HYDROXYliAPATITE BINDING WITH INCREASING TRACER FRAGMENT SIZE
JE labelled DNA of various single strand sizes was prepared as Methods.
a) Cot 100 Assay. DNA was used as a tracer in reassociations driven by a vast excess (greater than 1000:l) of short genomic driver DNA. DNA was allowed to reassociate in O.4IM NaPB pH6.8 at 69°C to driver E Cot of 100, calculated according to Britten et al (1973)* After reassociation the solutions were diluted to 0.12M NaPB using ice cold distilled water, before hydroxylapatite chromatography.
The hydroxylapatite bound fraction at each fragment size was corrected for zerotime binding (b) by
Corrected binding = Fraction bound at length,1-zerotime binding at length,!
1-zerotime binding at length,!
according to Davidson et al (1973)* Best fits to the data are described in the text.
b) Zerotime binding. 1-5 ng of labelled DNA was mixed with 50 ug sheared E.coli DNA in 1 ml 0.12M NaPB pH6.8. The solutions were boiled 5' at 100°C, quenched on ice and immediately assayed on hydroxylapatite at 60°C. The highest attainable Cot for the E'’ DNA was calculated to be 5 x K f 6 .
Fr ag me nt si ze (kb )
6?
from the slow rate of increase of binding to hydroxylapatite with increasing fragment length.
The data suggest at least two possible patterns of arrangement for the genome involving these sequences. The straight line fit to the data suggests that a fraction of the genome is arranged such that sequences of at least 4 kb are adjacent to Class I repeats. 17.9% of the slower reassociating sequences x looj would be involved based on figures of 57-5% binding at 4 kb and 48.2% binding at the y intercept. The majority of the Class I repeats would be interspersed among each other. Alternatively it is possible to deduce a short period interspersion pattern, with spacings between repeats of 0.6 - 1 kb. Under this organisation 13*9% 5 x of the slower reassociating sequences would be involved in the short period pattern. In addition a further
95% [5 ^ 5 x 100j would be involved in a longer period of interspersion with spacings of at least 4 kb. Again most of the Class I repeats would be interspersed amongst each other.
The alternative suggestion of Moyzis et al (1981 a + b) would be that this kind of distribution can be accounted for by assuming a spectrum of spacing lengths between Class I repeats. However in all of these cases it is inevitable that the majority of the Class I sequences will be inter spersed amongst each other. This type of arrangement of repeat sequences is not an isolated instance. A similar situation has been observed in the related Urodele Ambystoma tigrinum and in the high C value Anuran Rana berlandieri (Graham and Schanke, 1980), in several plant species (Plavell et al , 1977; Murray et al, 1978) and most recently, by analysis of recombinant DNA clones, in Xenopus laevis (Spohr et al , 1981), Drosophila melanogaster (Wensink, 1977). the chicken (Eden et al, 1981; Sob ieski and Eden, 1981; Musti et al , 1981) and the sea urchin Strongylocentrotus purpuratus (Anderson et al , 1981). The arrangement can also be inferred by the presence in most animal and plant genomes of long S.^ nuclease
resistant repetitive DNA isolated after reassociation of moderately sheared DNA to intermediate Cots (see for example, Galau et al, 1976; Pearson et a l , 1978; Moyzis et al, 1981 b). The possible development of such sequences has already been described in the Introduction.
Fig. 4*36 shows the binding of DNA at Cot 10 ^ . At such early Cot points only adjacent inverted repeats will have renatured. The increase in binding with fragment length is apparently linear over the fragment range analysed. Extrapolating to the y axis indicates that 5*5% of the genome is arranged as inverted repeats. At fragment lengths of 4 kb only 15% of the genome bound to hydroxylapatite at Cot 10 The rate of increase of binding with fragment length, together with the estimate of the fraction of the genome which occurs as inverted repeats, suggests that foldback sequences are clustered in the genome. These clusters may themselves be spread randomly through the genome, as suggested for Triturus (Wilson and Thomas, 1974)*
It has been shown by many groups that the repetitive sequence fraction of the genome displays varying degrees of homology in its reassociated products. This can be demonstrated by controlled thermal denaturation of the reassociated fraction. In most instances a broad spread of thermal stability can be observed in the order of 8 - 10°C. It has been shown, by Bonner et al (1973) that a 1°C reduction in Tm corresponds to 1% mismatch between the reassociated strands. Thus most repetitive sequences show an average divergence of around 8 - 10%, although exceptions do occur, (Wensink, 1977; Crain et al, 1976). The melting behaviour of the Class I repeats was examined in similar fashion and the results are plotted in
Fig. 4-4«
Sheared native axolotl DNA showed a T£ (point of 50% elution from HAP) of 86°C, whereas the same DNA denatured and allowed to reassociate to E Cot 100 showed a T£ of 76°C, a reduction of 10°C. By the criterion of Bonner et al (1973) this implies an average sequence divergence of 10%
LEG El U) TO FIG. 4-4