Isolation and coding sequence of the rat rod opsin gene.
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Amino Acid Sequence↗
Biomedical subjects
C J Barnstable
Publications and source records attributed to C J Barnstable.
Retinal ganglion cells express a cGMP-gated cation conductance activatable by nitric oxide donors.
We have identified a putative cGMP-gated cation conductance in rat retinal ganglion cells. Both in situ hybridization and polymerase chain reaction amplification detected transcripts in ganglion cells that were highly homologous to the cGMP-gated cation channel expressed in rod photoreceptors. Whole-cell patch-clamp recordings detected a current stimulated by cGMP due to activation of nonselective cation channels. This current had a reversal potential near 0 mV, showed some outward rectification, and could be blocked by Cd2+. The current could also be activated by a phosphodiesterase inhibitor and the nitric oxide donors sodium nitroprusside and S-nitrosocysteine. We propose that nitric oxide released from an identified subpopulation of amacrine cells may activate this channel to modulate ganglion cell activity.
1-Methyl-3-isobutylxanthine↗
A cis-acting element, T alpha-1, in the upstream region of rod alpha-transducin gene that binds a developmentally regulated retina-specific nuclear factor.
The G protein transducin (T) is an integral component of the signal transduction pathway in photoreceptors. We have identified a cis-acting element, T alpha-1, in the upstream region of the mouse rod alpha-T (Tr alpha) gene that may be important for tissue-specific expression. T alpha-1 binds a retina-specific nuclear factor of apparent molecular mass of 90 kDa. Binding to the T alpha-1 site is developmentally regulated and peaks between postnatal days 6 and 9. This corresponds to the time of rod photoreceptor maturation and the rise in Tr alpha gene expression. The sequence of T alpha-1 shows homology with RET-1, a cis-acting element in the proximal promoter of opsin gene that binds a distinct retina-specific factor. T alpha-1 and RET-1 sequences may have been derived from a prototype T alpha-1/RET-1 sequence, evolved to confer photoreceptor specificity on retina-specific genes.
Animals↗
Synaptic and photoreceptor components in retinal pigment epithelial cell transplanted retinas of Royal College of Surgeons dystrophic rats.
Plexiform layer synaptic and photoreceptor cell components were investigated in retinas of Royal College of Surgeons (RCS) dystrophic rats transplanted with normal retinal pigment epithelial (RPE) cells by immunocytochemistry using previously characterized monoclonal antibodies. In retinas of normal adult rats and RPE-cell transplanted retinas of 4 month-old RCS rats, HNK-1, a marker for a carbohydrate of the neural cell adhesion molecule (N-CAM), was detected immunocytochemically in the inner and outer plexiform layers and ganglion cell bodies and their axons. HNK-1 was also detected in the inner plexiform layer of nontreated retinas of 4 month-old RCS rats, but was reduced to scattered patches in the outer plexiform layer. In addition, immunoreactivity for the SVP-38 antibody recognizing synaptophysin was found in both plexiform layers of normal adult rat retinas and RPE-transplanted retinas of 4 month-old RCS rats. Furthermore, photoreceptor cell bodies and their inner and outer segments were immunostained for the opsin monoclonal antibody RET-P1 in retinas of normal adult rats and RPE-cell transplanted retinas of 4 month-old RCS rats. However, in nontreated retinas of 4-month-old RCS rats, only immunostained debris material was detected. These results strongly suggest that normal RPE transplants not only rescue photoreceptor cells in RCS rats, but also maintain an essential functional capacity, in this case, synaptic components in the plexiform layers.
Animals↗
Shared nuclear protein binding sites in the upstream region of the rat opsin gene.
DNase I protection and gel retardation assays have identified two sequences 5' to the rat opsin gene that interact with nuclear proteins from retina but not from a number of neuronal and non-neuronal tissues. These sites, Ret 2 and Ret 3, are over 1200 base pairs apart but seem to interact with the same protein(s). Synthetic oligonucleotides corresponding to each site were able to inhibit complex formation in a gel retardation assay using an oligonucleotide corresponding to the other site. The proteins binding to the Ret 2 and Ret 3 sites co-eluted in both ion exchange and gel filtration chromatography. The protein(s) were also present at adult levels at birth, suggesting that they may represent differentiation products expressed in the proliferating retinal epithelium.
Animals↗
Differential laminar expression of particulate and soluble guanylate cyclase genes in rat retina.
