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Investigative Ophthalmology & Visual Science, Vol. 31, No. 8, August 1990 Copyright © Association for Research in Vision and Ophthalmology

Distribution of Carbonic Anhydrase Among Human Photoreceptors T. Michael Nork, Steven A. McCormick,* Gung-Mei Chao, and J. Vernon Odom The distribution of carbonic anhydrase (CA) among human photoreceptors has been a topic of dispute. In our experiments, by modifying an established enzyme histochemical technique, reproducible staining was observed. Of the cones in the peripheral retina, 91% were positive for CA. The CA-negative (CA—) cones were absent within approximately 8 arc min of the foveal center and their density peaked at 2 arc deg. No CA activity was found in the rods. Morphologic differences were noted between the CA-positive (CA+) and CA— cones. Compared to the CA+ cones, the CA— cones had longer and more nearly columnar inner segments, more nearly spherical nuclei, and more generous amounts of perikaryal cytoplasm. In the peripheral retina, the distance between CA+ to CA+ nearest neighbors were larger compared to CA— to CA+ nearest neighbors (P < 0.0001). The frequency distribution and morphology of the CA— cones suggest that they are the blue-sensitive cones. As such, this study demonstrates a biochemical similarity between blue cones and rods that may provide insight into the function and phylogeny of the blue cones. Invest Ophthalmol Vis Sci 31:1451-1458,1990

cones may be similar to rods in this respect because, at least in tree shrew, they also contain the 48-kD protein.7 Cones may lack other enzymes found in rods, such as cyclic GMP and interphotoreceptor binding protein (IRBP).6 Although cones seem to lack some of the enzymes seen in rods, they may contain their own enzymes, one of which may be carbonic anhydrase (CA). CA is an enzyme found in most organs of the body, yet not all of its physiologic functions are fully understood. In the human neural retina, CA is present in the Miiller's glial cells.8'9 Previous investigators have placed the frequency of CA among human cones variously at 0%,8 10%,9 and 100%.l0 CA may" or may not1213 be present in the cones of nonprimate vertebrates. In our earlier work using CA as a marker for Miiller's cells, we noticed also that the cones occasionally were positive for this enzyme but that the percentage of positively stained cones varied greatly between eyes.1415 The purpose of this study was to determine the cause of this apparent variability in CA staining and to ascertain whether distribution of CA among human cones is a marker for certain spectral types.

In recent years, there have been exciting advances in our understanding of the biochemistry of vision, especially as it relates to phototransduction (for example, see the review by Stryer1). But these advances have been limited almost exclusively to rods, largely because most vertebrates have rod-dominated retinas that lend themselves to mechanical isolation of the outer segments for use in traditional biochemical analyses. It may be erroneous to conclude that cone and rod biochemistry is similar except for their chromophores. After all, cones mediate not only color vision but also photopic vision, since, in every vertebrate species except the skate, the rods saturate at fairly low light levels.2 Cones also contain greater numbers of mitochondria in their inner segments compared to rods, again suggesting differences in biochemistry. Investigators using immunocytochemical techniques have observed what may prove to be fundamental differences in the enzyme systems of rods and cones. Specifically, most cones seem to lack the 48-kD protein (also known as S-antigen and as arrestin) that is so abundant in rods.3"7 The blue-sensitive

Materials and Methods

From the Departments of Ophthalmology and *Pathology, West Virginia University Health Sciences Center, Morgantown, West Virginia. Submitted for publication: June 21, 1989; accepted August 14, 1989. Reprint requests: T. Michael Nork, MD, Department of Ophthalmology, West Virginia University Health Sciences Center, Morgantown, WV 26506.

