RESTRICTION ENZYME ANALYSIS AND CLONING OF HIGH MOLECULAR WEIGHT GENOMIC DNA ISOLATED FROM CHLORELLA SOROKINIANA (CHLOROPHYTA)1

Abstract

ABSTRACTHigh molecular weight (50–70 kb) genomic DNA was isolated from the eukaryotic green alga, Chlorella sorokiniana spec. nov, (formerly Chlorella pyrenoidosa Chick, strain 7‐11‐05), for restriction endonuclease digestion studies and for preparation of a genomic DNA library. Twenty restriction endonucleases were examined for their abilities to digest this DNA. Nine of the endonucleases gave nearly complete digestion of the DNA, whereas 11 gave only partial digestion. Additional purification steps to remove possible contamination by proteins, RNAs, or polysaccharides did not improve digestion. Digestion studies with pairs of endonuclease isoschizomers, of which one member was sensitive to base methylation, suggested that 5‐methylcytosine might be responsible Jor inhibition of certain endonucleases. Analysis of the DNA showed it to contain 63% GC and to have a high content (5.1 mol %) of 5‐methylcytostne but no other methylated or unusual bases. Evidence indicates that this high 5‐methylcytosine content, which is a characteristic of higher plant genomic DNA rather than of eukaryotic microorganisms, interfered with the cloning of restriction fragments (or fragments produced by mechanical shearing) of C. sorokiniana genomic DNA in standard bacterial host‐strains. Escherichia coli strain K803, which is a permissive host for cloning highly methylated DNA from higher plants, also permitted the cloning of a complete genomic library of 15–20 kb Mbol restriction fragments inserted into the BamHI site of the γ vector, EMBL 3. This C. sorokiniana genomic library appears to be the first genomic‐library constructed for any species of Chlorella.