Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events

• Published in: Genetics (Austin) Vol. 126; no. 4; pp. 875 - 888
• Main Authors: Newman, S.M. (Duke University, Durham, NC), Boynton, J.E, Gillham, N.W, Randolph-Anderson, B.L, Johnson, A.M, Harris, E.H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Genetics Soc America 01.12.1990; Genetics Society of America
• Subjects: ADN; ANTIBIOTICOS; ANTIBIOTIQUE; ARN RIBOSOMAL; ARN RIBOSOMIAL; Biological and medical sciences; Brackish; Chlamydomonas; Chlamydomonas - genetics; CHLOROPHYCEAE; CHLOROPLASTE; Chloroplasts - metabolism; Cloning, Molecular; CLOROPLASTO; DNA, Ribosomal - genetics; Drug Resistance - genetics; FENOTIPOS; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genic rearrangement. Recombination. Transposable element; GENOMAS; GENOME; Investigations; MEDICAMENT; MEDICAMENTOS; Molecular and cellular biology; Molecular genetics; MUTACION INDUCIDA; Mutation; MUTATION PROVOQUEE; PHENOTYPE; POLIMORFISMO; Polymorphism, Restriction Fragment Length; POLYMORPHISME; RESISTANCE AUX PRODUITS CHIMIQUES; RESISTENCIA QUIMICA; Restriction Mapping; RIBOSOMAS; RIBOSOME; RNA, Ribosomal, 16S - genetics; RNA, Ribosomal, 23S - genetics; Spectinomycin - pharmacology; Streptomycin - pharmacology; TRANSFORMACION GENETICA; TRANSFORMATION GENETIQUE; Transformation, Genetic; Integration; Recombination; Genetic transformation; Algae; Chlorophycophyta; Chloroplast DNA; Ribosomal DNA; 23S-»RNA; Resistance; Antibiotic; Gene; Chlamydomonas reinhardtii; 16S-RNA; Chloroplast; Thallophyta
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/126.4.875

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying 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 of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector: insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular 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 the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas