51] Cloning and identification of ribosomal protein genes in chloroplast DNA

Abstract

This chapter describes various methods which are suitable for isolating chloroplast DNA, cloning it into commercially available plasmid vectors, and for screening, identification, and fine mapping of clones which carry r-protein genes. A wider treatment of the cloning and physical mapping problem with respect to plastid DNA is given in this chapter. Chloroplast DNA is a convenient source for isolating clones of ribosomal protein genes. Many restriction enzymes cut chloroplast DNA into a relatively small number (∼20) of fragments of suitable length for cloning. Nearly all the chloroplast-encoded r-protein genes have been identified in several plants and, therefore, a newcomer to the field can obtain clones from other laboratories for use as gene probes. Because of their relatively conserved nucleotide sequence, such gene probes from one plant are suitable for isolating the corresponding genes from any other plant. Finally, chloroplast DNA is a rich source of r-protein genes: there is, on the average, one r-protein gene per 7 kilobase (kb) of chloroplast DNA, as compared to one r-protein gene/100 kb of Escherichia coli DNA.