Acta Biologica 30. (1979)
4. szám - Fehér Zs.–Schablik M.–Szabó G.: Hybridization of DANN's from Neurospora crassa strains may indicate base sequence alterations as a consequence of genetic transformation
388 G. SZABÓ et ai. The markedly increased frequency of aberrant tetrads can be interpreted either as the result of gene conversions or as a consequence of diploidization and haploidization events occurring during meiosis [15, 16, 17]. Both interpretations presume base sequence alterations in the recipient DNA caused by exogenous donor DNA integrated at several sites. Based on this supposition we made an attempt in the present studies to demonstrate by means of DNADNA hybridization if there is any detectable difference between transformant inl+ and recipient ini DNA’s. Material and Methods Strains The following strains were obtained from the collection of the Rockefeller University, New York, USA: R 2506-5-101 (ini, rg, mta), which was used as recipient and RL-3-8 (mtA), which was the source of the transforming DNA. Transformed (T5) and spontaneous revenant (K2) strains were obtained in our Institute [14]. The T5 transformant and K2 spontaneous revenant strains were crossed with 89601-5-5 (ini, rg + , mtA) in order to produce hornokaryotic inl+ progenies. DNA was isolated from these clones. The maintenance and the cultivation of the strains were described earlier [141. Isolation of DNA Both the labelled and the unlabelled DNA’s were prepared according to Marmur’s procedure [11] completed with T, RNase (Serva, FRG) and proteinase К (Merck, FRG) digestion [6, 7]. Labelling of DI\A was performed by growing the myceliaof the recipient strain in inositolsupplemented Vogel’s minimal medium with a 100-fold reduced phosphate concentration [4], to which :l2P-labelled Na-orthophosphate (carrier free, Isotope Institute of the Hungarian Academy of Sciences) had been added to a concentration of 100 /tCi/ml. Mycelia were harvested from 48-h cultures for the extraction of labelled (and unlabelled) DNA. The prepared DNA had a specific activity of 45 000 cpm//tg, as measured by Cherenkov counting. Hybridization and thermal elution DNA-DNA hybridization was carried out in solution [3, 5]. A small amount of sheared, denatured (SD) 32P-DNA (about 2000 cpm) was mixed with an excess amount of unlabelled SD-DNA in 0.14 M equimolar phosphate buffer (PB). Shearing of DNA was performed by ultrasonic treatment in an ultrasonic disintegrator (iVISE Ltd. London, Power Unit No. 3000) at 1.4—1.5 A Ammeter position for six times 15 s at 45 s intervals in an ice bath [6J. DNA was denatured by I boiling in a water bath for 5 min, it was then adjusted to 0.48 M PB and incubated at 65 °C until maximum reassociation (Cot = 1600—1900). Cot values were obtained as follows: A260X incubation time in h/2. A correction was applied for salt concentration according to Britten et al. [1]. After incubation, the mixture was diluted to 0.14 M PB concentration, it was then passed through a column of hydroxyapatite (DNA- Grade, BIO-RAD, USA) equilibrated at 60 °C in 0.14 M PB. The single-stranded DNA was washed off the column by ten bed volumes of 0.14 M PB, 0.4% sodium dodecyl sulphate (SDS). The DNA hybrids were then eluted by increasing the temperature of the column at 5° intervals and then eluting the resulting single-strands with 0.14 M PB, 0.4% SDS. The radioactivity of the fractions was measured with Cherenkov counting. The percentage of hybridization was calculated as follows: radioactivity of the double-stranded fraction ^ total radioactivity recovered from the column The UV absorbance of the fractions was determined at 260 nm. The thermal stability of the hybrids was characterized by plotting the percentage of eluted 32P-DNA as a function of increasing temperature. The optical density (A2G(J) elution profile of the excess unlabelled homologous duplexes was used as internal control. The data obtained in these experiments were Ada Biologien Academiae Scientiarum Hungaricae 30, 1979