AN AMPLIFIABLE DNA REGION FROM THE MYCOPLASMA-HYORHINIS GENOME

A novel amplifiable genomic region that displays variability in the number of tandem copies of a 1,368-bp DNA sequence (designated RS-2) was discovered among individual clonal derivatives within Mycoplasma hyorhinis broth-grown cell populations. Clonal isolates representing variant subpopulations from the original broth culture were of a single size variant, and although continued culture under a variety of growth conditions did not result in further amplification of RS-2, evidence for deletion events which reduced RS-2 copy number, presumably by homologous recombination, was obtained. RS-2 homologous sequences were identified in all M. hyorhinis strains tested, but only the tissue culture-derived strains GDL-1 and GDL-2 showed variability in genomic dosage. The RS-2 nucleotide sequence established that each tandem copy is flanked by direct repeats of a 20-bp sequence and suggested a possible mechanism for its original duplication as the initial phase of a genetic amplification process. The coding strand was defined by PCR amplification of a reverse transcriptase-generated cDNA, and its sequence revealed that RS-2 encodes a 456-residue internal, highly cysteine-rich domain of a larger M. hyorhinis protein whose coding sequence initiates and terminates in unique genomic sequences several hundred base pairs from 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 size-variant protein.