examined by electron microscopy (in collaboration 
with Dr. William Brinkley), but no dynein defects 
were detected. 
Mouse geneticists had previously identified the 
mutation situs inversus viscerum (or iv), which 
maps to mouse chromosome 12. Mice that are ho- 
mozygous for the iv mutation exhibit a random spec- 
ification of left-right polarity. Fifty percent of the 
mutants show normal polarity, the other 50% situs 
inversus. By contrast the homozygous OVE210 
mice exhibit 100% situs inversus. To test for possi- 
ble allelism between the two mutations, homozy- 
gous iv mice (strain Sl/Col from Dr. Robert Collins, 
Jackson Laboratory) were mated to heterozygous 
OVE210 mice. The transgenic offspring all showed 
normal left-right polarity, indicating that the two 
mutations were not allelic and suggesting that the 
transgenic insert inactivates a previously unidenti- 
fied gene essential for the normal specification of 
left-right polarity. 
A XEMBL3 genomic library was generated using 
OVE210 DNA, and four overlapping clones of the 
transgenic integration site were isolated. One of the 
clones contained the entire transgenic insert 
flanked on both sides by genomic sequences. These 
sequences have been used to confirm by restriction 
fragment length polymorphism (RFLP) analysis that 
the situs inversus mutants are homozygous for the 
transgenic DNA. 
The flanking sequence probes have also been 
used to identify homozygous embryos for studies of 
early embryonic development. These studies have 
revealed that the situs inversus embryos rotate in 
the opposite direction from normal embryos during 
early development. The direction of embryonic ro- 
tation is established even before folding of the car- 
diac tube, which was previously assumed to repre- 
sent the first step in the establishment of left-right 
asymmetry during mammalian development. 
The flanking probes have recently been mapped 
onto the mouse genome in collaboration with Drs. 
Neal Copeland and Nancy Jenkins. Both of the 
probes map to the proximal region of mouse chro- 
mosome 4 . Two candidate genes that had previously 
been mapped to the same region were Pax- 5 (a 
paired-box gene) and Ggtb (a glycoprotein galacto- 
syltransferase) . Probes for these genes were ob- 
tained from Drs. Peter Gruss and Gregory HoUis, 
respectively. The homozygous OVE210 mice 
showed no changes by RFLP analysis for either gene. 
The probes flanking the transgenic insert map 
~ 10 cm apart on mouse chromosome 4, suggesting 
the possibility of a large deletion or a rearrangement 
at the site of the transgenic insert. Two other genes 
had previously been mapped to the region of chro- 
mosome 4 that lies between the transgenic flanking 
sequences. These genes {Tal-2 from Dr. Richard 
Baer; Gt4-2 from Dr. Alexandra Joyner, HHMI Inter- 
national Research Scholar) showed normal patterns 
of hybridization to OVE2 10 DNA, indicating that an 
intrachromosomal inversion accompanies the inte- 
gration of the transgenic DNA. Portions of the geno- 
mic sequences flanking the transgenic insert have 
been subcloned and sequenced. The sequences are 
not identical to any previously reported in the com- 
puter database. 
In order to test for epistatic interaction between 
the iv and transgenic mutations, mating studies 
were performed to generate mice that were homo- 
zygous for both mutations {iv/iv, 210/210). The 
double mutants show random determination of left- 
right polarity, indicating that the OVE2 1 0 gene may 
function earlier than iv in the cascade that deter- 
mines left-right polarity. Inactivation of iv random- 
izes the polarity regardless of the status of OVE2 10. 
The double-mutant mice still exhibit jaundice 
and a failure to thrive, suggesting that the transgenic 
integration may have inactivated more than just one 
essential gene. Nonetheless, the new situs inversus 
mutation may allow the first positional cloning of a 
gene that specifies normal mammalian polarity. 
Dr. Overbeek is also Associate Professor of Cell 
Biology and in the Institute for Molecular Genet- 
ics at Baylor College of Medicine. He has adjunct 
appointments in the Department of Ophthalmol- 
ogy and the Division of Neuroscience. 
Books and Chapters of Books 
Liou, G.I., Matragoon, S., Overbeek, P. A., and 
Yang, J. 1992. Expression of mouse interphotore- 
ceptor retinoid-binding protein gene during de- 
velopment. In Methods in Neuroscience: Gene 
Expression in Neural Tissues (Conn, P.M., Ed.). 
San Diego, CA: Academic, vol 9, pp 101-115. 
Articles 
Al-Ubaidi, M.R., Hollyfield, J.G., Overbeek, P.A., 
and Baehr, W. 1992. Photoreceptor degeneration 
induced by the expression of SV40 T antigen in 
the retina of transgenic mice. Proc Natl Acad Sci 
USA 89:1194-1198. 
Fletcher, F.A., Moore, K.A., Ashkenazi, M., De Vries, 
P., Overbeek, P.A., Williams, D.E., and Bel- 
mont, J.W. 1991. Leukemia inhibitory factor im- 
proves survival of retroviral vector-infected hema- 
topoietic stem cells in vitro, allowing efficient 
long-term expression of vector-encoded human 
GENETICS 245 
