Characterization of inverted repeated sequences in Ascaris nuclear DNA

Abstract

The inverted repeated sequences of the chromatin-eliminating nematode Ascuris lumbricnides var. suum have been examined by electron microscopy and by hydroxyapatite chromatography, both in the germ-line and in the somatic DNA. 38% of the inverted repeats of the germ-line DNA analysed in the electron microscope have a single-stranded loop, in comparison to about 50% of looped structures in the somatic DNA. The loops are on average 2.3 x lo3 base pairs (bp) long. The rest of the foldback DNA consists of simple hairpins. The average length of looped and unlooped inverted repeats is of the order of 300 -400 bp in the germ-line and in the somatic DNA. The content of S1-resistant foldback duplexes isolated by hydroxyapatite chromatography amounts to 1.3% in spermatids, with an average length of 350 bp, and to 1 .I % in intestinal or larval cell nuclei, with a lcngth of about 320 bp. We estimate by two different methods that there exist approximately 12500 inverted repeats per haploid germ-line genome and approximately 8000 in the haploid somatic genome. A statistical analysis of the data indicates that the great majority of the foldback sequences are randomly distributed in the Ascuris genome, with a spacing of about (40-80) x lo3 bp, both in the germ-line and in the somatic DNA.A portion of the eucaryotic genome is composed of inverted repeated DNA sequences, which are capable of forming intrastrand base-pairing upon denaturation and renaturation [I]. DNA containing these sequences, also called foldback or hairpin DNA, can be separated from the remaining fractions of genomic DNA because these sequences reanneal with rapid kinetics independent of the DNA concentration following denaturation and incubation in annealing conditions. This fraction of DNA can be observed in the electron microscope as single-stranded molecules containing an intramolecular duplex segment, or can be isolated by hydroxyapatite chromatography.Inverted In view of the development of the chromatin-eliminating nematode Ascaris lurnhricoidcs var. suum, it is important to determine how individual sequences are arranged in the genome before and after chromatin diminution in order to elucidate the elimination process. The purpose of the present investigation is to characterize the inverted repeated sequences in the genome of A . lumhric~~id~s using the techniques of ('orrespoIt~~,nce to 1-1. 'I'obler, Zoologisches Institul, Universilat Fribourg. I'erolles. CH-1700 Fribourg, Switzerland Ahbrcviu/ions. r,,t, producl of initial concentration of DNA and time, in molcs of nucleotides x seconds/liter; bp, base pairs; kb lo3