Amplified Ribosomal DNA from Xenopus laevis Has Heterogeneous Spacer Lengths

Abstract

Eco R, restriction endonuclease makes two cuts in each repeating unit of amplified ribosomal DNA (rDNA) from Xenopus laevis. The locations of these cuts have been established by comparison of the secondary structure of single-stranded Eco RI fragments (as visualized in the electron microscope) with that of X. laevis rRNAs and of long strands of uncut rDNA. Of the two classes of fragments generated, the smaller one contains 90% of the 28S rRNA gene, has a duplex molecular weight of 3.0 X 106 and is homogeneous in size. Eukaryotic cells contain families of closely related repetitious DNA sequences. Some of these nucleotide sequences are highly species specific, such as those present in satellite DNAs of unknown function or the spacer sequences which separate genes of known function. A variety of evolutionary mechanisms has been proposed to account for the tandem evolution of genes and their spacers (1). One means to distinguish these evolutionary mechanisms depends upon the ability to detect heterogeneity within a family of DNA repeats and to map the location of this heterogeneity. The DNA containing the genes for 18S and 28S rRNA (rDNA) in Xenopus laevis has been characterized by thermal denaturation criteria (2, 3) and by the regularity of its denaturation map (3,4) Eco R1 Restriction Endonuclease was purified from E. coli strain RY 13 carrying the RI drug resistance plasmid (a generous gift from D. Nathans). The purification procedure followed the method of Greene et al. (7) through step F (second hydroxyl apatite concentration). Purity and specificity of the enzyme were demonstrated by its ability to make a single duplex cut in closed circular SV40 DNA; no singlestrand breaks were observed by electron microscopy of denatured SV40 DNA. Restriction endonuclease Hind III was prepared by the method of Smith (8), from a streptomycin supernate generously provided by H. Smith.Digestion of rDNA was carried out in a final volume of 0.5 ml containing 10 mMiMgCl2, 50 mM\ Tris-HCl (pH 8), 0.5-2 ug of rDNA with enough enzyme to give a limit digest. Reactions were stopped by the addition of EDTA (ethylenediaminetetraacetate), extracted with phenol, and DNA was precipitated from the aqueous phase with ethanol.Electrophoresis. DNA fragments were electrophoresed in 1% agarose disc gels and photographed after staining with ethidium bromide (9). Phage X DNA fragments produced by restriction endonuclease Hind III were run in parallel as molecular weight standards. Hind III makes six cuts in linear X DNA; the molecular weights of the seven resulting fragments were determined by electron microscopy to be 14.6, 5.84, 4.05, 2.67, 1.40, 1.21, and 0.34 X 106. The 14.6 and 2.67 X 106 fragments contain the ends of the X DNA with their short single-stranded termini.Electron Mlicroscopy. Duplex DNA was spread for electron microscopy from 50% formamide (10). Single-stranded DNA was prepared for electron microscopy as follows: The DNA was denatured in 0.1 N NaOH for 30 min, then neutralized with 0.1 N HCl. Concentrated stock solutio...

The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes

“…A review of some of the frequently cited papers that propose homogeneous rRNA operons revealed that their conclusions were largely based either on techniques which would not reveal relatively low levels of sequence variations (2,16,18,27,34,35) or on analyses of a fraction of a gene or a few genes of an organism (18,27). For example, endonuclease restriction analysis of genomic DNA or cloned rDNA would fail to detect nucleotide substitutions that did not destroy or create restriction sites for the enzymes in use (2,18,34); electron microscopic observation of heteroduplex DNA formation is limited to detection of large unpaired regions or loops (34); oligonucleotide cataloguing would reveal only a maximum of 45% of the information of a 16S rRNA (28); and the reverse transcription-based sequencing of rRNAs suffers from quite extensive ambiguities in sequence results (16,28), which could be, at least in some cases, due to the presence of different rRNA species. Taken together, the presence of distinct types of an rRNA species in a single organism could be a common rather than unusual phenomenon in nature.…”

Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis

“…Each repeating unit contains one sequence coding for the precursor molecule to the rRNAs (pre-rRNA), and an adjacent more or less extended sequence, which is not complementary to pre-rRNA (for references see Birnstiel etal., 1971; Weil au er and Dawid, 1974;Schi bier et al , 1975 ;Spring et al , 1976). Miller and Beatty (1969) and Miller and Bakken (1972) have shown that this subdivision of the repeating unit can be directly visualized in the electron microscope after dispersion and spreading of the transcriptionally active nucleolar chromatin.…”

In vitro replication of recombinant plasmids carrying chromosomal segments of Xenopus laevis.

“…Recently, Zakian (33) described a recombinant plasmid carrying the origin of the X. laevis mitochondrial DNA which can replicate in yeast cells. Our cleavage map of the 3.2-kb BamHI DNA segment in pXY65 shows a pattern different from that ofthe mitochondrial DNA (34) or ribosomal DNA (35,36) and, therefore, the segment probably is not derived from the mitochondrial DNA or ribosomal DNA of X. laevis. Moreover, the result of the DNA-DNA hybridization suggests that pXY65 contains a repetitive sequence(s) from the X. laevis chromosomes.…”