Three-Dimensional Structure of Eukaryotic Chromosomes 
ciated in interphase, when the essential processes 
of gene transcription and DNA replication occur. 
We used in situ hybridization techniques to 
determine specific chromosomal regions within 
a nucleus. The location of the hybridization sig- 
nals is revealed by labeling these embryos with a 
fluorescently tagged molecule. High-resolution 
three-dimensional optical sectioning microscopy 
of such embryos reveals the location of the nu- 
clear DNA sequences. 
We first analyzed the nuclear location of the 
histone gene cluster that lies close to the centro- 
meric heterochromatin on chromosome 2L. Our 
results, using a histone gene probe, revealed that 
homologous chromosomes are also separated in 
the majority of interphase nuclei at syncytial blas- 
toderm-stage embryos up to the 1 3th nuclear cy- 
cle. In dramatic contrast, at the 14th nuclear cy- 
cle when cellularization begins, homologous 
chromosomes are associated in the majority of 
nuclei. The frequency of homologue pairing 
reaches the maximum (about 95 percent) by the 
time of gastrulation. 
Analysis of the three-dimensional location of 
histone hybridization signals showed that histone 
genes are located on the nuclear midline at the 
1 3th nuclear cycle and move toward the centro- 
meric cluster on the apical side of nuclei at the 
l4th nuclear cycle. This can perhaps be ex- 
plained by the formation, at the l4th nuclear cy- 
cle, of heterochromatin, which process may be 
involved in initiating the pairing of homologous 
chromosomes. Translocations further define ho- 
mologue pairing, with strong effects on homolo- 
gous chromosome association. 
We are currently using a wide range of DNA 
probes for specific chromosomal sequences to 
study processes of homologue association. Our 
preliminary results suggest that frequency and 
timing of association vary from one chromosomal 
locus to another. For example, the gene en- 
grailed exhibits homologue association at the 
1 4th nuclear cycle with high frequency, but the 
gene Ubx does not. A generalized picture will be 
obtained once a larger number of genetic loci are 
analyzed. 
A Molecular Dissection of the Nuclear 
Periphery 
Recently we showed that the lamin proteins of 
the nuclear envelope (NE) form a highly discon- 
tinuous network in somatic interphase nuclei. 
Several obvious questions arise. First, where are 
the other known components of the nuclear pe- 
riphery (pore complexes, chromatin) relative to 
this network? Second, what, if anything, occupies 
these large, lamin-empty regions? Third, how are 
these structures assembled as the NE reforms dur- 
ing telophase? 
We are localizing the other two well-known 
components of the nuclear periphery relative to 
the lamin network, using monoclonal antibodies 
directed against a major glycoprotein component 
of nuclear pore complexes (GP-190) and the 
DNA-specific stain DAPl. Chromatin in the nu- 
clear periphery displays an interesting structural 
paradox in that a large fraction appears to be 
aligned beneath the lamin network, but with very 
little directly contacting lamins. The majority of 
it seems to be at a distance of about 0.2 ^m. This 
result is consistent with much indirect evidence 
of a strong interaction between chromatin and 
the nuclear lamina, but suggests strongly that a 
direct physical contact is not involved. 
We are injecting lamins and lamin-specific 
monoclonal antibody Fab fragments, both fluores- 
cently labeled, into early Drosophila embryos to 
study four-dimensional lamin-NE dynamics. In 
these experiments, the embryos develop nor- 
mally and hatch on time. We observe a highly 
discontinuous lamin network in vivo, with inter- 
lamin fiber spacings at least as large as those ob- 
served in fixed samples. If, however, we inject 
fluorescently labeled interphase lamins, a very 
different picture results. Arrested nuclear struc- 
tures leading to chromosomal/nuclear aggregates 
are seen. These studies suggest that functional as- 
says will be required for proper interpretation of 
the biochemistry. 
This general methodology has a number of po- 
tential applications to problems of cell lineage, 
neural architecture, and pattern formation in de- 
velopment. We are pursuing some of these inter- 
ests in collaboration with other laboratories. 
396 
