Role of tyrosine-sulfated proteins in retinal structure and function.

The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins' tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases.

Neuroprotectin D1 is synthesized in the cone photoreceptor cell line 661W and elicits protection against light-induced stress.

Docosahexaenoic acid (DHA), an omega-3 fatty acid family member, is obtained by diet or synthesized from dietary essential omega-3 linolenic acid and delivered systemically to the choriocapillaris, from where it is taken up by the retinal pigment epithelium (RPE). DHA is then transported to the inner segments of photoreceptors, where it is incorporated in phospholipids during the biogenesis of outer segment disk and plasma membranes. As apical photoreceptor disks are gradually shed and phagocytized by the RPE, DHA is retrieved and recycled back to photoreceptor inner segments for reassembly into new disks. Under uncompensated oxidative stress, the docosanoid neuroprotectin D1 (NPD1), a potent mediator derived from DHA, is formed by the RPE and displays its bioactivity in an autocrine and paracrine fashion. The purpose of this study was to determine whether photoreceptors have the ability to synthesize NPD1, and whether or not this lipid mediator exerts bioactivity on these cells. For this purpose, 661W cells (mouse-derived photoreceptor cells) were used. First we asked whether these cells have the ability to form NPD1 by incubating cells with deuterium (d4)-labeled DHA exposed to dark and bright light treatments, followed by LC-MS/MS-based lipidomic analysis to identify and quantify d4-NPD1. The second question pertains to the potential bioactivity of these lipids. Therefore, cells were incubated with 9-cis-retinal in the presence of bright light that triggers cell damage and death. Following 9-cis-retinal loading, DHA, NPD1, or vehicle were added to the media and the 661W cells maintained either in darkness or under bright light. DHA and NPD1 were then quantified in cells and media. Regardless of lighting conditions, 661W cells acquired DHA from the media and synthesized 4-9 times as much d4-NPD1 under bright light treatment in the absence and presence of 9-cis-retinal compared to cells in darkness. Viability assays of 9-cis-retinal-treated cells demonstrated that 34 % of the cells survived without DHA or NPD1. However, after bright light exposure, DHA protected 23 % above control levels and NPD1 increased protection by 32 %. In conclusion, the photoreceptor cell line 661W has the capability to synthesize NPD1 from DHA when under stress, and, in turn, can be protected from stress-induced apoptosis by DHA or NPD1, indicating that photoreceptors effectively contribute to endogenous protective signaling mediated by NPD1 under stressful conditions.

Transgenic Bcl-2 expressed in photoreceptor cells confers both death-sparing and death-inducing effects.

To examine its potential role within the retina as a modulator of cell death and photoreceptor degeneration, bcl-2 expression was targeted to the photoreceptors of transgenic mice by the human IRBP promoter. Three transgenic families were established, with levels of transgene expression between 0.2 and two-fold relative to that of endogenous bcl-2. The effect of bcl-2 expression on genetically programmed photoreceptor degeneration was evaluated by crossing these transgenic mice with mice that develop a rapid degeneration of rod photoreceptors due to expression of a distinct transgene, SV40 T antigen (Tag). Transgenic Bcl-2 was localized to photoreceptor inner segments and was capable of abrogating the activation of caspase activity and the resulting cell death associated with ectopic expression of Tag. However, Bcl-2 itself ultimately caused photoreceptor cell death and retinal degeneration. Several proteins not expressed normally in Tag or other transgenic retinas undergoing photoreceptor degeneration were induced in the Bcl-2 transgenic retinas. Analysis by mass spectroscopy identified one of these proteins as alphaA-crystallin, a member of a protein family that associates with cellular stress. Since Bcl-2 can promote as well as spare cell death in the same photoreceptor population, its potential utility in ameliorating photoreceptor death in human hereditary blinding disorders is compromised.

Bcl-2 overexpression protects photooxidative stress-induced apoptosis of photoreceptor cells via NF-kappaB preservation.

