Reports
Assignment of the Rhodopsin Gene to Humon Chromosome 3
Robert 5. Sparkes,*§ T. MohandasJ Sammye L. Newman,§ Camilla Heinzmann,* Daniel Kaufman, || Susan Zollman,*
Paula J. Leveille,§ Allan J. Tobin, || and James F. McGinnisf§
9pter-9q34 by virtue of an X/9 translocation 3 was also
analyzed.
DNA from the parental cell lines and somatic cell
hybrid clones was purified from isolated nuclei4 by incubation in 10 mM EDTA pH 8, 0.2% SDS, 600 fig/
ml proteinase K (Sigma, St. Louis, MO) at 37°C for
24 hr, followed by phenol extraction and ethanol precipitation.
Four independent rhodopsin cDNA clones were
isolated immunologically by screening a lambda gt-11
mouse retina cDNA expression library. The identity
of these clones was confirmed by demonstrating reactivity of the plaques with monospecific anti-rhodopsin
serum and with monoclonal antibody RholD4 (Dr.
Robert Molday). The cDNA insert from each of the
clones cross-hybridized with the others and all crosshybridized with bovine rhodopsin cDNA R1116 (from
Dr. Wolfgang Baehr through Dr. Toshimina Shinohara). None of the rhodopsin clones reacted with preimmune serum nor with monospecific sera against
other retinal proteins, and none cross-hybridized with
control lambda nor with inserts from clones which were
immunologically negative. Each of the clones gave the
same human chromosomal assignment (see below).
Nucleotide sequence analysis indicates that this probe
codes for the carboxy-terminal end of rhodopsin, has
the polyadenylation site, the phosphorylation site, the
retinal binding site, and good homology with the bovine
cDNA (unpublished observations, W. Baehr, P. J.
Leveille, M. Applebury, and J. F. McGinnis).
The 1200 base mouse probe for the rhodopsin gene
cross-hybridizes with human DNA. This probe was
used to detect the presence or absence of the human
rhodopsin DNA. The enzymes were obtained from
Bethesda Research Laboratory and radiolabelled nucleotides were obtained from Amersham Corporation.
The rhodopsin probe was radiolabelled with 32P to a
specific activity of approximately 1-3 X 109 cpm//xg
by random priming.5 Genomic DNAs from the parental cell lines and the hybrid clones were digested with
the restriction endonuclease Hind III (8 U//xg). Approximately 10 ng of DNA from each sample were
The human rhodopsin gene has been assigned to human chromosome 3 through the use of a mouse DNA probe and human/
mouse somatic cell hybrids. Invest Ophthalmol Vis Sci 27:
1170-1172, 1986
Rhodopsin, the major integral membrane protein
of the rod outer segement of photoreceptor cells, initiates the complex cascade of events associated with
the process of vision, by the absorption of a photon of
light. Because of its central role in vision, knowledge
of the gene map location for rhodopsin is important
for determining its relationship to hereditary blindness.
The isolation of the gene coding human rhodopsin has
recently been reported,1 but we are unaware of any
prior report on mapping the human gene. We here
report the assignment of the human rhodopsin gene to
chromosome 3 through the use of a mouse DNA probe
in the study of human/mouse somatic cell hybrids.
Materials and Methods. Somatic cell hybrids were
derived from the fusion of thymidine kinase (TK) deficient mouse cells (B82, GM0347A) and normal human male fibroblasts (IMR91), both obtained from the
Mutant Cell Repository (Camden, NJ). The cells were
fused in a mixed monolayer using a 50% solution of
polyethylene glycol (mol. wt. 1,000) in a balanced salt
solution.2 Multiple independent hybrid clones were
isolated and a preliminary cytogenetic analysis was
done on 10 Q-banded photographed chromosome
metaphases. Sixteen hybrid clones were selected from
an initial set of 40 clones, based on growth characteristics, human chromosome content, and a lack of detectable human chromosome rearrangements. As expected with this type of procedure, the hybrid clones
contained different human chromosome content. The
clones were then grown in multiple dishes and pooled,
and cell pellets were prepared for DNA extraction.
