AN AMPLIFIABLE DNA REGION FROM THE MYCOPLASMA-HYORHINIS GENOME

A novel amplifiable genomic region that displays variability in the nu mber of tandem copies of a 1,368-bp DNA sequence (designated RS-2) was discovered among individual clonal derivatives within Mycoplasma hyor hinis broth-grown cell populations. Clonal isolates representing varia nt subpopulations from the original broth culture were of a single siz e variant, and although continued culture under a variety of growth co nditions did not result in further amplification of RS-2, evidence for deletion events which reduced RS-2 copy number, presumably by homolog ous recombination, was obtained. RS-2 homologous sequences were identi fied in all M. hyorhinis strains tested, but only the tissue culture-d erived strains GDL-1 and GDL-2 showed variability in genomic dosage. T he RS-2 nucleotide sequence established that each tandem copy is flank ed by direct repeats of a 20-bp sequence and suggested a possible mech anism for its original duplication as the initial phase of a genetic a mplification process. The coding strand was defined by PCR amplificati on of a reverse transcriptase-generated cDNA, and its sequence reveale d that RS-2 encodes a 456-residue internal, highly cysteine-rich domai n of a larger M. hyorhinis protein whose coding sequence initiates and terminates in unique genomic sequences several hundred base pairs fro m RS-2 on either side of it. Changes in RS-2 copy number maintain the reading frame, and therefore the coding capacity, for this predicted s ize-variant protein.