groups. Genotype association with other clinical 
manifestations, such as lung and liver disease, are 
also being investigated, but no genotype correlation 
is immediately apparent. 
A full-length cDNA for CFTR was constructed and 
inserted into several expression vectors to facilitate 
direct biochemical and physiological analysis. Re- 
sults of DNA transfection studies showed that this 
cDNA could confer a cAMP-regulated chloride chan- 
nel activity de novo, suggesting that CFTR is a chlo- 
ride channel itself. Although most published studies 
involved the use of a transient assay, the expression 
system developed in Dr. Tsui's laboratory required 
the establishment of permanent transformed cell 
lines so that more subtle alteration of channel prop- 
erties could be detected. Preliminary data from the 
analysis of constructs reproducing some of the natu- 
rally occurring mutations show that they are in good 
agreement with data predicted from the severity of 
pancreatic involvement. 
Multiple transcription initiation sites, alternative 
splicing, and trans-splicing of CFTR transcripts were 
documented for the CFTR gene in different tissues, 
but a major initiation site could be identified within 
a shon distance downstream from an undermethyl- 
ated CpG-rich region. Deletion and transfection 
studies showed that the basal promoter element for 
the CFTR gene was within 250 bp of the major tran- 
scription initiation site. In addition, a negative regu- 
latory element could be located immediately up- 
stream of this sequence, and proper expression of 
CFTR in vivo might require additional cis- 
regulatory element (s) yet to be identified. 
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. Through re- 
placement of the structurally similar domain in 
STEGwixh CFTR sequences, hybrid expression vec- 
tors have been constructed whereby mutations in 
CFTR could be measured by efficiency of yeast mat- 
ing, the normal function of STE6. In addition, sec- 
ond-site mutations that could rescue the AF508 mu- 
tation have been found in adjacent regions of the 
deletion. A systematic survey of these second-site 
mutations should provide important information 
about the structure of NBFl and the possibility of its 
application in drug design. 
Lastly, to generate an animal model for the study 
of CF, work has been initiated to inactivate the 
mouse Cftr gene via homologous recombination in 
embryonic stem cells. The attempt to interrupt exon 
10 has so far been unsuccessful, and the current 
targets in this laboratory are exons 1 and 13- The 
availability of a mutant mouse strain should greatly 
facilitate studies to understand the pathophysi- 
ology of CF and improve means of treatment for the 
disease. 
Regulation of Gene Expression in 
Mammalian Lens Development 
Transparency of the vertebrate eye lens is at least 
in part conferred by the short-range ordering of 
water-soluble crystallin molecules in the lens fiber 
cells. To understand the role these proteins play in 
maintaining lens transparency. Dr. Tsui's laboratory 
has been interested in identifying the mutation re- 
sponsible for a dominant lens defect in the Elo (eye 
lens obsolescence) mouse strain. Through genetic 
linkage and subsequent DNA sequence analysis, a 
frame-shift mutation has been identified in the 7E 
gene, the last member of the six-membered gene 
cluster. This observation strongly argues that 7- 
crystallin plays a major rather than a generally 
assumed passive role in lens development. Experi- 
ments are currently under way to study the domi- 
nant effect of the Elo mutation and the effect of 
other 7-crystallin mutations in transgenic mice. 
Free-Chromatin Mapping 
Based on the use of specific cell-cycle blockers 
and an alkaline lysis buffer, a novel procedure has 
been established whereby chromatin fibers could be 
released from interphase nuclei for physical map- 
ping of the mammalian genome by fluorescent in 
situ hybridization. The hybridization signals from 
two DNA segments separated by <20 kb could be 
easily distinguished. This free-chromatin mapping 
technique has been applied in positional cloning 
strategies to determine the order and orientation of 
flanking markers and in fine structural analysis of 
complex sequence arrangement in the centromeric 
regions. 
Physical Characterization of Human 
Chromosome 7 
To generate a more complete set of reagents for 
the study of genes on human chromosome 7, a chro- 
mosome-specific yeast artificial chromosome (YAC) 
library has been constructed. Based on a human- 
hamster somatic cell hybrid with a single human 
chromosome 7, more than 1,200 YAC clones con- 
taining human DNA inserts with an average size of 
500 kb have been isolated. The clones are being 
mapped to specific chromosome regions by hybrid- 
ization with previously localized DNA segments and 
with a somatic cell hybrid mapping panel. More 
than 1 20 YAC clones and 1 5 overlapping contigs of 
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