INSTABILITY OF HIGHER PLANT CHROMOSOMAL DNA MOLECULARLY CLONED IN E. COLI VIA RECOMBINANT PLASMIDS

Abstract

This chapter studies the instability of higher plant chromosomal DNA molecularly cloned in E. coli via recombinant plasmids. High molecular weight DNA from Glycine max (soybean) and Arabidopsis thaliana was highly purified to remove polysaccharide contamination by banding in CsCl-PdI gradients. Restriction fragments were enriched 60-fold for the soybean rDNA genes by preparative agarose gel electrophoresis. Restriction fragments of total DNA from both plants and from fractions enriched for rDNA were inserted into the Hind III and Bam HI sites (tetracycline resistance) of plasmid pBR322 and the Eco RI site (chloramphenicol resistance) of plasmid pBR325. Hundreds of independently isolated transformants containing recombinant plasmids were screened. The vast majority of the plant DNA inserts was less than 10 6 daltons. The size of the input DNA varied from 10 6 to 10 7 daltons with a MW av of 4 × 10 6 daltons. It is shown that the cloned rDNA fragments result from a 60% loss of DNA from within the cloned rDNA fragment.