We recently showed that photooxidative stress on cultured photoreceptor cells results in down-modulation of NF-kappaB activity which then leads to apoptosis of cultured 661W photoreceptor cells. In an effort to further delineate the mechanism of photoreceptor cell death, we sought to determine the effects of Bcl-2 overexpression on cell survivability. Wild-type 661W cells were transfected with the plasmid construct pSFFV-neo-Bcl-2 and several clones were isolated. All clones demonstrated increased Bcl-2 mRNA and protein levels, with the B4 clone exhibiting the greatest enhancement. On exposure to visible light the B4 cells were protected from undergoing apoptosis when compared with the mock transfected cells, as ascertained by TUNEL apoptosis assay and formazan based estimation of cell viability. The Bcl-2 overexpressing cells also maintained a higher Bcl-2/Bax ratio, suggesting that this ratio is important in protection from photooxidative stress. Electrophoretic mobility shift assays for NF-kappaB demonstrated higher activity in both nuclear and cytosolic fractions of the B4 photoreceptors compared with the 661W wild-type cells at all light exposure time points. Furthermore, the findings of the gel shift assays were further supported by immunocytochemistry for NF-kappaB which revealed that protein levels of the RelA subunit of NF-kappaB were protected in the nucleus as well as in the cytoplasm of Bcl-2 overexpressing B4 cells exposed to light compared to the 661W cells. These results suggest that Bcl-2 overexpression protects NF-kappaB protein levels and activity in the nucleus, indicating that preservation of NF-kappaB binding activity in the nucleus may be essential for photoreceptor cells to survive photooxidative damage induced apoptosis.

The relationship between opsin overexpression and photoreceptor degeneration.

PURPOSE: To characterize the process by which overexpression of normal opsin leads to photoreceptor degeneration. METHODS: Three transgenic mouse lines were generated that express different levels of an opsin with three amino acid modifications at the C terminus. These modifications created an epitopic site that can be readily distinguished from the endogenous protein using a bovine opsin-specific antibody. Evidence of degeneration associated with opsin overexpression was provided by anatomic studies and electroretinogram (ERG) recordings. Western blot analysis was used to confirm the production of the transgenic opsin, and an enzyme-linked immunosorbent assay (ELISA) was used to determine the amounts of opsin overexpressed in each line. Immunocytochemistry was used to determine the cellular localization of transgenic opsin. Amounts of 11-cis retinal were determined by extraction and high-performance liquid chromatography (HPLC). RESULTS: Opsin expression levels in the three lines were found to be 123%, 169%, and 222% of the level measured in nontransgenic animals, providing direct correlation between the level of transgene expression and the severity of the degenerative phenotype. In the lower expressing lines, ERG a-wave amplitudes were reduced to less than approximately 30% and 15% of normal values, whereas responses of the highest expressing line were indistinguishable from noise. In the lowest expressor, a 26% elevation in 11-cis retinal was observed, whereas in the medium and the high expressors, 11-cis retinal levels were increased by only 30% to 33%, well below the 69% and 122% increases in opsin levels. CONCLUSIONS: The overexpression of normal opsin induces photoreceptor degeneration that is similar to that seen in many mouse models of retinitis pigmentosa. This degeneration can be induced by opsin levels that exceed by only approximately 23% that of the normal mouse retina. Opsin overexpression has potential implications in retinitis pigmentosa.

Degeneration of cone photoreceptors induced by expression of the Mas1 protooncogene.

Although transgenic expression of oncogenes typically leads to tumorigenesis, oncogene expression directed to the rod photoreceptors leads to cell death without tumor formation. To evaluate the cellular and functional changes induced in cone photoreceptors by an oncogene, the Mas1 protooncogene was targeted to the cones of transgenic mice by the human red/green opsin promoter. Mas1 was chosen because of its exclusive expression in the nervous system and its homology to opsin. The overall histologic appearance of the transgenic retina was normal and retinal tumors were never observed. While rod-mediated electroretinograms were normal in all respects, cone-mediated responses were diminished in direct relationship to the level of transgene expression as determined by Northern blot analysis. Responses of UV- and green-sensitive cones were reduced equivalently, and Northern analysis and immunocytochemistry indicated that cone photoreceptor densities were markedly diminished throughout transgenic retinas. These results indicate that oncogene expression in cones induces cell death without tumor formation and support the possibility that aberrant oncogene expression may underlie some forms of hereditary retinal diseases. The Mas1 transgenic mice may be useful in understanding the cone photoreceptor degeneration that occurs in cone dystrophies and age-related macular degeneration and in evaluating potential therapies for these disorders.

Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain.

Retinal neurons of virtually every type are coupled by gap-junctional channels whose pharmacological and gating properties have been studied extensively. We have begun to identify the molecular composition and functional properties of the connexins that form these 'electrical synapses,' and have cloned several that constitute a new subclass (gamma) of the connexin family expressed predominantly in retina and brain. In this paper, we present a series of experiments characterizing connexin36 (Cx36), a member of the gamma subclass that was cloned from a mouse retinal cDNA library. Cx36 has been localized to mouse chromosome 2, in a region syntenic to human chromosome 5, and immunocytochemistry showed strong labeling in the ganglion cell and inner nuclear layers of the mouse retina. Comparison of the developmental time course of Cx36 expression in mouse retina with the genesis of the various classes of retinal cells suggests that the expression of Cx36 occurs primarily after cellular differentiation is complete. Because photic stimulation can affect the gap-junctional coupling between retinal neurons, we determined whether lighting conditions might influence the steady state levels of Cx36 transcript in the mouse retina. Steady-state levels of Cx36 transcript were significantly higher in animals reared under typical cyclic-light conditions; exposure either to constant darkness or to continuous illumination reduced the steady-state level of mRNA approximately 40%. Injection of Cx36 cRNA into pairs of Xenopus oocytes induced intercellular conductances that were relatively insensitive to transjunctional voltage, a property shared with other members of the gamma subclass of connexins. Like skate Cx35, mouse Cx36 was unable to form heterotypic gap-junctional channels when paired with two other rodent connexins. In addition, mouse Cx36 failed to form voltage-activated hemichannels, whereas both skate and perch Cx35 displayed quinine-sensitive hemichannel activity. The conservation of intercellular channel gating contrasts with the failure of Cx36 to make hemichannels, suggesting that the voltage-gating mechanisms of hemichannels may be distinct from those of intact intercellular channels.

Retina-derived microglial cells induce photoreceptor cell death in vitro.

In animals with retinal degeneration, the presence of activated microglial cells in the outer retina during the early stages of injury suggests that they may be involved in the ensuing photoreceptor cell death. In the following study, we investigated the effects of rat retina-derived microglial cells on a photoreceptor cell line (661w) using cell culture techniques. The difficulty of obtaining pure populations of photoreceptor cells necessitated our use of the 661w photoreceptor cells generated from retinas of transgenic mice. 661w Cells were incubated for 24-48 h in basal medium or basal medium conditioned by activated microglial cells (MGCM) or Müller cells (MCCM), and tested for cell death using lactate dehydrogenase (LDH) assay. The induction of apoptosis in the 661w cells by MGCM was investigated using Terminal deoxynucleotidyl Transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and DNA laddering. Treatment of 661w cells with MGCM for 48 h resulted in approximately 73% of cells dead as compared with 19-20% of cells grown in either basal medium or MCCM. Serum supplementation or pretreatment with heat did not abolish the cytotoxicity of MGCM. More TUNEL-positive cells were observed in MGCM-treated cultures as compared with those in basal medium. Bands in multiples of approximately 180 bp formed DNA ladders in MGCM-treated but not in basal medium-treated samples. Our study shows that microglial cells release soluble product(s) that induce degeneration of cultured photoreceptor cells. Moreover, the mechanism of microglia-induced photoreceptor cell death may involve apoptosis similar to that observed in animals with retinal degeneration.

