Two-dimensional DNA gel electrophoresis as a method for analysis of eukaryotic genome structure: evaluation using Tetrahymena thermophila DNA.

There is growing interest in mapping and analyzing complete eukaryotic genomes. Yee and Inouye (in Experimental Manipulation of Gene Expression, pp. 279-290, Academic Press, New York) demonstrated that bacterial chromosomes can be resolved into interpretable patterns of DNA fragments by means of restriction enzyme digestion and electrophoresis in two dimensions. We have begun to explore applications of this procedure to analysis of eukaryotic genomes, which are far more complex. Tetrahymena thermophila was selected as a model organism because its genome is small, roughly equivalent to that of a single human chromosome. In addition, each Tetrahymena cell contains two nuclei which differ in sequence composition and methylation. Our results demonstrate that the Tetrahymena genome can be resolved into complex patterns of fragments in two dimensions. Hybridization to Southern blots of these gels with a multiply repeated sequence probe yielded analyzable patterns of a subset of the genome. The blots reveal alterations in genome structure due to methylation and rearrangement. Future extensions of the method are discussed.

Fluorescent in situ hybridization (FISH) analysis of the relationship between chromosome location and nuclear morphology in human neutrophils.

Human neutrophil nuclei typically consist of three of four large heterochromatic lobes joined by thin, DNA-containing filaments. In addition, some lobes exhibit appendages of various sizes and shapes. Classical genetic and cytological studies suggest that some appendages contain specific chromosomes. The studies reported here provide the first detailed analysis of the spatial relationship between individual chromosomes and recognizable structures in neutrophil nuclei using fluorescent in situ hybridization. Analysis of DNA sequences in chromosomes 2, 18, X, and Y demonstrate that specific lobes in a population of neutrophil nuclei do not have a fixed chromosome content. This result implies that chromosomes partition randomly among lobes during neutrophil differentiation. However, neutrophil nuclear topography is not entirely fortuitous. For instance, none of the sequences probed in this study mapped to a filament and most centromeres lie in clusters near the nuclear periphery. In addition, one of the X chromosome centromeres in females and the Y chromosome centromere in males consistently associate with specific nuclear appendages found in a subset of neutrophil nuclei. Chromosomes 2 and 18 occupy discrete nd separate territories within individual lobes and neither territory ever extends into a filament. Surprisingly, the sizes of these territories are not proportional to chromosome length, suggesting that individual neutrophil chromosomes vary in their degree of compaction. These results are discussed in the light of models that attempt to explain nuclear morphology in terms of chromosome spatial organization.

Efficient plasmid DNA replication in Xenopus egg extracts does not depend on prior chromatin assembly.

Small plasmids replicate efficiently in unfertilized Xenopus eggs provided they are injected before rather than after activation of the cell cycle. Here we use Xenopus egg extracts to test the hypothesis that efficient replication results from chromatin assembly prior to activation giving preloaded plasmids a head start toward the formation of a replicating pseudonucleus (Sanchez, J.A., Marek, D., and Wangh, L.J. (1992) J. Cell Sci. 103, 907-918). As in ovum, plasmid DNA preincubated in unactivated egg cytoplasmcytostatic factor extracts) replicate more efficiently after extract activation than does the same DNA added to the same extract after activation. Unlike in ovum, however, plasmids that replicate efficiently in vitro do not assemble into chromatin during preincubation and become topologically knotted instead. But even DNA knotting does not explain subsequent efficient replication. Also, plasmids preassembled into chromatin in vitro do not replicate efficiently in activated egg cytoplasm unless first preincubated in a CSF extract. We conclude that unactivated eggs contain replication-enhancing activities that can act independently of plasmid chromatin assembly and DNA topology. These postulated "preloading" factor(s) may be related to licensing factor, an activity that controls initiation of DNA replication in eukaryotic cells. The experimental conditions described here will permit characterization of preloading/licensing factor(s) in the context of a small plasmid substrate.

Efficient human sperm pronucleus formation and replication in Xenopus egg extracts.

We have achieved efficient in vitro reactivation and replication of human sperm nuclei in frog egg extracts by constructing a 4-step protocol that mimics the events of fertilization and pronucleus formation in mammalian eggs. With use of this protocol, 78-97% of human sperm nuclei from fertile donors synchronously swelled and completed full genome replication in about 2 h. We document the changes in nuclear structure that accompany efficient DNA synthesis and discuss future research and potential clinical implications of this new system.