phase, ~60% of the enzyme present in the late in- 
terphase nucleus leaves it and diffuses into the cyto- 
plasm. The remaining 40% is associated with the 
chromosomes throughout prometaphase and meta- 
phase. After anaphase, 25% of the metaphase topoll 
leaves the chromosomes. 
Control four-dimensional studies, using fluores- 
cently labeled histones as chromosomal labels, have 
shown that the topoll loss, at specific times, is not 
the result of chromosomal condensation or segrega- 
tion. These movies also show that topoll localizes, 
with temporal regulation, to specific sites within 
the interphase nucleus. Metaphase chromosomes do 
not have an axial core along the chromosome 
length. The localization of the enzyme is thus highly 
regulated, spatially and temporally, revealing novel 
behavior as well as a role in chromosome condensa- 
tion and segregation. 
A comparable four-dimensional study was under- 
taken to dissect, structurally and functionally, the 
nuclear envelope (NE) . Dr. Sedat and his co-workers 
have shown that the major NE structural protein, 
lamin, is organized as a highly discontinuous fibril- 
lar network, leaving in the nuclear periphery large 
voids containing little or no lamin. They have also 
correlated this structure with the underlying 
chromatin. 
To study the dynamics of the lamins during mito- 
sis, fluorescently labeled lamin-specific MCA-F, to- 
gether with labeled histones (to follow chromo- 
somes), were injected into early Drosophila 
embryos. A highly discontinuous lamin network was 
observed in vivo, with interlamin fiber spacings ap- 
proximating 1 nm. New four-dimensional data show 
a surprisingly complex series of NE and lamin struc- 
tural dynamics. Laminar structures do not com- 
pletely disassemble at the onset of mitosis, but per- 
sist until well into mitosis. Further lamin structural 
changes, including NE breakdown and reassembly, 
take place at anaphase and telophase. Additional 
studies indicate that lamins are essential for the nu- 
clear structural reorganizations that occur at all 
points of the cell cycle. 
Homologous Chromosome Pairing: 
Three-Dimensional Nuclear Studies 
It has long been debated whether interphase 
chromosomes follow ordered paths, whether there 
are special associations between the homologous 
chromosomes in diploid nuclei, and what roles such 
associations might play in regulating nuclear organi- 
zation and function. This group's development of a 
three-dimensional in situ hybridization technique, 
which allows localization of specific chromosomal 
sequences under conditions of chromosome struc- 
ture preservation in intact tissues, provides a new 
avenue for exploring these questions. 
The issue of homologous association has re- 
mained particularly significant in Drosophila biol- 
ogy, because genetic evidence has shown that ex- 
pression of certain alleles of a few genes (such as 
bx-c, dpp-c, and sgs-4) can be affected by the allelic 
state of the homologous locus. These genetic ef- 
fects, which appear to depend on interactions in 
trans between homologous sequences, have been 
grouped into the phenomenon known as transvection. 
The locations of chromosomal loci on the two ho- 
mologues were probed by in situ hybridization to 
chromosomal DNA in whole-mount embryos, using 
a DNA sequence specific for the histone gene clus- 
ter. Up to the nuclear cycle 13, homologous loci of 
the cluster were separated during syncytial mitoses. 
In dramatic contrast, at cycle 14, homologues at the 
site were found to be associated at a high frequency. 
Furthermore, the three-dimensional location of the 
histone gene cluster at the 1 3th cycle is in a defined 
plane about halfway between the apical and basal 
sides of the nucleus, but changes toward a position 
in the apical side at the l4th cycle, when transcrip- 
tion and differentiation start. It is intriguing that 
chromosome reorganization is concomitant with 
profound changes in embryonic development. 
The group's recent investigations of homologue 
associations have focused on single-copy loci, as ex- 
amples, the engrailed and Bithorax genes or Re- 
sponder of SD, a heterochromatic locus. Use was 
made of new procedures to label the DNA probes 
fluorescently, making possible direct multiple-label 
in situ experiments. Improved hybridization proto- 
cols now allow chromosomal probes as small as 1 2 
kb to be detected, very reproducibly, with high 
signal-to-noise ratios. Each of these loci is also in a 
spatially defined nuclear plane but is homologously 
paired much less frequently than the histone locus, 
a result implying differential control of homologue 
associations. 
To assay homologous chromosome pairing on a 
whole-chromosome basis, the laboratory is using 
polymerase chain reaction (PCR) amplification of 
microdissected polytene chromosomes to develop 
whole-chromosome DNA probes. 
To test whether gene expression and regula- 
tion require the association of homologous chro- 
mosomes, this group made use of chromosomal 
rearrangements. In the homozygous LTX13 translo- 
cation strains, the association frequency of homolo- 
gous histone loci stays low, with a perturbed nu- 
clear location at the time of gastrulation, yet these 
embryos develop normally to fertile adults. There- 
fore, full homologous association at the time of gas- 
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