Functional characteristics of skate connexin35, a member of the gamma subfamily of connexins expressed in the vertebrate retina.

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the gamma subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials >/= 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the alpha or beta subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 microm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1. 6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Photo-oxidative stress down-modulates the activity of nuclear factor-kappaB via involvement of caspase-1, leading to apoptosis of photoreceptor cells.

The mechanisms of photoreceptor cell death via apoptosis, in retinal dystrophies, are largely not understood. In the present report we show that visible light exposure of mouse cultured 661W photoreceptor cells at 4.5 milliwatt/cm2 caused a significant increase in oxidative damage of 661W cells, leading to apoptosis of these cells. These cells show constitutive expression of nuclear factor-kappaB (NF-kappaB), and light exposure of photoreceptor cells results in lowering of NF-kappaB levels in both the nuclear and cytosolic fractions in a time-dependent manner. Immunoblot analysis of IkappaBalpha and p50, and p65 (RelA) subunits of NF-kappaB, suggested that photo-oxidative stress results in their depletion. Immunocytochemical studies using antibody to RelA subunit of NF-kappaB further revealed the presence of this subunit constitutively both in the nucleus and cytoplasm of the 661W cells. Upon exposure to photo-oxidative stress, a depletion of the cytoplasmic and nuclear RelA subunit was observed. The depletion of NF-kappaB appears to be mediated through involvement of caspase-1. Furthermore, transfection of these cells with a dominant negative mutant IkappaBalpha greatly enhanced the kinetics of down modulation of NF-kappaB, resulting in a faster photo-oxidative stress-induced apoptosis. Taken together, these studies show that the presence of NF-kappaB RelA subunit in the nucleus is essential for protection of photoreceptor cells against apoptosis mediated by an oxidative pathway.

Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family.

We have cloned cDNAs for two closely related connexins (Cx), Cx35 and Cx34.7, from a perch retinal cDNA library. Sequencing of PCR products from genomic DNA revealed that both connexins have an intron 71 bp after the translation initiation site; in Cx35, the intron is 900 bp in length, whereas in Cx34.7 it is approximately 20 kb. Southern blots of genomic DNA suggest that the two connexins represent independent single copy genes. In Northern blots, Cx35 and Cx34.7 transcripts were detected in retina and brain; Cx34.7 also showed a weak signal in smooth muscle (gut) RNA. Antibodies against Cx35 labeled a 30 kDa band on a Western blot of retinal membranes, and in histological sections, the pattern of antibody recognition was consistent with labeling of bipolar cells and unidentified processes in the inner plexiform and nerve fiber layers. When expressed in Xenopus oocytes, Cx35 and Cx34.7 formed homotypic gap junctions, but the junctional conductance between paired oocytes expressing Cx35 was 10-fold greater than that recorded for gap junctional channels formed by Cx34.7. The homotypic gap-junctional channels were closed in a voltage-dependent manner but with relatively weak voltage sensitivity. Heterotypic gap junctions formed by Cx35 and Cx34.7 displayed junctional conductances similar to those of Cx34.7 homotypic pairs and showed a slightly asymmetric current-voltage relationship; the side expressing Cx35 exhibited a higher sensitivity to transjunctional potentials. An analysis of the sequence and gene structure of the connexin family revealed that perch Cx35 and Cx34.7, skate Cx35, and mouse Cx36 constitute a novel gamma subgroup.

Isolation and characterization of a skate retinal GABA transporter cDNA.

PURPOSE: The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina. METHODS: cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution. RESULTS: Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene. CONCLUSIONS: We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.

Light exposure induces ubiquitin conjugation and degradation activities in the rat retina.

