Genetic Sequences by Electron Microscopy

Year: 1973 DOI: 10.7907/YNNT-V341
Part I: Sequence homology between the DNA molecules of the two temperate Bacillus subtilis bacteriophages, SPO2 and Ø 105, has been mapped by electron microcope observation of heteroduplexes. There is a region of partial homology covering about 14 % of the genome (3.8 x 10⁶ daltons) close to the center of the heteroduplex molecule, flanked by completely non-homologous regions of lengths about 1.2 x 10⁷ and 1.0 x 10⁷ daltons on the two sides. Within the central homologous region there is a characteristic pattern of duplex regions and single-strand loops. The amount of duplex decreases as the denaturing power of Ue solvent used for preparing the electron microscope grids increases; this indicates that the DNA molecules of the two phages are only partially homologous w ithin the homology region. The heteroduplex patterns show that there are no completely homologous nor completely non-homologous gene size sequences within the central region of partial homology. Since the phages are serologically related, we conclude that the antigenic determinants for serological cross-reactivity in the phage tails are coded for by genes in the central region of homology. This conclusion is consistent with available genetic data. Comparison of genetic and physical data indicates that the genes for DNA synthesis and for clear plaque formation in the two phages are non-homologous. The molecuiar weights of SPO2 DNA and Ø 105 DNA are both calculated as 26.3 (± 0.3) x 10⁶ daltons, from length measurements relative to Øx 174 RF II DNA. Both DNA's have cohesive ends and are capable of reversible cyclization the joined ends dissociate more readily that do those of λ DNA. Our physical studies show that each phage DNA consists of a unique linear sequence and is not circularly permuted, in agreement with the conclusion from genetic studies that both phage maps are linear.

Part II: Circular duplex structures of the correct length are observed in the electron microscope in hybridization mixture of lysogen DNA and mature phage DNA for the case of the temperate Bacillus subtilis SPO2. This result shows that the sequence order of the prophage is a circular per mutation of that of the mature phage. By making heteroduplexes of prophage DNA with that of the SPO2 deletion mutants R90 and S25, the att site of the phage has been mapped at 61.2 ± 0.6 % from one end of the mature phage DNA, which has a length of 38,600 base-pairs. In the same coordinate system, the R90 deletion extends from 58.9 ± 0.7 to 66.8 ± 0.8 % on the SPO2 chromosome whereas the S25 deletion extends from 63.2 ± 0.6 to 66.9 ± 0.7 % In similar experiments with lysogen and mature phage DNA's of the temperate B. subtilis phage, Ø 105, no circular structures were seen. This result shows that the sequence order in the prophage and the phage are collinear, without circular permutaion. Short duplex segments, of length 4830 ± 250 base pairs, with two single-strand arms at each end are seen at a low frequency after denaturation and renaturation of B. sublitis DNA. Several lines of ecidence support the hy pothesis that these duplex segments are formed by out-of-register renaturation of the 16s+23s ribosomal RNA genes (rDNA) of B. subtilis. They are of the correct length. Their formation is inhibited if homologous but not if heterologous ribosomal RNA is added to the hybridization mixture. The frequency of occurrence of the duplex structures is consistent with the rDNA hypothesis. Heteroduplex molecules are seen with two or three rDNA duplex segments separated by single-strand substitution loops with specific lengths for each of the two single-strand arms of any one loop. On the basis these structures, linkage groups containing 7 to 9 rDNA sets (each set containing one 16s and one 23s rDNA gene) separated by spacer DNA's are proposed. The evidence indicates that if 5s rDNA is present in the set it is located near one end to give a gene order 16s-23s-5s. All of the 16s rDNA genes are linked to 23s rDNA and vice versa with little or no spacer DNA between a 16s and a 23s sequence. The spacer DNA between 23s and 5s must also be short. The propnage SPO2 bacterial att site maps at a distance 6200 bases away from a 16s+23s rDNA set which is itself separated by a very short spacer (less than 600 bases) from a second rDNA set.

Part III: The genetic sequences of seventeen different λdv's obtained Dr. D.E. Berg have been mapped on λ DNA by the electron microscope heteroduplex method. The physical results are in agreement with the genetic analyses of the contents of the λdv's. Different λdv's exist predominantly either as monomers, or as dimers, or as trimers in recA⁻ carrier cells. Higher order oligomers are also found in allλ λdv preparations, Most of the λdv's originated from λ phage carrying the nin deletion are observed to contain inverted partial duplications (and are called amphimers). That is, the λdv's carry a duplication of all or some of the sequences in an inverted order on the same strand. There are four types of inverted repeat structures: (1) complete inversion and duplication, (2) partial inversion and duplication with a long (5.7 % of λ⁺) non- inverted, non-duplicated (or unique) sequence on the left end covering the immunity region (oriented according to the λ map), (3) partial inversion and duplication with a long (5.1 %) and a short (2.2 %) unique sequence on the left and the tight ends respectively, (4) partial inversion and duplication with a very short (<0.7 %) unique sequence on the right end of the λdv's near the nin deletion. When the closed circular molecules of these inverted λdv's are nicked lightly, denatured by alkali, and then reneutralized, the complementary sequences on the same DNA strand "snap back" to form double-stranded linear molecules. The unique sequences, if any, appear as single-stranded loops at one or both ends when the above treated DNA preparations are mounted by the formamide technique. Lambda phage that does not carry the nin deletion does not produce λdv's with inverted repeats. Denaturation of the open circular molecules of these non-inverted λdv's generates, as expected, only single-stranded circular and single-stranded linear molecules when mounted by the formamide technique. The nin deletion is believed to have caused somehow the formation of the λdv's containing inverted repeats. The left end points of many λdv's map around 73 % on the λ map while the right boundaries of many λdv's map in the vicinity of the nin deletion which extends from 83.8 to 89 .6 % on λ⁺, indicative of two regions of high recombination frequency.