Suspecting a problem related to preparation of the tissue, we placed 15 adult human retinas in 4% phosphate buffered paraformaldehyde at times ranging from 1 min to 24 hr after cessation of retinal blood flow (prefixation time). Three of the eyes (with prefixation times of 1-5 min) were obtained as surgical

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polymorphisms (RFLPs) for DNA markers, and progeny from such crosses have been most useful for genetic analysis with molecular probes in other studies.12"14 The first experiment was a three-point intersubspecific backcross designed to position the gene corresponding to zr.408 on mouse chromosome 5. In the second experiment, C57BL/6J mice made congenic for the normal allele of rd, derived from the wild mouse, Mus spretus, were analyzed for the segregation of the putative rd gene and the segregation of rd disease expression. Materials and Methods Probes The putative rd cDNA (zr.408) was cloned as a 3.45-kb fragment in the EcoRI site of the plasmid pBluescript SK(—). Because of an internal EcoRI site, two fragments were released from the plasmid after EcoRI digestion; the sizes of the fragments were 1.65 and 1.8 kb. Both fragments gave the same hybridization pattern in Northern blots of mouse retinal RNA. However, only the 1.65-kb fragment was used in the experiments, and the 1.8-kb fragment was not tested further. The cDNA corresponding to the Gus gene (kindly provided by Dr. Gordon Watson, University of California—Berkeley, Berkeley, CA) was a 1.45-kb PstI insert in pBR322 and is referred to as pGAl.15 The cDNA corresponding to the gene Afp (obtained from Dr. Shirley Tilghman, Princeton University, Princeton, NJ) was a 960-bp Hindlll insert in pBR322 and is referred to as pBR322-AFPl.16 Labeling of all cDNAs was done with [«-32P]dCTP (3000 Ci/mmole) (New England Nuclear, Boston, MA) by the random priming method.17 All restriction enzymes were obtained from Promega Biotec, Madison, WI. Intersubspecific Backcross

Mus musculus mice of the NFS/N strain were obtained from the National Institutes of Health, Division of Natural Resources, Bethesda, MD. Mice from the subspecies Mus musculus musculus were obtained from a laboratory colony derived from mice originally trapped in Skive, Denmark and maintained by Dr. M. Potter (NCI contract NO1-CB25584) at Hazelton Laboratories, Rockville, MD. NFS/N females were mated with M. m. musculus males and the Fl females backcrossed with M. m. musculus males to produce the experimental animals. DNAs were extracted from mouse livers by standard procedures,18 cleaved with the appropriate restriction enzyme, run on 0.4% agarose gels for 48 hr at low voltage, and transferred to nylon membranes (Amersham, Arlington Heights, IL) by the technique of Southern.19 The membrane blots were prehybri-

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dized for 3-6 hr at 65°C in a solution containing 7.0% sodium dodecyl sulfate (SDS), 0.5 M phosphate buffer, pH 7.0, 1 mM EDTA, and 1% bovine serum albumin. Hybridization was carried out at 60°C overnight in the same solution as that for prehybridization but with the addition of labeled probe. After hybridization, all blots were washed for 15 min at 37°C in 2X SSC (IX SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7.0) + 0.1% SDS. When probing with zr.408, blots were washed an additional two times for 20 min at 57°C in 2X SSC + 0.1% SDS, and two times for 20 min at 57°C in 0.4X SSC + 0.1% SDS. When probing with pGAl or pBR322AFP1, blots were washed an additional two times for 20 min at 47°C in 2X SSC + 0.1% SDS, and two times for 20 min at 47°C in 0.2X SSC + 0.1% SDS. After washing, the blots were exposed at —80°C to Kodak XAR-5 film with intensifier screens (DuPont Cronex Lightning Plus) for 5-14 days. Interspecific Backcross

