Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events
1990
Newman, S.M. | Boynton, J.E. | Gillham, N.W. | Randolph-Anderson, B.L. | Johnson, A.M. | Harris, E.H.
Transformation of chloroplast ribosomal RNA (rRNA) genesin Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragmentscarrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination ofantibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment lengthpolymorphisms that flank these genes. Complete or nearly complete replacement of a region of thechloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the mostcommon integration event. Exchange events between the homologous donor and recipientsequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genesand flanking sequences into one inverted repeat of the recipient genome was followed byintramolecular copy correction so that both copies of the inverted repeat acquired identical sequences.Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of thechloroplast genome in the recipient cells and by decreasing the heterology between donor and recipientDNA sequences flanking the selectable markers. In addition to producing bona fide chloroplastrRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin,typical of nuclear mutations in Chlamydomonas.
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