From the same pooled cells of each clone, an analysis
of chromosome content was made on a minimum of
30 Q-banded photographed metaphases per hybrid
clone. Because these mouse-human hybrids do not retain human chromosome 9, DNA from a Chinese
hamster-human hybrid clone selectively retaining
1170
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1171
Reports
No. 7
A
Fig. 1. Southern blot results using the rhodopsin
probe and the Hind III restriction endonuclease. The
numbers to the left indicate
the size of the fragments in
kilobases. Channel F is from
human control and channel
E is from mouse control.
Channels A, C, and D are
from somatic cell hybrids
positive for the human 10 kb
human band and channel B
is from a hybrid negative for
the human band.
B
C
D
E
3.8-
shaking. The filter was then washed twice, first in 2X
SSC, 0.1% SDS and then in 0.1X SSC, 0.1% SDS, each
time for 20 min at 55°C. The filter was dried briefly
and exposed to Kodak (Rochester, NY) XAR-5 film.
Results. The DNA probe detects a 10 kb band in
the human genomic DNA following digestion with
Hind III (Fig. 1). This band is readily distinguishable
electrophoresed through a 1.2% agarose gel in TAE (40
mM Tris acetate, 1 mM EDTA) buffer pH 7.4 and
transferred by blotting to nitrocellulose by the method
of Southern.6 Hybridization was performed in 45%
formamide, 4.6X SSC, 5X Denhardt's solution, 20 mM
NaH 2 PO 4 pH 6.5, 250 Mg/ml denatured salmon sperm
DNA and 10% dextran sulfate for 16 hr at 42°C with
Table 1. Segregation of rhodopsin gene with human chromosomes in mouse-human somatic cell hybrids
Human chromosomesf
Hybrid
clone
Rhodopsin*
84-3
84-4
84-5
84-7
84-13
84-26
+
+
+
+
+
+
I
2
+
+
+
+
3
4
+
9 10 11
-
+
+
13 14 15
+
+
-
+
+
- - -
84-38
84-39
+
+
+ - + + ( + ) + +
- - + - - _ +
+
-
+ + - +
+
- - - - - + +
+ - +
_ _ _ + _ +
- - _ +
+
8
9
0
7
-
+
++
16
17
18
19
20
21
22
X
Y
+
+
+
84-35
84-2
84-21
84-25
84-27
84.34
84-37
No. of Discordant
Hybrids
12
+
+
+
+
8
+ ) +
+
+
+ + + + +
+
+
7
+
+
+
+
- - - _
+
+
+
+
_
_
_ _
+
+
+
+
+
+ +
+
+
- +
- - - +
+
+
+
+
- - +
+
+
+
+
+
+
+
+
+
_
6
+
_ + _ _
+ + + + + - - - - (
- - + + - + +
+ - - +
- - + - - + - - - + + - + + + + . + + - + '-
-
+
5
+
+
-
+
+
-
+
+
-
-
+ - + + + + _ _ - _ _ + _
+
+
• + + ( + ) _ _ - -
+
-
+ +
+ - - +
+
+ +
+
+ - - + +
+ - + + - + + ( + ) - - - + + - - +
+ +
_ + +
_ _
+ _
_
+
+
+ + ( + ) _ - +
+ _ + _ -
10 7 7 7 1 1 8
* + = presence of the rhodopsin sequence in the hybrid clones determined
by the presence of the 10 kb band. - = the absence of the gene.
I + = presence of the human chromosome in greater than 30% of metaphases
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14
9
3
6
5
10
+
+
+
+
+
-
-
-
-
- - _
+
+
+
+
+
- - +
+ - - ( + )
- - +
- - +
+ • +
- _ ( + ) +
_
+
_ _
_ _
+
+
+
- _
6
5
+
8
8
10
9
9
10
analyzed; (+) indicates presence of the chromosome in 10-30% of metaphases
analyzed; — = absence of the human chromosome.
1172
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1986
from the mouse bands of 3.8 and 12 kb (Fig. 1). Table
1 summarizes the studies of the individual somatic cell
hybrids and shows that the human rhodopsin band
maps to human chromosome 3. There were no discordant clones for the co-segregation of the rhodopsin
band and chromosome 3. All other chromosomes
showed at least 3 or more discordancies. None of the
mouse/human clones retains the chromosome 9; analysis of a single Chinese hamster/human hybrid clone
selectively retaining most of chromosome 9 was negative for the presence of human rhodopsin.