Expression of both particulate and soluble forms of guanylate cyclase have been identified in adult rat retina using reverse transcriptase-PCR amplification of retinal RNA and sequencing of the cloned cDNAs. Over a 267-amino acid region, the retinal particulate guanylate cyclase was found to be identical in sequence to the GC-A form of the enzyme found in many tissues. No expression of the closely related GC-B or GC alpha forms of the enzyme was found. mRNA corresponding to both subunits of the soluble form of guanylate cyclase were detected in retinal. The sequence corresponding to the 70-kDa subunit was identical to that from rat lung over a 304-amino acid region and the sequence corresponding to the 82-kDa subunit showed only one amino acid difference over a 275-amino acid region. From Northern analyses the level of expression of the soluble guanylate cyclase in retina was higher than that in lung. In situ hybridization to sections of adult retina indicated that the particulate guanylate cyclase was expressed predominantly in rod photoreceptors. Although the soluble form of the enzyme was detected in all retinal layers, the level of expression was much higher in the inner nuclear layer. The results suggest that multiple enzymes and hence multiple regulatory pathways may control cGMP levels in rod photoreceptors and that cGMP may play an important role in neurons of the inner retina.
Animals↗
Renal epithelial cells show nonselective cation channel activity and express a gene related to the cGMP-gated photoreceptor channel.
Nonselective cation channels have been found in various parts of the nephron and represent a heterogeneous group of channels. We briefly review their putative physiological function. Renal epithelial nonselective cation channels may play a role in volume regulation, calcium entry, cell proliferation, and sodium reabsorption. In some renal epithelia cGMP seems to be involved in the regulation of nonselective cation channels. Furthermore, there is evidence that a gene related to the cGMP-gated photoreceptor channel, a well-characterized, nonselective cation channel, is also expressed in whole rat kidney tissue. In the context of these observations, we review recent findings from our own work on a nonselective cation channel in the M-1 mouse cortical collecting duct cell line. We could demonstrate that M-1 cells show nonselective cation channel activity in inside-out patches and express a gene related to the cGMP-gated photoreceptor channel (Proc. Natl. Acad. Sci. USA 89:10262-10266, 1992). The possibility of a relation between the kidney channel and the photoreceptor channel is discussed.
Adenosine Triphosphate↗
Cyclic nucleotide-gated nonselective cation channels: a multifunctional gene family.
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Amino Acid Sequence↗
Glutamate and GABA in retinal circuitry.
Glutamate and GABA have been identified as the major neurotransmitters in the radial and lateral synaptic pathways, respectively, of the vertebrate retina. Over the past year or so new information has appeared that has significantly increased the knowledge of how these compounds can elicit a range of responses. Key features of this new information are the identification and localization of many receptor subtypes within the retina, the recognition that glutamate can modulate membrane potential through cGMP-gated ion channels, and the finding that GABA can be released through non-vesicular pathways.
Animals↗
RET-PE10: a 61 kD polypeptide epitope expressed late during vertebrate RPE maturation.
PURPOSE: This study sought To learn more about the mechanisms that determine and maintain the differentiated state of the retinal pigment epithelium (RPE). METHODS: Monoclonal antibodies were raised against human RPE and used in conjunction with other antibodies. Immunocytochemical and biochemical analyses were performed on tissue sections and cells in culture. RESULTS: An RPE-specific epitope, RET-PE10, has been detected as a 61 kD cytoplasmic polypeptide in a variety of mammalian, amphibian, and avian species. In the rat, RPE-PE10 was expressed late in eye development, with a faint initial labelling of the RPE in central regions at postnatal day 9 (PN9) that increased to adult levels and extent of staining by PN14. RET-PE10 expression initially was present in overnight cultures of dissociated rat RPE cells but was lost rapidly from these cultures during the first week. Comparison of the staining patterns of RET-PE10 with those of various cytoskeletal elements suggests that RET-PE10 may be associated with part of the intermediate filament network. Culture of whole eyecups also resulted in a loss of RET-PE10 expression. RET-PE10 expression was normal in eyes of adult rd/rd mutant mice. CONCLUSIONS: RET-PE10 is a late-appearing marker of RPE differentiation. The results also suggest that the maintained expression of RET-PE10 depends upon extrinsic factors but that these do not include maintained contact with Bruch's membrane or light-induced retinal activity.
Animals↗
Mouse cortical collecting duct cells show nonselective cation channel activity and express a gene related to the cGMP-gated rod photoreceptor channel.
Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors.
Animals↗
Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth.
Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-1 glycoprotein.
Animals↗
Molecular properties of GABAergic local-circuit neurons in the mammalian visual cortex.
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Animals↗
A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules.