Laboratory mice of the C57BL/6J-rd/rd strain were used from our (MML) breeding colony. This line of mice is identical to the congenic C57BL/6J-rd le strain described elsewhere,3 except that the light ear (le) gene was eliminated by a series of crosses. Thus, the mice are homozygous for the rd gene (rd/rd) and homozygous wild-type (+/+) at the le locus. M. spretus mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Over a period of several years these mice were bred according to the following pattern: C57BL/6 J-rd/rd females were mated with M. spretus males and the Fl females (also termed Nl when used for repeated backcross matings) were backcrossed to C57BX/6J-rd/rd males to produce the N2 offspring. A single eye from N2 mice was enucleated under ether anesthesia and was examined histologically3 for the presence or near-absence of photoreceptor cells to identify mice of rd/+ or rd/rd genotypes, respectively. Mice of either sex with the rd/+ genotype were backcrossed with C57BL/6J-rd/rd mice to produce N3 progeny. This process was repeated until the N8, N9, and N10 generations were reached, and these animals were used for the study. Mice from the parental lines (C57BL/6J-rd/rd and M. spretus) also were examined. Mice were sacrificed between 17 days and 5 months of age by cervical dislocation; the eyes were enucleated and prepared for routine eye histology;3 and the brains were dissected and rapidly frozen on dry ice. Tissue DNA was extracted by a modification of the method of Fodor and Doty.20 Individual brains were pulverized in liquid nitrogen and the resulting powder mixed with 3 ml of a solution containing 0.3 M sucrose, 10 mM Tris buffer at pH 7.5, 10 mM

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MgCl2 and 1% Triton X-100, and kept on ice for 10 min. The suspension was then centrifuged at 5-10°C for 10 min at 8000 RPM in the SS-34 rotor in a Sorvall RC-5B centrifuge (7649 g) and the pellet resuspended in 3 ml of a solution containing 75 mM NaCl, 24 mM EDTA at pH 8.0, 0.5% SDS, and 600 itg/m\ protease K. Incubation of the suspension was carried out at 44°C overnight or until all particulate matter disappeared. RNAse was added to a concentration of 300 /ig/ml, and a second overnight incubation was carried out at 37°C. The mixture was extracted once with an equal volume of P/C (50% equilibrated phenol and 50% solution containing 24 parts chloroform to 1 part isoamyl alcohol), and once with an equal volume of the chloroform-isoamyl alcohol mixture. DNA was precipitated overnight at room temperature in 0.1 volume of 3 M sodium acetate and 1.5 volumes of 95% ethanol. After washing in 75% ethanol the centrifuged DNA pellet was dissolved in TE buffer (10 mM Tris buffer and 1 mM EDTA) at pH 7.4. For blot hybridization studies, the extracted mouse genomic DNAs were digested with the appropriate restriction endonuclease, loaded at 6 jug per lane in 1.2% agarose gels, and electrophoresed for 16-20 hr. Transblotting c r the DNA to nylon membranes, and prehybridization, hybridization with the zr.408 probe, washing, and exposure of the membranes to X-ray film, was performed as described above. All procedures with animals conformed to the ARVO Resolution on the Use of Animals in Research and the guidelines of the committees on animal research at our respective institutions.

1

2

t

HI 12.5 kb

.1 kb

Fig. 1. Autoradiogram of a Southern blot of DNAs from interspecies backcross parental controls M. m. musculus (lane I) and NFS/N (lane 2) hybridized with the zr.408 cDNA; each lane has 6 Mg DNA. The DNAs were digested with Seal restriction endonuclease. The arrows point to t*ie 12.5-kb NFS/N fragment and the 11.1 -kb M. m. musculus fragment.

Results • - • *

Intersubspecific Backcross

DNAs from the NFS/N and M. m. musculus mice were examined for RFLPs that hybridize to the zr.408 probe. When digested with Seal, DNA from NFS/N mice showed a 12.5-kb fragment that was absent from M. m. musculus, and M. m, musculus DNA showed an 11.1-kb fragment that was absent from NFS/N (Fig. 1). DNAs were extracted from 62 progeny of the backcross ([NFS/N X M. m. musculus^ \ X M. m. musculus) and scored for inheritance of the NFS/N fragment (Fig. 2). This inheritance pattern was compared with the inheritance patterns of two genes that flank the rd locus on mouse chromosome 5, Gus and Afp. The NFS/N allele for Gus was followed as a pGA1-hybridizing Hindlll RFLP of 3.7 kb (the M. m. musculus RFLP was 3.9 kb), and the NFS/N allele for Afp was followed as a pBR322AFP1-hybridizing EcoRI RFLP of 6.0 kb (the M. m. musculus RFLP was 3.8 kb).