PURPOSE: To evaluate the consequences of light exposure on retinal ubiquitin (Ub) conjugation and degradation. METHODS: Two-month-old Long Evans pigmented rats were exposed to constant light (180 foot-candles) or were left in complete darkness for 18 hours. Rats used for cyclic light and diurnal rhythm experiments were removed from their light cycles at different times (24-hour clock): 0700 (before the light was turned on), 1000 (3 hours into the light cycle), 1000D (continued in the dark cycle), 1900 (before the light was turned off), 2200 (3 hours into the dark cycle), and 2200L (continued in the light cycle). The retinas were examined for Ub conjugation, adenosine triphosphate-Ub-dependent degradation, levels of Ub messenger RNA, and localization of Ub immunocytochemistry. RESULTS: There was a statistically significant increase in Ub conjugation and degradation in retinas isolated from light-exposed animals compared with degradation in retinas of dark-adapted animals. However, no significant differences were observed in the levels of Ub messenger RNA from cyclic light, or light-exposed or dark-adapted retinas, suggesting that light-stress-induced changes do not reflect increased transcriptional activity. The daily variations observed in Ub conjugation and degradation suggest that these processes are probably the result of a circadian rhythm. Results of immunohistochemical studies revealed that Ub and its conjugates were uniformly distributed throughout the retinal cell layers in light- and dark-adapted retinas. However, in light-exposed retinas, a strong positive immunoreactivity was observed in the inner retina, specifically in horizontal and ganglion cells. CONCLUSIONS: These results suggest that light exposure may play a role in inducing Ub-conjugating activity in certain retinal cells. Furthermore, the results support the hypothesis that Ub is a stress protein that plays an important role in protecting cells under stress conditions.

A 221-bp fragment of the mouse opsin promoter directs expression specifically to the rod photoreceptors of transgenic mice.

Mutations in the human rod opsin gene have been shown to segregate with autosomal dominant retinitis pigmentosa (ADRP) and photoreceptor degeneration in transgenic mice. While these degenerations are characterized by the primary degeneration of rods, cones eventually die as well. To determine whether this subsequent cone degeneration is the result of expression of mutant rod opsin in the cones, the retinal cell-type specificity of a 221-bp fragment of the mouse rod opsin promoter was evaluated. Two transgenic mouse lines generated by injecting a fusion gene comprised of a 221-bp fragment of the mouse rod opsin promoter and the simian virus 40 large tumor antigen gene (Tag) were examined. The expression of Tag causes photoreceptor cell degeneration in members of both transgenic lines. However, the two lines differed with respect to the level of Tag expression and the rate and extent of photoreceptor cell degeneration. Immunocytochemical localization of opsin and Tag in surviving photoreceptor cells was determined and the results were confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Rod- and cone-mediated function was evaluated by electroretinography (ERG). In the higher Tag-expressing transgenic line only one row of nuclei remained in the outer nuclear layer at postnatal day (P) 150. While these nuclei showed no antigenicity for rod opsin or Tag, they did stain with an antibody that reacts with both rod and cone S-antigens (arrestins), indicating that these cells were surviving photoreceptor nuclei. Positive staining with peanut agglutinin, which uniquely decorates matrix domains surrounding cones in the normal retina, confirmed that the surviving photoreceptor nuclei were of cone origin. RT-PCR substantiated the results from immunostaining; amplification product was obtained using blue cone opsin transcripts but not from either Tag or rod opsin transcripts. The second transgenic mouse line exhibited a much slower photoreceptor cell death that was associated with low levels of Tag transgene transcript. At P120, approximately 50% of photoreceptors remained and an approximately 45% reduction in the rod ERG a-wave was observed. Cone-mediated ERGs, however, were normal. The results demonstrate the rod-specific expression of Tag as directed by the 221-bp fragment of the mouse rod opsin promoter and suggest that the cone degeneration in ADRP or transgenic mice associated with mutations in the rod opsin gene is a secondary effect of rod degeneration.

Molecular cloning of a rod opsin cDNA from the skate retina.

