MOLECULAR ANALYSIS OF BIRTH DEFECTS 
David M. Kurnit, M.D., Ph.D., Investigator 
I. Nondisjunction on Chromosome 21. 
Dr. Kurnit's laboratory wishes to explore why the 
aging of mothers is associated with an increased 
risk for nondisjunction. In a five year effort sup- 
ported by the National Institutes of Health, the cen- 
tromere of chromosome 2 1 will be sandwiched be- 
tween the cytogenetic markers on the shon arm 
and DNA polymorphisms on the long arm. As their 
part in this program, Dr. Kurnit's laboratory is re- 
sponsible for generating DNA sequence variation 
on 21q. In addition to the DNA polymorphisms for 
sequences on chromosome 21 that are available, 
the laboratory is also examining DNA sequence 
variation on chromosome 2 1 by using oligonucleo- 
tides that surround regions of local DNA sequence 
variation mediated by stretches of oligo 
d(A,C):d(G,T). Although it will take considerable ef- 
fort to generate sequence variation on chromosome 
2 1 using this strategy, once the oligonucleotides are 
constructed, DNA sequence variation in a large 
number of subjects can be examined rapidly. The 
two major advantages of this technique are that ra- 
dioactivity need not be used and only a small 
amount of DNA is consumed. The latter point is im- 
portant, because it obviates the need to transform 
white blood cells of subjects with Epstein-Barr virus 
to obtain sufficient quantities of DNA. 
In collaboration with Terry Hassold in Atlanta, 
Dr. Kurnit and his colleagues have performed pilot 
studies to determine the parental origin of nondis- 
junction events that cause Down syndrome. This 
work demonstrates that crossing over occurs in a 
significant plurality (and perhaps all) cases of non- 
disjunction. 
II. Genie Sequences on Chromosome 21. 
Strategies have been employed to isolate ex- 
pressed sequences on chromosome 21. Although 
the specific genes involved in Down syndrome have 
not been identified, their chromosomal location is 
known, as they have been mapped more precisely 
by other laboratories this past year to 21q22. The 
test for expression is presence of a homologous se- 
quence in a complex cDNA library. A system (based 
on the ttVX system of Brian Seed) has been devel- 
oped and used to screen DNA probes for expres- 
sion in an efficient manner. The irPlee series of vec- 
tors constructed in Dr. Kurnit's laboratory contain 
both the chloramphenicol gene and the supF gene. 
This allows harsher selection for integrates than 
was previously possible using supF alone. The 
amber plasmid vector Sumo 15A was designed by 
Dr. Kurnit for the purpose of constructing cDNA li- 
braries and can be screened by recombination 
against TrFlee clones containing pieces of DNA from 
regions of interest on chromosome 21. 
Flow-sorted chromosome 21 fragments (courtesy 
of Drs. P. Dejong and H. Lehrach) have been shot- 
gun cloned into cosmid vectors. After a cosmid is 
mapped to chromosome 21, it is fractionated and 
cloned into TiFlee plasmids. Individual clones are 
grown up, and their copy number is corroborated, 
using the recombination-based assay. Single-copy 
clones carrying fragments of genomic chromosome 
21 DNA are infected with Sumo 15A-cDNA phage. 
The lysate is plated on DK21, an Escherichia coli 
dnaB amber host that was constructed by Dr. 
Kurnit's laboratory and that selects for phage carry- 
ing supF. Only phage containing supF can grow in 
the cell line DK21, and the only way supF can have 
been acquired is by homologous recombination. 
Thus a method has been established for the isola- 
tion of expressed sequences on chromosome 21. 
III. Situs Inversus in Mouse. 
The iv mutation in mouse is an autosomal reces- 
sive mutation that causes random determination of 
situs for the heart and other internal organs. Dr. 
Kurnit's laboratory (with Dr. W Layton) is crossing 
inbred strains containing this mutation with other 
mouse strains to establish linkage with the muta- 
tion. A recent report {Proc Natl Acad Sci USA 
86:5035-5038) mapped this mutation to mouse 
chromosome 12. With a larger series of animals. Dr. 
Kurnit and his colleagues confirmed this report and 
showed that the iv mutation is very close (<1 cen- 
timorgan) to the immunoglobulin heavy-chain 
locus on this chromosome. Genomic strategies to 
walk to the iv locus from the mouse Ig-H locus can 
now be considered. 
IV Two-dimensional Protein Gels and Mouse 
Database. 
Major organogenesis takes place during days 7 to 
10 of gestation in the mouse. Dr. Kurnit has created 
a database of embryonic proteins synthesized dur- 
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