Action of Nalidixic Acid on Chloroplast Replication in Euglena gracilis

Abstract

The role of light in nalidixic acid bleaching of Euglena gracilis var. bacillaris was investigated. The kinetics of loss of the chloroplast-associated DNA and the sensitivity of chloroplast replication to ultraviolet light was followed during treatment with nalidixic acid. By using the mutant P4ZUL, and 3-(3,4-dichlorophenyl)-, 1-dimethylurea, it was demonstrated that the requirement for light was a functioning photosynthetic electron transport system. Ultracentifugal analysis showed a substantial decrease in chloroplast-associated DNA after 6 hours of treatment with nalidixic acid. Ultraviolet target analysis revealed that the number of chloroplast genomes per cell had been reduced. The possible role of light and implications of the reduction in chloroplast genomes for chloroplast replication are discussed.Chloroplast replication in Euglena gracilis is specifically inhibited by Na12 (5, 6, 9, 13), a potent inhibitor of DNA synthesis (2,7,8 and grown for experiments on a defined organic medium at pH 3.6 (18). Incubation was carried out in gyrotary water bath shakers (New Brunswick) at 26 C ± 0.5 C. The cultures were illuminated with a bank of four 40-w cool white Westingthouse fluorescent bulbs supplemented by cool beam tungsten 150-and 75-w bulbs (General Electric) to give an intensity of 3.6 X 10, ergs/ cm2 sec.Green colony-forming ability was assessed on plates using the above medium (18).Preparation of Nalidixic Acid. A fresh stock solution of Nal' (5 mg/ml) was prepared for each experiment as follows: 100 mg of Nal powder were dissolved in 0.8 ml of 1 N NaOH and distilled H20 added to 100 ml. The solution was sterilized by Millipore fitration. The final concentration of Nal for all experiments was 50 ,ug/ml of culture.Preparation of DCMU. For the stock solution DCMU was dissolved in 95% ethanol. The final concentration for all experiments was 10-sM.DNA Isolation. A total of 3 to 4 X 10' log phase cells was harvested by centrifugation at 3,000 rpm in a Sorvall refrigerated centrifuge at 4 C. The resultant pellet was washed once with distilled H.O followed by a second wash with saline-EDTA. Those cells not extracted immediately were fast frozen in Dry Ice and acetone then stored at -5 C.DNA was isolated using a modification of the Marmur method (12). The pellet was suspended in a 5-ml cold saline-EDTA (0.15 M NaCl, 0.1 M Na2EDTA, pH 8). SLS (25%'o, freshly prepared) was added to a final concentration of 0.2 mg/ml of suspension and lysis was determined by microscopic examination. If lysis was incomplete, the tube was warmed with the hands and/or a small additional amount of SLS added. The material was then incubated with 1 mg/ml pronase (stock solution 10 mg/ml, predigested) at 37 C. The lysate was placed in an ice bath, and 5 M sodium perchlorate was added to a final concentration of 1 M. One volume of chloroform-isoamnl alcohol (24: 1, v/v) was added, and the suspension was placed in an ice bath for 30 min. During this time the emulsion was agitated frequently with a gentle reciprocal motion. Followi...

Isolation and Characterization of Chloroplast DNA from the Marine Chromophyte, Olisthodiscus luteus: Electron Microscopic Visualization of Isomeric Molecular Forms

“…DNA supercoiling which results from the double stranded DNA break-and-reunion activity of these enzymes is suggested to be important to the process of DNA replication, recombination and transcription (10). The fact that ctDNA replication in Euglena is inhibited by naladixic acid (29), an inhibitor which specifically interferes (13) with subunit A activity (nicking-closing) of topoisomerase, supports this hypothesis.…”

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive γ-Irradiation of the Seed

“…Little information is available on the repair of single strand or double stranded breaks after ionizing radiation in plants (19). It seems that some spinach chloroplast division like that in tobacco (1) and Euglena (17) (12), and Possingham (20), an increase in cell size is probably a prerequisite for an increase in chloroplast numbers. The present radiation studies reiterate the relationship between the two processes.…”

“…It is now established that there are many DNA copies in chloroplasts (16). More recently, it has been shown that in Euglena plastids can replicate using a reduced number of genomes (17).…”

Chloroplast replication and loss of chloroplast DNA induced by nalidixic acid in Euglena gracilis.

“…Lyman (15) and Lyman et al (16) concluded from their results that chloroplast replication is inhibited as a result of the loss of the chloroplast DNA induced by NAL . Their studies, however, do not prove that the chloroplasts did not replicate and subsequently were diluted out after the loss of chloroplast DNA.…”

“…Nalidixic acid (NAL), a potent inhibitor of DNA replication that affects DNA gyrase in prokaryotes (6,7,23), is one agent that causes the bleaching of Euglena gracilis (15,16,19,20). Pienkos et al (20) and Lyman et al (16) used CsC1 equilibration analysis to show that NAL induced loss of chloroplast DNA, which resulted in the inability to form green colonies.…”