Skates (Raja erinacea and R. ocellata) are among the few animals that have an exclusively rod retina. However, skate rods are unusual in that they are capable of adapting to extremely high levels of illumination that initially saturate the rod photocurrent. This adaptive process restores the ability of the visual cells to respond to incremental photic stimuli and enables them to function under ambient conditions that are subserved by the cone mechanism in mixed (rod/cone) retinae. As a first step towards exploring the molecular basis of visual adaptation in the skate retina, we have cloned and analyzed the opsin cDNA from a skate retina library. The cDNA codes for a protein 354 amino acids (aa) long and 39.7 kDa predicted molecular mass, and labels a single abundant transcript of 1.7 kb in retinal RNA. Amino acid alignments and a parsimony analysis of nucleotide alignments show the skate opsin to be homologous to other rod opsins. An analysis of the aa sequence reveals a high degree of conservation of those residues thought to be important for most aspects of rhodopsin function. However, a few critical aa replacements may indicate alterations in the interactions of skate rhodopsin with other proteins in the phototransduction cascade. In particular, replacements of Glu150 with serine and Cys323 with leucine are in cytoplasmic domains thought to interact with transducin and rhodopsin kinase. The latter change eliminates one of the conserved acylation sites in the carboxyl terminal tail. These substitutions increase the similarity of the cytoplasmic domains of skate opsin to those of blue-sensitive visual pigments.

Structural and developmental analysis of the mouse peripherin/rds gene.

Mutations in the peripherin/rds gene have been reported to be associated with different forms of human autosomal dominant retinitis pigmentosa (ADRP) and macular degeneration (MD). To better understand the disruptive role of these mutations, knowledge of the structure-function relationship of the peripherin/rds gene is needed. To facilitate that, genomic clones encoding the mouse gene were isolated using bovine cDNA sequences as probes. Sequence analysis of clone lambda 6-1-1, that contained the entire coding sequence for the mouse peripherin/rds, yielded the exon-intron organization of the gene. The gene is composed of three exons (581, 247, and 213 bp) and two introns with the first and second introns 8.6 kb and 3.7 kb in size, respectively. Two major (1.6 and 2.7 kb) and three minor (4.0, 5.5, 6.5 kb) transcripts were detected on RNA blots. The major transcripts first appeared in the brain at embryonic day 13 and in the retina at postnatal day 1. Transcripts were missing in brain and eye of mice at embryonic day 15. Several transcription start sites were mapped within 26 nucleotides approximately 200 bp upstream from the translation initiation site. However, transcripts varied in the lengths of their 3' untranslated portion as a result of the utilization of different polyadenylation signals.

Unscheduled DNA replication precedes apoptosis of photoreceptors expressing SV40 T antigen.

Expression of simian virus 40 T antigen (Tag) in the rod photoreceptors of transgenic mice leads to cell death that is completed by the end of the third week of postnatal development. To understand the mechanistic link between Tag expression and the death of the expressing photoreceptors, cell cycle activity was followed in a transgenic mouse family that expresses Tag directed by the mouse opsin promoter. Tag-expressing photoreceptors also expressed rhodopsin suggesting that these cells were differentiated. The presence of Tag in the photoreceptors induced the expression of both proliferating cell nuclear antigen (PCNA) and thymidine kinase (TK). The abnormally high levels of PCNA and TK continued until the complete disappearance of the cells expressing Tag. Photoreceptor cell death was also associated with continued DNA synthesis that ceased shortly after postnatal day 16. The specific loss of the rod photoreceptors that re-entered the cell cycle accounted entirely for the loss of photoreceptors from the outer nuclear layer. The antiproliferative nature of the mature retina is directly involved in the apoptotic death of photoreceptors expressing Tag.

Use of a retroviral vector with an internal opsin promoter to direct gene expression to retinal photoreceptor cells.

