x 106 + 4.675 x 105 x 500_

5.4 x 1010

67.

First, in both of the distributions shown very little of the nuclease resistant material is much larger than 2 k bases in length, while most of the material appears to have been digested to low molecular weight duplexes. TJnsheared DNA was used in this experiment specifically to try to establish whether long nuclease resistant duplexes do exist (e.g. satellites). Under the digestion conditions used the majority of the highly repeated

nuclease resistant fraction (8% of the genome in this experiment) do not exhibit the characteristics of tandem repeats. Similarly the Class I repeats (48% of the genome in this experiment) show few long repeats. As the DNA is uniformly labelled these distributions measure weight average lengths, so the proportion of long repeat elements is very small.

Second, the Cot 100 resistant duplexes show a class of repeat lengths with a weight average length of 500 bases not seen in the Cot 0.01 fraction. This fraction can be demonstrated to increase progressively with increasing Cot, between Cot 0.01 and Cot 100 (data not shown). These duplexes

presumably represent the average fragment lengths of Cot 100 repeats, although it should be stressed that the absolute amounts of all the fragment size classes increases between Cot 0.01 and Cot 100.

These observations tend to suggest that Class I repeats are inter­ spersed in such a way that members of any particular family are located between members of different families.

Around 8 0 % of the axolotl Cot 100 sequences form short nuclease resistant repeats (Fig. 4•5"b)• This represents 0.8 x 0.43 or 54.4% of the axolotl genome, and might therefore suggest that 54% of the axolotl genome consists of Cot 100 repeats surrounded by a variable fraction of slower reassociating sequences. However it was shown in Fig. 4.5 that 8 % of the genome was slower reassociating sequences of around 1 kbp in length adjacent to Class I repeats, while at least 5-5% of the genome consisted of slower reassociating sequences at least 4 k*>p long adjacent to a Class I repeat. Therefore, taking 300 bp as a reasonable estimate for the average length of

68.

the short nuclease resistant repeats it can he estimated that a

maximum of 2.8% of the genome (6.4%» (0.028 x 0.43) of the Class I repeats) need to he associated with slower reassociating sequences to account for

the observations of Fig. 4-3- Of course this figure varies with the various assumptions made, being reduced as estimates of spacing lengths increase (and vice versa) and being increased as the estimate of the repeat length increases (and vice versa). Therefore an observable discrepancy exists between the observed 34-4% of the genome present as short repeats, and the value of 2.8% required, as a maximum, to account for the observed inter- spersion pattern. This discrepancy can be easily explained as being the result of reassociation of Class I repeats interspersed among themselves leading to the formation of accessible regions for nuclease action. These regions could occur for several reasons. Steric hindrance within networks of interspersed repeated sequences may prevent the reassociation of a

proportion of repeats e.g. those Cot 100 repeats with the lower copy numbers in the repeat range. Reassociation between members of divergent repeats may present accessible regions within the reassociated duplexes,

Posakony et al (1981) have shown by DNA sequencing of members of sea urchin repeat families that divergence may be due to small deletions, insertions and rearrangements (which may be nuclease accessible in stringent digestion conditions) as well as to simple base substitutions.

Recent work using recombinant DNA clones containing long repeat elements has confirmed that in many cases the long S.^ resistant repeats may consist of arrays of shorter repeats, these shorter repeats originating from different repeat families. This has been clearly shown in Drosophila melanogaster (Wensink, 1977). Xenopus laevis (Spohr et al, 1981). the sea urchin Strongylocentrotus purpuratus (Anderson et al , 1981; Scheller et al, 1981), the chicken Gallus gallus (Musti et al , 1981; Eden et al, 1981; Sobieski and Eden, 1981). Detailed analysis of the long Sj resistant repeats in the Syrian hamster (Moyzis et al , 1981 a + b) and the amphibians

69.

Ambystoma tirrlnum arid Rana berlandieri (Graham and Schanke, 1980) shows that the majority of these sequences are composed of shorter repeats. Moyzis et al, (1981 h), show that for the Syrian hamster most repetitive DNA sequences are present in both long and short S^ nuclease resistant repetitive DNA duplexes, at approximately the same concentrations, Graham and Schanke (1980) have shown that the long S^ resistant repeats in A. tigrinum and R. berlandieri are criterion sensitive suggesting that shorter repeat families with different degrees of divergence are inter­ spersed amongst each other. This situation probably occurs in the axolotl and it may be proposed that a proportion, in some cases a substantial portion, of the short S^ nuclease resistant repeats observed in many species may be of the same source.

An alternative method of analysing the organisation of a particular genome is to shear the DNA to moderate fragment lengths of several thousand bases (single stranded) and allow the DNA to reassociate. The fraction bound at each Cot point is determined by the reassociation of the most highly repetitive component present on each fragment. Hence by comparison with the reassociation of short DNA fragments relative interspersion patterns can be deduced. To reduce errors in analysis caused by networks of DNA being retained on hydroxylapatite the duplex DNA is eluted at 95°C (Kiper and Herzfeld, 1978).

When axolotl DNA, sheared to a weight average single strand length of 9*5 kb was allowed to reassociate and was fractionated as described, the Cot curve shown in Fig. 4.6 was generated. Several features are apparent. The proportion of the genome assigned to each sequence class is altered compared to the curve shown in Fig. 4-1 (the fitted curve from Fig. 4.1 is shown in Fig. 4.6 as the broken line). Each of the repetitive fractions has increased in proportion in the genome. The highly repetitive fraction has increased from 5 to 9% of the genome. This value is similar to that obtained from Fig. 4.3b for 2.5 kb DNA. The major reassociating class, which most probably

LEGEND TO FIG. 4.6