Molecular Size and Conformation of Chloroplast Deoxyribonucleic Acid from Pea Leaves

Richard Kolodner,K.K Tewari

doi:10.1016/s0021-9258(19)44804-7

Richard Kolodner, K.K Tewari

Open Access

https://doi.org/10.1016/s0021-9258(19)44804-7

Copy DOI

Abstract

Abstract The molecular size and conformation of chloroplast (ct-) DNA has been studied by renaturation kinetics and electron microscopy. Ct-DNA of buoyant density 1.698 ± 0.001 g per cm3 showed a Tm of 84° and a homogeneous melting pattern. Denatured and sheared ct-DNA renatured as a single kinetic class with no suggestion of repeating sequences. Ct-DNA is found to have a molecular weight of 95 x 106 by renaturation rate, assuming a value of 106 x 106 for T4 DNA. The lysed chloroplasts are shown to contain 37% of the total DNA in circular molecules of 37 to 42 µ. The remaining DNA is found to be in linear form ranging from 1 to 30 µ. Ct-DNA has been isolated by deproteinization and shown to contain 25% of the DNA in circular form. Supercoiled molecules constitute 30% of the circular molecules. Three per cent of the circular DNA is found in circles of dimer lengths. φX174 RF II DNA and circularized phage λ DNA have been used as internal standards, and the molecular weight of ct-DNA is calculated to be 91 x 106.

Highlights

We have studied ct-DNA from pea leaves by electron microscopy of lysed chloroplasts and of deproteinized DNA
Our results conclusively show that DNA molecules in the chloroplast exist as circular molecules with a contour length of 39 p
Our results show the occurrence of circular c&DNA in higher plants and agrees with the molecular weight of 83 X lo6 reported for c&DNA from Euglena grucilis [10]

Summary

Methods

Isolation of Chloroplust DNA-In a typical experiment, 1 kg of12- to 15-day-old pea leaves was homogenized with 4 liters of buffered medium (Medium A) containing 0.3 M mannitol, 0.05 MTris, pH 8.0, 3 mu EDTA, 0.1% bovine serum albumin, and1 mM mercaptoethanol.The homogenates were filtered through four layers of cheese cloth and centrifuged for 10 min at 100 X g in the Sorvall centrifuge. 12- to 15-day-old pea leaves was homogenized with 4 liters of buffered medium (Medium A) containing 0.3 M mannitol, 0.05 M. The supernatant was centrifuged at 1020 x g for 15 min and the chloroplast pellet (containing broken nuclei and some mitochondria) was suspended in 80 ml of Medium. At the end of the incubation, 240 ml of a medium (Medium B) containing. 0.15 M NaCl and 0.1 M EDTA were added and the mixture was. The pellet was washed twice by suspending it in 120 ml of Medium I~. The pellet was suspended in 10 ml of Medium I~, sodium dcdecyl sulfate was added to a final concentration of 2?(, and the lysed chloroplasts were extracted with an equal volume of phenol buffered with

Results

Discussion

Conclusion