sv ) was clearly higher (12± 11 μmol/L Si), suggesting a threshold value to initiate valve formation. In this respect one should notice that indeed a threshold of silicic acid controls the expression of DNA polymerase in diatoms [42]. It is important to find out whether these transcription and valve formation thresholds have a concerted mode of action to assure that cell division only proceeds when sufficient silicon is available.…”

“…It is possible that diatom possess some sensory or feedback mechanisms to control the Si concentration in the cell. Although this has to be further investigated, it has been shown that a Si sensory system is involved in regulation of diatom DNA transcription [42]. The transport rate constants could well obey Michaelis-Menten kinetics (Fig.…”