Restriction endonucleases were used to determine the degree of methylation at the sequences CCGG and GCGC in a wide range of animal DNAs. Both total DNA methylation and ribosomal DNA methylation were studied. Whole DNA methylation was indetectable in arthropods, fractional in other invertebrate phyla, and high in the vertebrates. Ribosomal DNA was predominantly unmethylated in all animals except fish and amphibia, where it was heavily methylated. We discuss the evolutionary and functional implications of these results, and suggest that the large differences between genome types are the result of evolutionary changes in the relative size of heavily methylated and unmethylated compartments.

Variable patterns of total DNA and rDNA methylation in animals

The diploid nuclear DNA contents in eight species of fish have been found to differ over a 178-fold range. Among these, lungfish have the highest values, and teleosts have the lowest. Shark and coelacanth have intermediate values, which are close to the highest teleost values. Such great variation in DNA contents among vertebrates indicates that fundamental differences in chromosome structure and DNA function have arisen during evolution.



In order to gain insight into the organization of genes in organisms with different DNA contents, the relative proportion of the genome coding for ribosomal RNA was determined for five species, winter flounder, goldfish, rainbow trout, dogfish shark, and Australian lungfish. The proportion of ribosomal DNA (r-DNA) was found to vary over a six-fold range from flounder to lungfish. In general, species with higher DNA contents have lower proportions of r-DNA. The r-DNA proportion of trout is consistent with the view that salmonids are recent tetraploids. If the goldfish is also considered a tetraploid, then its r-DNA proportion indicates a loss of ribosomal genes subsequent to polyploidization. Measurement of the ribosomal genes in fish DNA's was based on hybridization with yeast ribosomal RNA, which was assumed to be equally homologous with the r-DNA of all species studied.



The activity of multiple copies of genes was studied by measuring the volumes of blood cells in five species. It was found that erythrocyte volume correlated both with ribosomal gene multiplicity and with total DNA content. The size of goldfish and trout erythrocytes suggests that cell volume in these tetraploids has declined to the ancestral diploid level.



It is concluded that the multiplicity of specific repetitive genes varies out of proportion with the DNA content, contrary to expectation based on simple polyteny. This variation in the proportions of specific genes and the large variations in total DNA content among fish are thought to be due to a combination of tetraploidy and extensive linear replication of specific chromosomal regions during evolution.

DNA content, ribosomal gene multiplicity, and cell size in fish.

SUMMARY Early oogenesis in the toad Xenopus laevis has been investigated at the ultrastructural level, with particular reference to the formation of extrachromosomal DNA. Thymidine incorporation was localized by electron microscope radioautography. In oogonia, the nucleus is irregular in outline and may contain several nucleoli. Oocytes, from premeiotic interphase to late pachytene, are found in cell nests which are estimated to consist of about 16 cells each. Adjacent oocytes within a nest are connected by intercellular bridges and develop synchronously. Each premeiotic interphase-leptotene oocyte has a round nucleus which contains one or two centrally located, spherical nucleoli. Electron-microscope radioautography showed that all nuclei in a cell nest incorporate thymidine synchronously during premeiotic S-phase. In zygotene oocytes, axial cores and synaptonemal complexes are observed in the nucleus and abut against the inner nuclear membrane in the region nearest the centre of the cell nest. The nucleolus is still moreor-less round in outline, but is asymmetrically positioned in the nucleus. It lies near the nuclear envelope on the side of the nucleus furthest away from the attachment of the chromosome ends, that is, nearest the outside of the cell nest. Each nucleolus is surrounded by a fibrillar ' halo' of nucleolus-associated chromatin into which a low level of thymidine incorporation occurs during zygotene. This is thought to represent the start of the major period of amplification of the ribosomal DNA. Pachytene is characterized by the presence of synaptonemal complexes in the nucleus. The nucleolus becomes very irregular in outline. The fibrillar area around it, which represents the extrachromosomal DNA, increases in size and thymidine is incorporated over the whole of this region. In late pachytene, many small fibrogranular bodies, the multiple nucleoli, are formed in it. The members of a cell nest become separated from one another at this time and begin to develop asynchronously. In diplotene, synaptonemal complexes are no longer observed in the nucleus. The most prominent structures in the nucleus are now the multiple nucleoli, which increase greatly in number in early diplotene. A large increase in cytoplasmic volume occurs and the oocyte grows in size.

An Ultrastructural and Radioautographic Study of Early Oogenesis in the Toad Xenopus Laevis

Gene amplification proceeds by a chromosome copy mechanism.

Publisher Summary This chapter discusses the organization and transcription of the rRNA genes of eukaryotes with an emphasis on the nucleotide sequences in relation to function. The rRNA genes (rDNAs) are organized in repeating pre-rRNA transcription units separated by nontranscribed spacers (NTS). The pre-rRNAs transcription unit consists of coding sequences corresponding to mature 18-S, 28-SY, and 5.8-S rRNA, or their equivalents, and both external and internal transcribed spacer (ETS and ITS) sequences, which are transcribed as parts of the pre-rRNA molecule, but are lost during maturation. One transcription unit and the adjoining NTS constitute one rDNA repeating unit. The tandem head-to-tail arrangement of the repeated units is the basic feature in the organization of rRNA genes in most eukaryotes. The determination of nucleotide sequences at the transcription initiation and termination sites and at the gene-spacer boundaries has revealed some general features regarding sequence conservation and divergence in rDNAs. In general, the structural and functional domains of rRNA have been conserved in evolution in spite of wide divergence in the transcribed and nontranscribed spacers.

Vincent Ws

Papers

A comparison of ribosomal gene amplification in uni- and multinucleolate oocytes.