Sequence organization of the spacer DNA in a ribosomal gene unit of Xenopus laevis

Abstract

A detailed restriction map was constructed for a cloned Xenopus laevis rDNA fragment containing the nontranscribed spacer (NTS) and external transcribed spacer (ETS) together with a portion of both the 18S and 28S rRNA genes. The NTS was found to contain at least three distinct repetitious areas. Region 1 has a repeating unit of approximately 100 bp. The primary structure of this unit has been determined by DNA sequencing. Region 2 is very similar in organization to region 3, and both have an alternating 81 60 bp arrangement as revealed by restriction with Alu I and DNA sequencing. It can be shown that the 81 and 60 bp canons are virtually identical to one another excepting a deletion/insertion of a 21 bp segment. Region 3 differs from region 2 in having sites for Sma I within its 81 bp units. Between these repeated DNA sequences there are two identical, nonrepetitive DNA sequences, each of which is centered around a Bam HI site. Most of the ETS has been sequenced. It was found to be nonrepetitive and extremely rich in Cs. Close to the 5′ end of the 18S coding sequence there is a DNA stretch very rich in purines. About 2.25 kb upstream from the Eco RI restriction site bisecting the 18S structural gene there is a unique sequence which may be homologous to the 5′ end of the 40S precursor RNA. Present evidence suggests that the boundaries between NTS and ETS occur farther downstream than was suggested by electron microscopic data. Sequencing has revealed that the spacer DNA of X. laevis contains different kinds of simple DNA sequences, but no evidence has been found that spacer DNA once arose by saltation of a 15 bp segment. The most surprising finding was that the spacer sequences around the Bam restriction sites (the Bam islands) show high homology with a sequence near the NTS/ETS interface. From the restriction and sequencing analyses it can be deduced that in recent evolutionary times the DNA sequences near the 5′ end of the ribosomal transcription unit were reduplicated twice and displaced into spacer by saltation of an intervening short DNA sequence (the 60 81 bp canons). Possible implications of these evolutionary events for spacer functions are considered. The sequencing has also provided a molecular basis for a whole range of conclusions arrived at previously by indirect approaches, and these are discussed.