Composition and structure of chromosomal and amplified ribosomal DNA's of Xenopus laevis

Abstract

Ribosomal DNA of Xenopus laevis is a homogenous DNA component of high buoyant density which contains genes for ribosomal RNA. This DNA comprises about 0.2% of the chromosomal DNA of somatic cells (chromosomal rDNA † † Abbreviations used: rRNA, ribosomal RNA, including 18 s RNA, 28 s RNA and 40 s RNA (ribosomal precursor RNA), but not 5 s RNA; rDNA, ribosomal DNA, the homogenous DNA component of high density which includes gene sequences homologous to rRNA; chromosomal rDNA, rDNA from somatic cells; amplified rDNA, the extrachromosomal rDNA present in germinal vesicles (nuclei) of oocytes; H-strand, the transcribed strand of rDNA; L-strand, the non-transcribed strand of rDNA; cRNA, complementary RNA synthesized by E. coli RNA polymerase; T m melting temperature, the temperature at which 50% of a thermal transition has taken place; G, (deoxy) guanylic acid; C, (deoxy) cytidylic acid; A, (deoxy) adenylic acid; T, thymidylic acid; U, uridylic acid; 5-MeC, 5-methyl deoxycytidylic acid; GC content, mole% of G plus C plus 5-MeC (when present). ). In oocytes of X. laevis, rDNA is amplified about 1000-fold and the additional rDNA copies (amplified rDNA) are located extrachromosomally. Both chromosomal and amplified rDNA's have been isolated and several of their properties compared. The single unequivocal difference between these DNA's is in their content of 5-methyl deoxycytidylic acid. Chromosomal rDNA contains 4.5% of its residues as 5-MeC, whereas amplified rDNA contains no detectable 5-MeC. The 5-MeC residues are located on both strands of chromosomal rDNA, including the gene regions which are transcribed in vivo. The base composition of the rDNA's is otherwise very similar; both contain 67% deoxyguanylic deoxycytidylic acids (with 13% of the latter methylated in chromosomal rDNA). Both rDNA's exhibit a biphasic melting curve reflecting two regions of the DNA with markedly different base compositions. Differences in their buoyant density and thermal stability are most likely due to their different contents of 5-MeC. The two rDNA's were compared by their ability to hybridize with rRNA and complementary RNA, which had been transcribed from either rDNA by Escherichia coli RNA polymerase. The stability of such hybrids and the nucleotide composition of the cRNA's transcribed from either rDNA were indistinguishable. The two rDNA's are therefore considered to be identical except for their difference in 5-MeC content. A model of rDNA is presented which includes data from several laboratories. The rDNA consists of a repeating unit of about 9 × 10 6 daltons which recurs about 450 times at each nucleolar organizer region. A gene sequence which is transcribed as the 40 s precursor rRNA molecule alternates with a sequence of about equal length which is not transcribed into RNA (“spacer region”). The model presents our estimates for the lengths and base composition of each region and for their arrangement in rDNA.