Isolation and molecular cloning of the pneumolysin gene from group 19 chromosomal DNA were performed, using 32P-labeled cloned pneumolysin gene from type 1 strain as a probe (type 2 pneumolysin gene was found to show similar nucleotide sequence as type 1 pneumolysin gene, having 6- location difference among 1,530 nucleotides). The group 19 chromosomal DNA samples were isolated and treated with various restriction enzymes. DNA samples were blotted to nitrocellulose filters and hybridized with a 32P-labeled probe. Type 19F, 19A, 19B and 19C chromosomal DNA exhibited 2 bands in the autoradiograph pictures, following treatment with EcoRI, Hind III, and EcoRV enzymes. In contrast, except for type 19A, these DNA samples showed only 1 band, when treated with ClaI; 5 kb for 19F and 19C, and 9 kb for 19B. Type 19A DNA showed 2 bands, which were 2.2 and 3.5 kb. Thus, group 19 pneumococci have produced pneumolysin molecules, which show the same biological activity to lyse erythrocytes, but may have differeces in their pneumolysin genes. A gene library of ClaI generated 19F DNA was constructed by cloning into the ClaI site of a phagemid vector derived from pUC19. A 5 kb fragment of 19F DNA was isolated, ligated into a ClaI site of the vector, and cloned into E. coli DH5a. The transformed DH5a cells containing 19F 5 kb DNA expressed high pneumolysin activity. Similar studies were continued to ligate the DNA fragment from ClaI-treated chromosomal DNA of other group 19 to the vector and clone into E. coli cells. Genetic characterization of restriction enzyme sites and pneumolysin gene DNA fragments, isolated from ClaI-treated chromosomal DNA samples of group 19 will be studied further.