Discussion. Based upon the characterization of the
DNA probe used in this study, there is high certainty
that this represents rhodopsin. This probe used in conjunction with mouse/human somatic cell hybrids has
assigned the rhodopsin gene to human chromosome
3. These studies demonstrate the power of this technique, especially in the use of a non-human probe to
map a human gene. These studies suggest that there is
only a single human rhodopsin gene; or, if there are
more than one, they all map to chromosome 3.
There has been no specific human disease related to
a mutation affecting the gene for rhodopsin. It would
seem that this autosomal gene is not involved in mutations related to color vision defects, at least those of
the X-linked variety. The availability of this DNA
probe will make it possible to study inherited retinal
diseases and to investigate whether a mutation of rhodopsin is responsible for the disease. This may be possible by direct study of the rhodopsin gene from individuals with retinal diseases or through the use of genetic linkage looking for probe co-segregation of the
disease with the rhodopsin gene. This latter approach
would use the DNA restriction fragment length poly-
Vol. 27
morphisms for the rhodopsin gene in affected families.
This approach is underway in our unit.
Key words: rhodopsin gene, DNA probe, somatic cell hybrids,
human chromosome 3, retina
Acknowledgment. We thank A. J. Lusis, PhD, for helpful
discussions at several stages of this work.
From the Departments of *Medicine, fAnatomy, ^Obstetrics &
Gynecology, and the §Mental Retardation Research Center, UCLA
Center for the Health Sciences; the || Department of Biology, UCLA;
and the HDi vision of Medical Genetics, Harbor-UCLA Medical Center, Torrance, California. Supported in part by Grants HD 05615,
NS 20356, NS 22256, and GM 07185 from the NIH; Dystonia Medical Research Foundation. Submitted for publication: November 21,
1985. Reprint requests: James F. McGinnis, PhD, MR Unit, Neuropsychiatric Institute, UCLA Center for the Health Sciences, Los
Angeles, CA 90024.
References
1. Nathans J and Hogness DS: Isolation and nucleotide sequence
of the gene encoding human rhodopsin. Proc Natl Acad Sci USA
81:4851, 1984.
2. Davidson RL and Gerald PS: Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol.
Somatic Cell Genet 2:165, 1976.
3. Mohandas T, Sparkes RS, Sparkes MC, Shulkin JD, Toomey
KE, and Funderburk SJ: Regional localization of human gene
loci on chromosome 9: studies of somatic cell hybrids containing
human translocations. Am J Human Genet 31:586, 1979.
4. Fodor EJB and Doty P: Highly specific transcription of globin
sequences in isolated reticulocyte nuclei. Biochem Biophys Res
Comm 77:1478, 1977.
5. Feinberg AP and Vogelstein B: A technique for radiolabeling
DNA restriction endonuclease fragments to high specific activity.
Anal Biochem 132:6, 1983.
6. Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503, 1975.
Isolation ond Biochemical Characterization of Frog
Retinal Pigment Epithelium Cells
Rocio Solcedo
The structure and function of the photoreceptor cell depends
on the renewal of its outer segment. Phagocytosis of the rod
outer segments by RPE is an essential part of the renewal
process. Several methods have been reported in order to isolate RPE cells; however, the isolated cells are heavily contaminated by other cell types, mainly erythrocytes and rod
outer segments. The primary aim of this study was the isolation of pure and viable frog RPE cells. Cells were dissociated
in a calcium-free Krebs bicarbonate medium and purified by
centrifugation in a ficoll density gradient. Viability of the purified cells assessed by trypan blue dye exclusion was 95%.
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The metabolic activity of the cells was tested by several parameters: RPE cells consume oxygen at a rate of 11.5 ngatoms/min/mg protein, transport and metabolize l4C-glucose
by a sodium dependent mechanism, and are able as well to
accumulate l4C-leucine and incorporate it into proteins. Results obtained in this study indicate that our isolation procedure yields a more intact preparation of RPE than those
described previously; hence, it may be helpful in elucidating
the biochemical and metabolic parameters involved in pigment
epithelium physiology. Invest Ophthalmol Vis Sci 27:11721176,1986