PURPOSE: Viral-mediated gene transfer to retina, as well as to other tissues, is evolving rapidly. We have evaluated the potential of a retroviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells. METHODS: Two recombinant retroviral vectors were prepared; in each Vector, a 1.4 kb fragment of the mouse opsin promoter was placed downstream from the neoR gene in the Moloney murine leukemia virus-based vector G1Na. The opsin promoter fragment was linked either to the cDNA for mouse rod photoreceptor phosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expression after transduction of 3T3 and Y79 cells, or of dissociated retinal cell cultures or retinal explants from neonatal mice. RESULTS: As expected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. Northern blot analysis indicated that expression was derived from the viral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in which beta-galactosidase is driven by the viral LTR, resulted in expression in many cell types, while the opsin-lacZ vector mediated the expression of the lacZ reporter gene specifically in photoreceptor cells. CONCLUSIONS: The internal opsin promoter fragment appears capable of selectively directing gene expression to photoreceptor cells after retroviral-mediated gene transfer. These findings serve as a basis for future studies using the opsin promoter-beta PDE retroviral vector to rescue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells during the early postnatal period.

Trilateral tumors in four different lines of transgenic mice expressing SV40 T-antigen.

PURPOSE: A line of transgenic mice containing the simian virus (SV) 40 T-antigen (T-ag) gene driven by the beta-luteinizing hormone (BLH) promoter developed bilateral retinoblastoma and primitive neuroectodermal tumors (PNET) of the midbrain. Midbrain tumors arose from the subependymal layer of the cerebral aqueduct. Bilateral ocular and brain tumors ("trilateral") were found in three other SV40 T-ag transgenic murine lines containing different promoters (murine interphotoreceptor retinoid-binding protein (IRBP), human IRBP, and alpha A-crystallin). To gain insight into the regulatory mechanisms involved in central nervous system tumorigenesis, the authors examined brain tumors from four lines of SV40 T-ag mice with different promoters. METHODS: Formalin-fixed brain tumors were examined from four lines of transgenic mice containing different promoters linked to the protein coding region of the enhancerless SV40 T-ag oncogene. Transgenes contained the following promoters: BLH, mouse 1.8-kb IRBP, human 1.3-kb IRBP, and alpha A-crystallin. RESULTS: Mice with a 1.8-kb IRBP promoter develop retinal photoreceptor and pineal tumors. Intracranial tumors arising from the subependymal layer of the third ventricle also were observed. Mice with a 1.3-kb IRBP promoter exhibit bilateral retinal PNET and PNET originating from the subependymal layer of the third ventricle. Mice with the alpha A-crystallin promoter exhibit bilateral lens tumors and PNET of the midbrain. CONCLUSIONS: Ocular tumors in these mice may be ascribed to the promoter-driven, tissue-specific expression of SV40 T-ag. The common finding of PNET arising from the subependymal layer of the diencephalon is unlikely to be promoter related. These findings indicate that a regulatory region specific to the subependymal layer of the cerebral aqueduct and third ventricle resides in the structural region of the SV40 T-ag gene.

Connexin 35: a gap-junctional protein expressed preferentially in the skate retina.

We have used low stringency hybridization to clone a novel connexin from a skate retinal cDNA library. A rat connexin 32 clone was used to isolate a single partial clone that was subsequently used to isolate seven more overlapping clones of the same cDNA. Two clones containing the entire open reading frame have a consensus sequence of 1456 bp and predict a protein of 302 amino acids length and molecular mass of 35,044 daltons, referred to as connexin 35 or Cx35. Southern blot analysis suggests that the cloned sequence lies in a single gene with one intron. Polymerase chain reaction amplification from genomic DNA and partial sequencing of this intron showed that it was approximately 950 bp in length, and located within the coding region 71 bp after the translation start site. Hydropathy analysis of the predicted protein and alignments with previously cloned connexins indicate that Cx35 has a long cytoplasmic loop and a relatively short carboxyl terminal tail. Multiple sequence alignments show that Cx35 has similarities to both alpha and beta groups of connexins and suggests that its origins may be near the divergence point for the two groups. Consensus sequences consistent with sites for phosphorylation by protein kinase C and by cAMP - or cGMP -dependent protein kinase were identified. Two transcripts were detected in Northern blot analysis: a 1.95-kb primary transcript and a 4.6-kb minor transcript. In RNA samples from 10 tissues, transcripts were detected only in the retina.