Three-Dimensional Structure of Eukaryotic 
Chromosomes 
John W. Sedat, Ph.D. — Investigator 
Dr. Sedat is also Professor in the Department of Biochemistry and Biophysics at the University of Califor- 
nia, San Francisco. He received his Ph.D. degree in biology from the California Institute of Technology. 
His postdoctoral work with Fred Sanger was done at the Medical Research Council in Cambridge, England. 
Before joining the faculty at UCSP, Dr. Sedat was a research associate at Yale University. 
THE three-dimensional structure of chromo- 
somes, both in the nucleus and during cell 
division, remains a major unsolved problem in 
biology. Our laboratory, in collaboration with 
David Agard (HHMI, University of California, San 
Francisco), has investigated chromosome struc- 
ture from the perspective of a series of interlock- 
ing questions. 1) What are the levels of chromo- 
some architecture in the intact diploid nucleus? 
How does the three-dimensional structure 
change as a function of development, or progres- 
sion through the cell cycle? 2) What is the chro- 
mosome architecture of a given gene in the nu- 
cleus? Do the structural attributes reflect the 
detailed molecular information? 3) How do in- 
terphase chromosomes condense to form the in- 
tricate mitotic structure at cell division? The fruit 
fly Drosophila melanogaster, well known for its 
genetics, development, and biochemistry, was 
chosen as a model biological system. Although 
the initial emphasis is structural, molecular ge- 
netics and biochemistry provide functional 
correlations. 
Although the UCSF three-dimensional optical 
microscope has been developed to the point that 
data at several wavelengths can be routinely col- 
lected, even as a function of time (four-dimen- 
sional microscopy), and can be used without 
computer experience, we continue to perfect 
and enhance the instrumentation. We have im- 
proved the time resolution for data collection in 
the four-dimensional image work, permitting 
analysis of much information on biological struc- 
tures. We continue to write software, with exten- 
sive mathematical analysis, to correct systematic 
image acquisition problems, to display results in 
a variety of formats, and to model and analyze, 
often quantitatively, the intricate three- or four- 
dimensional data. 
Four-Dimensional Optical Microscopy 
We have continued to study the structure of the 
cell nucleus in living Drosophila embryos. Nu- 
clei were labeled by microinjection of fluores- 
cent histones or other abundant chromosomal 
proteins. Nuclear and chromosome structures 
were followed throughout the cell cycle during 
embryonic development. In addition to discern- 
ing structural changes, we could now infer 
function. 
This approach has shown several discrete chro- 
mosome sites attached to the nuclear envelope. 
These sites are the last to decondense during telo- 
phase (the stage in the nuclear division cycle that 
follows chromosome separation). Similar sites 
are observed at the beginning of prophase (the 
stage of nuclear division involving chromosome 
condensation) , with condensation proceeding bi- 
directionally away from these centers. 
During prophase, a remarkable wave of chro- 
mosome compaction started at the centromeres 
(chromosome regions at which mitotic spindles 
attach) and proceeded toward the opposite nu- 
cleus pole that contained the telomeres (ends of 
chromosomes) . We could also determine that the 
site for the start of the compaction wave coin- 
cided temporally and spatially with the initial 
breakdown of the nuclear envelope as well as the 
site of the mitotic spindle formation, suggesting 
that there is a high degree of temporal-spatial 
control and organization in the mitotic process. 
A major conclusion is that the structures in 
real-time living nuclei were very similar, if not 
identical, to the three-dimensional nuclear struc- 
tures determined in our ongoing studies at higher 
resolution in fixed embryos. 
Three-Dimensional In Situ Hybridization 
We have analyzed the spatial arrangement of 
chromosomes in embryos of Drosophila. One 
specific biological question is whether homolo- 
gous chromosomes (one from the male parent, 
the other from the female) have an ordered ar- 
rangement in a diploid nucleus. Our previous re- 
sults showed that homologous chromosomes are 
not associated from prophase to anaphase in syn- 
cytial blastoderm stage embryos. This apparently 
contradicts genetic evidence for transvection, 
which suggests that the homologous chromo- 
somes are associated. Thus we wished to test 
whether homologous chromosomes are asso- 
395 