In the 62 mice for which all three genes were scored, four recombinants were found to contain the NFS/N Afp aJlele but not the NFS/N fragment crossreactive with zr.408. This suggests a map distance of 6 ± 3.1 cM (recombination = 4/62 = 6 ± SE 21). For zr.408 and Gus, the corresponding map distance was 21 ± 5 . 2 cM, and for Afp and Gus, 27 ± 5.7 cM (Table 1). Considering the three genes taken together, all recombinants were single recombinants. That is to say, whenever there was a recombinant between Afp and zr.408, the allelic pattern of Gus was the same as that of zr.408, and whenever there was a recombinant between Gus and zr.408, the allelic pattern of Afp was the same as that of zr.408. There was no instance in which zr.408 was of one allelic pattern and both Afp and Gus were of the other (double recombinant). Therefore, the most likely gene order is Afp—zr.408 —Gus. The corresponding distances in the standard mouse linkage maps of Lyon9 and Davisson et al10 are

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1 2

*

3

*

4

5

I



Fig. 2. Autoradiogram of a Southern blot of interspecies backcross progeny DNAs of the cross (NFS/N X M, m. musculus)F\ X M. m. muscttlus. DNAs were digested with Seal and hybridized with the zr.408 probe; each lane has 10 tig DNA. Five representative backcross progeny are in tanes I-5. The lower arrow marks the 11. l-kb M. m. musculus fragment present in all backcross progeny, and the upper arrow marks the 12.5-kb NFS/N fragment present only in progeny heterozygous for the NFS/N putative rd allele (ie, lanes 3 and 5).

smaller in this region of chromosome 5 than are our recombination distances. However, the distances reported here between Afp and zr.408 and between zr.408 and Gus are not statistically different from expected distances between Afp and rd and between rd and Gus in this sample size (x2 = 2.06 and x2 = 3.59, respectively; P < 0.05 for both). Furthermore, our recombination distances fall within the range of recombination distances reported for rd, Gus, and Afp in earlier studies.22'23 Therefore, the location of zr.408 between Afp and Gus and its position relative to the two genes strongly suggests that zr.408 corresponds to the rd gene.

zr.408 probe. BamHI digestion revealed two prominent fragments of 12.5 and 7.5 kb present in M. spretus and absent from the C51BL/6J-rd/rd mice (Fig. 3, lanes I and 2). Histologically, M. spretus mice had normal retinas and C57BL/'6J-rd/rd mice had retinas nearly devoid of photoreceptors, as expected.3 DNAs were then extracted from mice from the N8, N9, and NIO generations and examined for the two M. spretus RFLPs, ie, the M. spretus allele (Fig. 3, lanes 3-9). These results were compared to the histologic results demonstrating the presence (rd/rd) or absence (rd/+) of retinal degeneration. Of 72 mice tested by blot hybridization with the zr.408 probe, 45 were heterozygous (contained the M. spretus allele) and 27 were homozygous (showed only the C57BL/ 6J hybridization pattern). Each of the 45 heterozygotes had normal retinas and each of the 27 homozygotes had degenerative retinal disease. Thus, no recombinants were detected between the putative rd gene and the appearance of retinal degeneration. These data can be used to estimate the maximum possible distance between rd and the zr.408-hybridizing sequences by the formula P = (I - R)n 2t, where R = recombination distance between zr.408-hybridizing genomic sequences and the gene responsible for the observed retinal degeneration; n = the number of mice; and P = probability. The expression (I — R)n represents the probability P that two genes will segregate together in a mating of n mice with zero recombinants. If P < 0.05, then the two genes are significantly linked within a distance of R. At the 95% confidence limit Pis 0.05. Since I mouse can be added to the total studied for each N backcross analyzed, a

