333The nucleotide sequence relationships between 18 species of Mycoplasma and 3 species of Acholeplasma were examined by solution DNA hybridization. Radiolabelled DNAs from strains representing 13 Mycoplasma and 2 Acholeplasma species were used as hybridization probes, The mycoplasmas were heterogeneous but displayed a low degree of conserved information of the order of 3 to 5 % of the genome. However, several species within each genus showed 13 to 15 % homology. There was no detectable homology between species from the two genera, and M . pneumoniae and M . neurolyticum appeared to be unrelated to any of the other Mycoplasma species or to each other. These results suggest that it may be possible to isolate genes common to most, if not all, Mycoplasma and Acholeplasma species. I N T R O D U C T I O NThe use of nucleotide sequence homology as a taxonomic tool for bacteria and viruses is accepted practice. Likewise, measurement of nucleic acid hybridization kinetics has become a widely employed technique for detecting viruses in infected and transformed cells (Sharp et al., 1974). Before this technique can be extended to the detection of fastidious microorganisms in tissue a good understanding of the nucleotide sequence relationships within the microbial group is necessary. Suitable micro-organisms for this type of study are the mycoplasmas, especially those closely cell-associated. Although the relationships between various mycoplasmas have been reported (Reich et al., 1966a, b ;Somerson et al., 1967;McGee et al., 1967), the work was not exhaustive and utilized either RNA-DNA hybridization or DNA-DNA hybridization in agar columns, which have limits of accuracy that impede the interpretation of low levels of hybridization.The present study utilized the more quantitative solution hybridization procedure followed by analysis of hybrid formation on hydroxyapatite columns (Britten & Kohne, 1966) in an attempt to provide a base of relationship data upon which to build a molecular detection system. In addition, this study utilized a large number of reciprocal hybridizations with 1251-labelled DNA allowing grouping of the micro-organisms into classes based on low degrees of homology.This study demonstrates that there is little homology among different Mycoplasma and Acholeplasma species. However, there is a partial conservation of nucleotide sequences between several different species that are found in the same or similar hosts. M E T H O D SGrowth of mycoplasmas andpurifcation of DNA. with 10 % (v/v) whole horse serum (Irvine Scientific, Santa Ana, Calif., U.S.A.), 0.5 % (w/v) yeast extract and 100 units penicillin ml-l. Confirmation of growth and the absence of contamination were checked by plating 0.1 ml of the broth culture on PPLO agar supplemented as above. Before harvesting, cultures wereGram-stained to check for contamination. Uncontaminated cultures were centrifuged (40 min, 33 OO0 g) and the pellet was resuspended in 0.15 M-NaCl (1Oml) with sodium dodecyl sulphate (SDS, 1 %, w/v, h a 1 concn), 1 m-EDTA and 100...
SUMMARYFour cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22 ~o of the HSV genome. Of five revertant clones selected for aH-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to I. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from I2 to 28 %. The number of virus DNA fragments per cell ranged from I to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-I transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone I39 is not entirely composed of the HSV-I and HSV-2 common sequences.
The thymidine kinase activities present in nine strains (including four type strains and one neotype strain) of nine species of the genus Mycoplusmu and two strains (including one type strain) of two species of the genus Acholeplusmu were characterized with respect to migration in polyacrylamide gels and isoelectric point as determined by electrofocusing. There was a marked heterogeneity in the enzymes, not only between the two genera but also within each genus.Previous investigators have shown that certain mycoplasmas have thymidine kinase (TK) activity (7, 9), and it has been suggested that these organisms might use the scavenger pathway to supply thymidine nucleotides for deoxyribonucleic acid synthesis.The purpose of this study was to examine other mycoplasmas and some acholeplasmas for the presence of TK activity and to characterize the TKs by polyacrylamide gel electrophoresis and isoelectric focusing. MATERIALS AM) METHODSThe sources and designations of the strains of Mycoplasma and Acholeplasma used in this study are listed in Table 1.Cells were grown at 37°C in 40 ml of a medium consisting of beef heart infusion supplemented with 20% whole horse serum, 10% yeast extract, 100 U of penicillin per ml, and 0.002% phenol red. Growth of the organisms and possible contamination were checked by inoculating 0.1 ml of the broth culture onto PPLO agar plates supplemented as described above. The growth from a 40-ml broth culture was harvested by centrifugation of the culture for 30 min at 20, OOO x g in a Ti 60 rotor in an L5-50 Beckman ultracentrifuge; the pellet was then resuspended in 1 ml of 0.1 M tris(hydroxymethy1)aminomethane-hydrochloride (pH 8.0) containing 0.5% Triton X-100. The resulting lysate was centrifuged for 15 min at 35,000 x g, and 100 p1 of the supernatant was assayed for TK activity. TK activity was assayed as described by Munyon et al. (3). The 200-pl TK reaction mixture consisted of 0.07 M KCl, 1.3 mM 2-mercaptoethanol, 2 mM MgC12, 4 mM adenosine triphosphate, 4.5 pCi of [3H]thymidine (60 Ci/mmol; New England Nuclear Corp.), 0.1 M tris(hydroxymethy1)aminomethane-hydrochloride (pH 8.0), and 100 pl of the supernatant from the centrifuged lysate. This was allowed to react for 20 min at 38°C. The reaction was stopped with 35 pl of 50% trichloroacetic acid, and 30-p1 portions were spotted onto DE-81 filters. After drying, the filters were washed four times in 0.001 M ammonium formate buffer (pH 3.6), four times in water, and once in 95% ethanol. The papers were dried and counted in 5 ml of toluene scintillation fluid; 5% polyacrylamide gel electrophoresis and isoelectric focusing were then run on the lysate supernatants which contained TK activity.Electrophoresis was carried out as described by Munyon et al. (3); 5% polyacrylamide was used for the separating gels. Electrophoresis was carried out in 7-cm gel columns at 4OC for approximately 2 h. A 100-pl portion of the enzyme lysate plus 20 pl of 50% glycerol and 0.2% bromophenol blue were added to the top of the gel. A constant current power suppl...
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