Cystic Fibrosis, Gene Expression in the Mammalian Lens, and Mapping 
of Chromosome 7 
activity and tissue specificity. Our current data 
suggest that the basal promoter element is within 
250 bp of the major transcription initiation site. 
There also seems to be a negative regulatory ele- 
ment immediately upstream of this sequence, 
and proper expression of CFTR in vivo may re- 
quire additional cis-regulatory element(s) yet to 
be identified. A thorough understanding of the 
regulation of CFTR transcription may provide ad- 
ditional means for treatment of CF, particularly in 
some mild cases where an increase of CFTR syn- 
thesis may compensate for the partial defect. 
Experiments are also in progress to exploit the 
yeast STE6 gene as a genetic system to gain some 
insight into the structure and function of the first 
ATP-binding domain (NBFl) in CFTR. A system- 
atic survey of second site mutations (which may 
rescue the primary mutation) should provide im- 
portant information about the structure of NBFl 
and the possibility of its application in drug 
design. 
Lastly, in order to generate an animal model for 
the study of CF, we have been trying to inactivate 
the mouse Cftr gene via homologous recombina- 
tion in embryonic stem cells. Our attempt to in- 
terrupt exon 1 0 has so far been unsuccessful, and 
our current targets are exons 1 and 13. The avail- 
ability of a mutant mouse strain should greatly 
facilitate studies to clarify the pathophysiology of 
CF and improve means of treatment. 
Regulation of Gene Expression 
in Mammalian Lens Development 
Transparency of the vertebrate eye lens is at 
least partly conferred by the short-range ordering 
of water-soluble crystallin molecules in the lens 
fiber cells. In order to understand the role these 
proteins play in maintaining lens transparency, 
one of our interests has been to identify the muta- 
tion responsible for a dominant lens defect in a 
mouse strain called Elo {Eye lens obsolescence) . 
We have performed genetic linkage analysis to 
show that the Elo mutation is closely linked to the 
7-crystallin gene cluster and have excluded the 
first five of the six genes in the cluster as being 
the location of the mutation. Through subse- 
quent sequence analysis, we have identified a 
frameshift mutation in the gene, the last 
member of the six-membered cluster. Although 
the significance of the latter observation is pres- 
ently unknown, it strongly argues that 7-crystal- 
lin plays a major rather than a generally assumed 
passive role in lens development. 
Physical Characterization of Human 
Chromosome 7 
In order to generate a more complete set of 
reagents for the study of genes on human chro- 
mosome 7, a chromosome-specific yeast artificial 
chromosome (YAC) library has been constructed. 
Using a human-hamster somatic cell hybrid with 
a single human chromosome 7, we have isolated 
more than 1,000 YAC clones containing human 
DNA inserts averaging 475 kb. The clones are be- 
ing mapped to specific chromosome regions by 
hybridization with previously localized DNA seg- 
ments and with a somatic cell hybrid mapping 
panel. Over 100 clones have thus far been identi- 
fied. In addition to the generation of a long-range 
physical map of chromosome 7, we are develop- 
ing efficient techniques for detecting gene se- 
quences based on YAC cloning. 
We have also developed a novel in situ hybrid- 
ization procedure to facilitate our physical map- 
ping effort. Using chromatin fibers released from 
interphase nuclei with specific reagents (mostly 
known as topoisomerase II inhibitors) and an al- 
kaline buffer, we have been able to perform fluo- 
rescent in situ hybridization to order DNA 
segments less than 20 kb apart. Immediate appli- 
cation of this technique (named free chromatin 
mapping) should allow us to estimate physical 
distance between any given DNA segments and to 
study complex sequence arrangement, such as 
that in the centromeric regions. 
532 
