chromosome sites. By following the early conden- 
sation sites at early prophase, Dr. Sedat and his col- 
leagues could see bidirectional chromosome con- 
densation growth from these nuclear envelope 
attached sites. Study of the prometaphase nuclear 
transition revealed that a 1.7 (jun/min wave of chro- 
mosome-nuclear compaction started at the centro- 
mere end of the nucleus and proceeded toward the 
telomere end. These investigations are proceeding 
with loci identification and efforts to quantify the 
long-range chromosome movements observed dur- 
ing the nuclear cycles. 
II. Structural Characterizations of a Highly Discon- 
tinuous Nuclear Lamina Localization Pattern. 
What is the distribution on the nuclear surface of 
the major nuclear envelope protein structural 
lamin? Two-color, separate fluorescent labels (using 
a lamin-specific monoclonal antibody and a DNA- 
specific dye, DAPI) have been used to investigate 
this problem. The three-dimensional images were 
aligned so that comparisons were possible. The 
lamin distribution on the nuclear envelope was a 
highly discontinuous fibrillar pattern that left large 
voids in the nuclear periphery. This pattern did not 
change by varying fixation conditions or through 
use of polyclonal antibodies and was seen in a vari- 
ety of tissue types, including mammalian cells. 
Careful quantitation using Fourier and real-space 
methods shows 0.25 |jum fibers with spaces of 0.5 
|jim between the fibers, with approximately half of 
the nuclear surface occupied by the lamin struc- 
tures at OM resolution. EM experiments using 10 A 
gold-antibody beads and silver detection have con- 
firmed the optical studies. Software written for this 
project was used to "unfold" the nuclear surface 
and to study the association of the lamin structures 
with the underlining chromosomal DNA. The vast 
majority of the chromosome regions that come to 
the nuclear surface are not associated directly with 
the lamin but are either significantly below the 
lamin structures or lie in the 0.5 iJum gap between 
the lamin fibers. These studies are continuing. 
III. Three-dimensional DNA In Situ Hybridization. 
Until recently the structure of chromosomes was 
only revealed at certain times in the diploid cell 
cycle when chromosomes were condensed, namely 
prophase through telophase, or in certain special- 
ized tissues, e.g., those containing polytenized 
giant chromosomes. To gain a complete under- 
standing of the structure of diploid chromosomes 
during interphase (when the essential processes of 
gene transcription and DNA replication occur) as 
well as the precise relationship between specific 
chromosomal regions during the entire cell cycle, 
Dr. Sedat and his colleagues have developed new 
three-dimensional OM techniques. 
The three-dimensional structure of the diploid 
nucleus is now being dissected, using high-resolu- 
tion in situ hybridization to whole-mount Drosoph- 
ila embryos. In this procedure, biotinylated probes 
homologous to 8-20 kb of single-copy chromo- 
somal sequences are hybridized in situ to embryos 
that were previously fixed to preserve chromosome 
structure. The location of the hybridization probes 
is revealed by staining these embryos with a 
fluorescently tagged avidin molecule. Optical sec- 
tioning of such embryos reveals the three-dimen- 
sional location of the DNA sequence relative to 
chromosomal structures within the nucleus. 
Preliminary results, using a limited number of hy- 
bridization probes to preblastoderm embryos, sug- 
gest that the chromosomes in diploid interphase 
are arranged much like those in polytene nuclei, 
with the centromeric regions near the embryo sur- 
face and the telomeres diametrically opposed (the 
Rabl orientation). Only one spot is present for each 
hybridization probe, indicating that the homolo- 
gous loci are paired at interphase. Examination of a 
small number of mitotic embryos reveals an unex- 
pected degree of ordering during cytokinesis. 
Currently a wider range of hybridization probes 
is being used to generalize these initial observa- 
tions and to determine how chromosome conden- 
sation proceeds during mitosis and how homo- 
logue pairing redevelops after the necessary 
structural disruptions of mitosis. 
rv Multiple-Label DNA In Situ Hybridization. 
Last year Dr. Sedat and his colleagues completed 
a high-resolution molecular and structural investi- 
gation of specific polytene band/interbands. Results 
of experiments using these newly defined fluores- 
cent in situ hybridization methods show that 1) the 
transcription unit of the Notch gene is located in 
the polytene band; 2) the interband contains the 5' 
region of the gene, in particular the untranscribed 
putative regulatory sequences; 3) the interband is 
short (~3 kb) and extended, hence likely to have 
less higher-order chromosome organization. 
In preparation for the application of these tech- 
niques to other genes, these laboratories have de- 
Continued 
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