Table I. Segregation of a zr.408 hybridizing fragment (putative rd gene) with Afp and Gus among 62 backcross progeny between NSF/N and Mus musculus musculus (Skive)* Inheritance of NFS/N allele Mice

Afp-1

Putative rd

Gus

Nonrecombinants (parental genotypes) Single recombinants

Number ofprogeny 21 24

4 0 6 (Afp, putative rd) (Afp, Gus) (putative rd, Gus)

= 4/62 = 17/62 = 13/62

Analysis of Segregation of rd and zr.408

Gene order: centromere—Afp—putative

rd—Gus

DNAs from C51BL/6J-rd/rd and M. spretus mice were examined for RFLPs that hybridized with the

* Distances in cM between hybridizing fragments and standard errors were calculated according to Green21 from the number of recombinants. t All recombination values are significant to the 0.05 level.

Recombination

= 6±3.If = 27 ± 5 . 7 = 21 ± 5.2

MAPPING AND CO-SEGREGATION OF A PUTATIVE rd cDNA / Donciger er ol

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3

4 5 6 7 8 9

23.1kb» 9.4 kbmi

6.6 kb-

4.4 k b -

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Gus in our three-point cross was comparable to the position of the rd gene in standard mouse gene maps. The second study demonstrated linkage between the putative rd gene and rd expression (in this case, photoreceptor degeneration). With zero recombinants in the equivalent of 80 mice, we were able to establish with a >95% probability that the zr.408 cDNA hybridized to DNA sequences that are within 4 cM of the rd gene. Taken together, the data from the two studies position the genomic sequences corresponding to zr.408 to a site on chromosome 5 at or near the rd mutation, and demonstrate its co-segregation with retinal degeneration in genetic crosses. Thus, the data are consistent with our characterization of zr.408 as the normal product of the rd locus. Key words: rd mouse, rd gene, interspecies backcross, cosegregation,finemapping

Acknowledgments

2.3 kb» Fig. 3. Autoradiogram of a Southern blot of progeny of the N8 and N9 generation of crosses (described in the text) that were derived from the original backcross (C57BL/6i-rd/rd X M. spretus)Fl X C51BL/6J-rd/rd. DNAs were digested with Bam HI and hybridized with the zr.408 probe; each lane has 6 fig DNA. The C57BL/6J-rdfrd mouse control is in lane 1 and the M. spretus control is in lane 2. Representative progeny from the N8 and N9 generations are in lanes 3-9. The arrows on the right mark the 12.5and 7.5-kb RFLPs found in the M. spretus control (lane 2) and in some of the backcross progeny (lanes 4, 5, and 7-9). The backcross progeny in lanes 3 and 6 show only the C57BL/6J-rd/rd pattern. The numbers on the left (in kilobase pairs) mark the positions of X DNA fragments produced by digestion with Hindlll.

minimum of 8 can be added to the 72 mice tested, such that n = 80. Then, (1 - R ) 8 0 = 0.05, and R = 0.0368. Therefore, there is a 95% probability that the mouse genomic DNA sequences to which zr.408 hybridizes are within 3.68 cM of the knownrafgene. Discussion We tested a putative rd cDNA, zr.408 (recently cloned in our laboratory) by two methods, to verify that this cDNA corresponds to the rd gene. With a three-point intersubspecific backcross we showed that zr.408 hybridizes to mouse DNA sequences that lie between the genes Afp and Gus. The position of the putative rd gene relative to Afp and

The authors thank Drs. Robert Sparkes and Bronwyn Bateman for helpful discussions; Dr. Lawrence Pinto for the initial suggestion to produce the interspecific crosses with M. spretus; and Edwin Mar, Gregg Gorrin, and Douglas Yasumura for expert technical assistance.

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