EVIDENCE OF TANDEM DUPLICATION OF GENES IN A MERODIPLOID REGION OF PNEUMOCOCCAL h!T.UTANTS RESISTANT

A Pneumococcal mutant, sulr-c, resistant to sulfonamides, and three transformants bearing associated d or d+ resistance markers have earlier been reported to be unstable and show distinct patterns and frequencies of segregating stable progeny lacking the c marker. Each of the four stra ins showed a characteristic dosage of the genes involved in the merodiploidy. Complementary strands of DNA's from these stable and unstable strains were resolved and homoduplex and heteroduplex hybrids made from the separated DNA strands were used as donors in genetic transformations. Activities of a normal marker (streptomycin resistance) and those involved in the heterozygosity (c, d and d') were quantitatively measured. From those heteroduplexes made up of opposite strands derived from a heterozygote and a stable strain, the normal marker is transferred efficiently, but the heterozygous markers are not. On the other hand, if both strands of a heteroduplex are derived from different heterozygotic strains, all markers can be transferred with usual efficiency to a stable recipient strain. The lowered efficiency in the former type of heteroduplex is attributed to an inhomology resulting from a tandem duplication in the merodiploid strains, and a postulated DNA repair process stimulated by it while in the form of the donor duplex. The inhomology probably includes (a) a microheterogeneity between the c site and the wild type locus, and (b) a more extensive incompatibility attributable to an extra segment of genome in a tandem duplication covering the c and d sites. The first of these inhomologies produces a lowered efficiency of transfer from all configurations of the particular d allele associated with the mutant c marker, and therefore accounts for the characteristic transfer patterns even from the native merodiploid DNA's. T was suggested in a recent report that the heterozygosity and instability of I a certain mutant of Pneumococcus and four transformants derived from it might be attributed to the tandem duplication of the genes in a region of merodiploidy. The unstable mutant (SUP-c) ,a persistent heterozygote, is resistant to sulfonamides and its first progeny contain about 3% drug sensitive cells. In the highly resistant cd transformants that arose through the introduction of (SUP-c) into a stable sulfonamide resistant strain, S U P d , the heterozygosity extends to the closely linked locus d. The marker-transfer frequencies from several different strains bearing c, d and d' markers suggested configurational differences, in the corresponding 1 Present address: Litton Bioneties, Bethesda. Maryland 20014. Genetics 81: 21-31 September, 1975. 22 S. V. S. KASHMIR1 A N D R. D. HOTCHKISS DNA’s. However, no physical evidence was found that these markers were in any way different from other markers carried by DNA. Thus, the marker activities themselves, or the relative marker-to-marker ratios of activity underwent no perturbation or fractionation as the DNA’s were sedimented, melted, denatured or renatured (KASHMIRI and HOTCHKISS 1975) . In this article we explore the degree of homology and inhomology between the DNA of merodiploid c-bearing and of stable strains by measurements of transforming activity of DNA heteroduplexes obtained by hybridization of separated DNA strands. MATERIALS A N D METHODS Strains of Pneumococcus, transformation, and scoring of markers A description of the Pneumococcal strains used and their designations has been given in an accompanying article (KASHMIRI and HOTCHKISS 1975). The stable strains RI-26 (“wild”) and RF6-7 (d) were used as recipients. The unstable merodiploid strains in order of increasing d donor marker activity are: RF3-7, o r c (containing d’ only, therefore ‘“lo”); RF63-10 (cd,); RF63-13 (cd,) and RF63-11 (cd,). Each of these was also available as a streptomycin-and micrococcin-resistant (S,K) doubly transformed strain. Transformation and scoring of markers were accomplished as outlined in the same article. The final concentration of DNA used for transformation of thawed frozen cultures was 0.45 pg DNA/ml. All scoring here was done in agar media. Preparation of homoand hetero-duplex transforming DNA’s Preparation, denaturation and resolution of strands were performed as described in the accompanying article (KASHMIRI and HOTCHHIS 1975) which presents a representative profile of density-fractionated merodiploid DNA. Fractions from two or three independent resolutions were employed for all of the strains utilized. Several fractions at and near each of the density peaks were pooled and dialyzed against 0.15 M NaCl buffered with 0.02M Na phosphate (pH 6.8) to give heavy (H) and light (L) fractions. Equal amounts of the respective fractions were mixed, sodium ion molarity was adjusted t o 0.5M and the mixture, in stoppered tubes, was immersed in refluxing methanol (65”) for an hour followed by chilling in ice. Homoduplexes were made by mixing H and L fractions of the same DNA, whereas complementary-strand fractions derived from two different strains were mixed to make heteroduplexes. For brevity, the heteroduplexes made by mixing complementary-strand fractions of DNA’s from a merodiploid and a stable strain will be referred to as c/non-c heteroduplexes. Likewise, c/c and non-c/non-c will be the respective designations of the heteroduplexes that are made by mixing H and L fractions of DNA’s from two different heterozg gous strains or from two different nonheterozygous strains. Calculation of the recovery of marker aclivities from heteroduplexes The data are presented for each marker in terms of the total activity of that marker summed up for the two reciprocal heteroduplexes relative to the summed activities of the same marker in the two homoduplexes. This way of presentation of the data, rather than the display of the recovery of marker activity in each individual reciprocal heteroduplex, averages the strand-bias effects due to the different transfer efficiencies of the same markers when in their two complementary aspects (GABOR and HOTCHKISS 1966, 1969). Having performed the four different transformations under identical conditions in equivalent aliquots of competent cells. one is permitted to combine and relate the observed activities. To illustrate the method of calculation: Let m and n be the respective activities of a certain marker in the homoduplexes (H, + L,) and (H,, 3Ly), made up of the complementary strands of the DNA’s from any two different strains z and y respectively. Let p and q be the respective TANDEM DUPLICATIONS IN PNEUMOCOCCAL MERODIPLOIDS 23 activities of the heteroduplexes (H, 4L,,) and (HY + L,); then p 4q X 100 represents the combined-marker activities of the two heteroduplexes as percent of the combined activities of the homoduplexes. In case a particular marker is carried by only one strain, say z, then the relationship is simply p 4U X 100; but the magnitude remains of the same order. m + n