ul in the buffer suggested by the supplier The reaction was

terminated by the addition of l/lOth volume of lOOmM Tris pH7.5; lOOmM EDTA; 0.3% agarose beads; 0.01% orange G; 50% glycerol. The samples were

electrophoresed in a 1.5% agarose gel containing 1 jig/ml ethidium bromide for 16 hours at 30 V. The gel was illuminated with u.v. light (360 nm) and photographed.

a. Msp-I b. Hpall c. Hae-III

The negative was scanned using a Joyce-Loebl microdensitometer

a-c as above

d. background fluorescence

Molecular weight markers were Hindlll digested A DNA and Hinfl digestedpBR322 DNA.

g- f v i o f s c o CN O ' <3 2 go-'o •<*' CN —

was preventing total hydrolysis. In later experiments, which gave the

same result (see Figs. 5-13 - 5*15) a small amount of phage \ DNA was included as an internal control to ensure that complete digestion had occurred.

The size distribution of restriction fragments (3.3B) indicates that while a large fraction of the genome contains Hpa II resistant regions there are regions in which the DNA is not as heavily methylated, as seen by the presence of low molecular weight fragments in the Hpa II digest (b, in Fig. 3*4B). A similar conclusion was reached by Bird and Taggart (198O) for both Xenopus laevis and Triturus cristatus. They suggest that the animal genome can be divided between methylated and non-methylated "compartments" interspersed among each other through the genome. The amplification of sequences from within compartments is postulated to have brought about the different vertebrate methylation patterns seen. In this regard it is notable that repeated sequences tend to maintain their methylation pattern even after translocation to

different locations within the genome (Eden et al , 1981), probably as a result of the mechanism of methylation after DNA synthesis (see Ehrlich and Wang, 1981).

The Msp I digestion of genomic LNA (Fig. 3*3A a) also shows a fraction of the genome to be relatively devoid of the sequence 5 ,-CCGG. Recent data on dinucleotide frequencies in animal DNA has shown that the dinucleotide CpG is present at around one third of the frequency expected on a random basis (Nussinov, 1980, 1981). Bird (1980) has shown a correlation between low CpG levels and high levels of methylation and suggests that 5MeCpG tends to mutate to TpG. Indeed TpG is the most frequently observed dinucleotide (Nussinov, 1980, 1981). Thus the Msp I resistant stretches of DNA may contain mutated Msp I sites within the sequence. An alternative is that these regions are simply of low ?fc+C so that Msp I sites would be rare in any case. This is not thought to

was preventing total hydrolysis. In later experiments, which gave the same result (see Figs. 5«13 - 5*15) a small amount of phage \ DNA was included as an internal control to ensure that complete digestion had occurred.

The size distribution of restriction fragments (3»3B) indicates that while a large fraction of the genome contains Hpa II resistant regions there are regions in which the DNA is not as heavily methylated, as seen by the presence of low molecular weight fragments in the Hpa II digest (b, in Fig. 3-4B). A similar conclusion was reached by Bird and Taggart (198O) for both Xenopus laevis and Triturus cristatus. They suggest that the animal genome can be divided between methylated and non-methylated "compartments" interspersed among each other through the genome. The amplification of sequences from within compartments is postulated to have brought about the different vertebrate methylation patterns seen. In this regard it is notable that repeated sequences tend to maintain their methylation pattern even after translocation to

different locations within the genome (Eden et al, 1981), probably as a result of the mechanism of methylation after DNA synthesis (see Ehrlich and Wang, 1981).

The Msp I digestion of genomic DNA (Fig. 3*3A a) also shows a fraction of the genome to be relatively devoid of the sequence 5 ,-CCGG. Recent data on dinucleotide frequencies in animal DNA has shown that the dinucleotide CpG is present at around one third of the frequency expected on a random basis (Nussinov, 1980, 1981). Bird (198O) has shown a correlation between low CpC levels and high levels of methylation and suggests that 5MeCpG tends to mutate to TpG. Indeed TpG is the most frequently observed dinucleotide (Nussinov, 1980, 1981). Thus the Msp I resistant stretches of DNA may contain mutated Msp I sites within the sequence. An alternative is that these regions are simply of low 9&J+C so that Msp I sites would be rare in any case. This is not thought to

46

be the case. Hae III, which cute within the sequence 5'GGCC, the reverse of the Msp I sequence but containing two of the three possible dinucleotide pairs found in the Msp I sequence does not show these high molecular weight fragments when used to digest axolotl DNA (Fig. 3*4A, c).

Table 3.2 shows some data on % G+C content in animals collected from various sources. Some of the data has been derived from buoyant density measurements and some from thermal denaturation data, the

different methods used to obtain the data are noted, and the significance of the differences in result obtained by the two methods is discussed later.

From the table it can be seen that a spread of % G+C value exists with a modal value around 42%. Species with low C values appear to be more A + T rich (insects, echinoderms), however sufficient exceptions exist so that little can be definitely concluded as to whether the

C value and % G+C content are correlated. It may be however that a general increase in % G+C content with increasing C value may be a factor in reducing the skewed distributions noted by Thiery et al (1976). It is of interest that of the Urodeles which have been studied, all have

% G+C contents of 45% or more compared to the value of 4 0 - 9 % for X. laevis an Anuran. Whether this represents a true reflection of the phylogenetic relationship remains to be seen.

From the melting profile (Fig. 3.2b) it can be seen that a small fraction of the DNA appears to melt at a higher temperature than the main body of DNA. This may be due, as has been suggested above, to local regions of heavily methylated DNA which may affect the Tm of a fraction of the genome. Alternatively a GC rich satellite fraction, undetected in the analytical centrifugations may exist. The possible existence of such a satellite was investigated.

Walker defined satellite DNA as a native fraction of the chromosomal DNA which after isolation by any method gives a narrow unimodal band in

TABLE 3.2