An Analysis of the Bovine Genome by Density‐Gradient Centrifugation

Abstract

The dG + dC-rich fractions obtained by density gradient centrifugation of bovine DNA in Cs2S04/ BAMD [J. Cortadas, G. Macaya & G. Bernardi (1977) Eur. J. Biochem. 76, were centrifuged in Cs2S04/Ag+ density gradients. These experiments led to the preparation of the DNA Components which had been detected (by analytical centrifugation in CsCl) in the Cs2S04/BAMD fractions, and also of DNA components which had identical behaviors in CszSO4/BAMD gradients and identical buoyant densities in CsCl. A total of eight satellite components and 11 minor components, accounting for 23 % and 4 % of the bovine genome, respectively, were thus isolated and characterized in their relative amounts and buoyant densities. The implications of these results on the interpretation of renaturation kinetic data on the bovine genome are discussed.Centrifugation of calf thymus DNA in preparative Cs2S04/BAMD density gradients has led to the separation of dG + dC-rich fractions which have been analyzed in terms of DNA components by analytical centrifugation in CsCl [l]. In the present work, we have prepared these components by submitting the CszS04/BAMD fractions to further centrifugations in Cs2S04/Ag' density gradients. This has also led to the preparation of DNA components having the same behavior in CSZSO~/BAMD density gradients and the same buoyant density in CsCl. A precise assessment of the buoyant density and relative amount of eight satellite components and 11 minor components so isolated from the bovine genome has thus been possible.As in other recent papers from this laboratory, (quoted in [I]), we call here DNA components the populations of genome fragments which can be separated from each other by density gradient centrifugation techniques. We distinguish three groups of DNA components according to their nucleotide sequence patterns and relative amounts in the genome as follows. (a) Satellite components are formed by short repeated nucleotide sequences ; each satellite component usually represents a small percentage of the genome. (b) Minor components each account for less than 3 % of the genome; they are not formed by short repeated nucleotide sequences, yet they may contain rf, molar ratio of ligand to DNA phosphate. The different features of nucleotide sequences in these three classes of DNA components allow a distinction to be made on the basis of a number of physical and chemical properties. Suffice it to mention here that the electrophoretic patterns of fragments produced by different restriction enzymes provide a rapid criterion for such a distinction (see following paper). Satellite components are characterized by banding patterns in which the molecular weights of the fragments reveal the underlying repetitive organization of the nucleotide sequences. Major components show continuous distributions of fragment sizes, in which no bands are detected. Minor components either show simple banding patterns due to the tandem repetition of gene-spacer units or continuous distributions of fragments. A detailed discussion of the prope...

The primary structure of bovine satellite 1.715

“…Previous investigations from our laboratory, involving density gradient centrifugation in the presence of DNA ligands (1)(2)(3), have shown that 23% of the bovine genome is formed by 8 satellite DNAs (3,4). This allowed us to study their long-range periodicities (5), extending previous results (6)(7)(8)(9)(10), and to show that the bovine satellites share identical repeat lengths, a fact suggesting a common evolutionary origin.…”

The Major Components of the Mouse and Human Genomes

“…The cOt1/2 values of the slowly reassociating sequences of the latter showed the decrease expected on the basis on their enrichment as obtained upon fractionation and were used to provide an independent estimate of the percentages of different DNA components; this was in good agreement with that derived from their yields (compare the results of Table 1 with those of Table 1 in the preceding paper [4]). Incidentally, determining the amount of interspersed repeated sequences on isolated major components avoids mistaking satellite DNA for interspersed repeated sequences, an error which was made [12] in the case of the total bovine genome (see [13]). …”

Distribution of 5-methylcytosine in the DNA of somatic and germline cells from bovine tissues

“…Hydrolysis of multiple, small DNA samples to bases was performed as described earlier (20) (7,8) HaeIII; (9,10) HpaIT; (11,12) MspI.…”

“…Bovine satellite I, density 1.714 g/cm3 (12), which is cut by EcoRI into segments of about 1400 base pairs (13)(14)(15)(16), is very much undermethylated in sperm DNA compared to thymus DNA. The difference in methylation of the fragments from both tissues is large enough to suggest that reduced methylation of all highly repeated sequences could account for all or most of the difference in the level of methylation between total sperm and somatic cell DNA.…”