Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Abstract

The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease S1 from Aspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form S1 resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with S1 nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive S1 nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9 degrees C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.

The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes

“…The presence of reDetitive DN4A sequences in eukaryotic organisms is now well established (1)(2)(3)(4). Characterisation of the repeat elements by buoyant density centrifugation, renaturation kinetics and restriction enzyme digestion has demonstrated the existence of distinct families with little or no sequence homology between them.…”

“…The humn Alu family of repeats, for example, are scattered throughout the genome (7) whereas another set of complex repeat sequences, the human Alpha Rl family, and other "buoyant density" satellites appear to be localised near to the centromeric C IRL Pres Umited, 1 Falconb.rg Court, London W1V 5FG, U.K. Volume 9 Number 15 1981 regions of certain chromosomes (10).…”

Application of higher derivative techniques to analysis of high-resolution thermal denaturation profiles of reassociated repetitive DNA.

“…Reassociated repetitive eukaryotic DNA typically melts over a much broader range of temperature, and with a lower average thermal stability, than does the corresponding native DNA. It is generally accepted that such profiles reflect the presence of many imperfectly paired duplexes resulting from reactions between divergent members of repetitive sequence families (5). The reduction in average thermal stability (tm) relative to native DNA can provide an estimate of the average base pair mismatch in a given population of duplexes in vitro (5) and thus of the average sequence divergence characterizing the families of repetitive DNA in a given sample.…”

“…It is generally accepted that such profiles reflect the presence of many imperfectly paired duplexes resulting from reactions between divergent members of repetitive sequence families (5). The reduction in average thermal stability (tm) relative to native DNA can provide an estimate of the average base pair mismatch in a given population of duplexes in vitro (5) and thus of the average sequence divergence characterizing the families of repetitive DNA in a given sample. Recently, it has been possible to show that reassociated repetitive sequences from plants could be fractionated according to tm (by elution from hydroxyapatite), and each fraction then could be reassociated to yield duplexes with tm values similar to those of the original fraction from which they were derived (6,7).…”

Sequence relationship between long and short repetitive DNA of the rat: a preliminary report.

“…The calculations we presented earlier suggest that as much as 70% of rat repeated DNA may be longer than 1 kb. Britten et al (10) showed that almost 47% of isolated repeated sequence duplexes of sea urchin DNA are longer than 1 kb (mild S1 nuclease digestion). The smaller the fraction of true short repeats in a genome, the more difficult will be the isolation of the short repeat fraction free of long repeat contamination.…”