VC1.1 is a monoclonal antibody that stains the surfaces of neuronal subsets in the brain. Previous work showed that VC1.1 recognizes 3 polypeptide bands with molecular weights of 95-105 kDa, 140 kDa and 170 kDa and two high molecular weight proteoglycans with weights of approximately 680 and 650-700 kDa. The heterogeneity and molecular weight range of these bands suggested that VC1.1 might recognize a carbohydrate moiety associated with a family of cell surface molecules. It had been previously demonstrated that a separate monoclonal antibody, HNK-1 also recognized a cell surface associated epitope characterized as a sulfate- and glucuronic acid-containing N-linked carbohydrate. This epitope has been shown to be present on members of the N-CAM adhesion molecule family. In this report, we demonstrate that VC1.1 recognizes an N-linked carbohydrate group that is attached to myelin-associated glycoprotein and N-CAM. Immunocytochemical and biochemical comparisons of VC1.1 and HNK-1 staining in rat and cat brain indicate that these two antibodies probably recognize overlapping, or identical carbohydrate epitopes.
Animals↗
Characterization of developmentally regulated and retina-specific nuclear protein binding to a site in the upstream region of the rat opsin gene.
DNase I protection and gel retardation assays have identified a sequence 5' to the transcription start site of the rat opsin gene that interacts with nuclear proteins from mammalian retinas but not from a variety of other neural and non-neural tissues. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to nitrocellulose the protein(s) responsible for this binding were identified with an oligonucleotide probe and were found to migrate with an apparent molecular size of 40 kilodaltons. The binding complex eluted from fast protein liquid chromatography gel filtration as a peak centered at 100 kilodaltons, suggesting the presence of more than one subunit. Binding activity could be detected in postnatal day 1 retinal extracts and increased over the next 2 weeks of development, a time course coincident with opsin gene expression and maturation of rod photoreceptors. Synthetic oligonucleotides with altered sequences showed that the binding was dependent upon residues in a CTAAT motif and was facilitated by surrounding GGCCCC sequences. The specificity of the binding interaction was measured by inhibition of complex formation in a gel retardation assay. The unaltered sequence was over 2 orders of magnitude more effective at inhibiting complex formation than either an unrelated DNA sequence or a concensus sequence corresponding to a known CCAAT box binding protein NF1.
Animals↗
Expression of a unique 56-kDa polypeptide by neurons in the subplate zone of the developing cerebral cortex.
In the mammalian cerebral cortex, neurons destined for layers 2-6 are generated only after the period of genesis for a group of transient neurons that populate the subplate and marginal zones. Although a number of molecular markers for the subplate zone exist, most are also expressed by other cell populations in the cortical plate. To begin to study molecular properties of the subplate, we generated monoclonal antibodies against homogenates of cat cortical subplate zone. One monoclonal antibody, termed subplate 1 (SP1), recognized a polypeptide of 56 kDa. This antigen was strongly expressed within the subplate neurons only during a 3-week period beginning at birth and extending until 3 weeks after birth. From postnatal day 1, the number of SP1-immunoreactive neurons below the visual cortex increased until the end of second postnatal week and then declined thereafter. This period coincides with the period when a majority of the subplate neurons undergo naturally occurring cell death. The antigen was not expressed by subplate neurons surviving in the adult white matter. At the peak of antigen expression, 14% or less of the immunoreactive neurons also coexpressed gamma-aminobutyric acid, somatostatin, or neuropeptide Y. Biochemical and immunocytochemical properties of the SP1 antigen were also compared with the Alz-50 antigen (A68), a marker for dying neurons. On Western blots, SP1- and Alz-50-reactive polypeptides were selectively enriched in cytosolic fractions of kitten cerebral cortex, but each marker recognized different molecular weight polypeptides. In tissue sections many subplate, cortical plate, and layer 1 neurons were Alz-50 immunoreactive. In contrast, a rarer subpopulation of neurons restricted to the subplate was labeled by SP1. We propose that the SP1 antigen is a protein expressed within dying cortical subplate neurons, at the commencement of cell death.
Aging↗
Developmental and tissue-specific expression of the rod photoreceptor cGMP-gated ion channel gene.
Probes against the retinal cGMP-gated cation channel were generated by PCR amplification of cDNA from rat and bovine retina. Southern and Northern analyses showed that the channel is encoded by a single gene that gives rise to a single mRNA species of 3.2 kb. Low levels of cGMP-gated channel RNA were detected in postnatal day 1 (PN 1) retinas and the amount increased to adult levels over the next two weeks of development. Screening of a number of tissues by Northern blot hybridization and by PCR amplification showed the channel to be expressed by heart and kidney as well as retina, but not by cerebellum, cerebral cortex, liver, muscle, olfactory bulb, spleen, testes or thymus.
Aging↗
Cell commitment and differentiation in explants of embryonic rat neural retina. Comparison with the developmental potential of dissociated retina.
The differentiation of presumptive neural retina following its isolation from rat embryos and growth in explant and monolayer culture has been studied to obtain information regarding the extent to which factors extrinsic and intrinsic to the retina participate in determining molecular and cytological differentiation. Explanted retinal epithelium retained the capacity for mitosis, as shown by [3H]thymidine incorporation, and from the undifferentiated neuroepithelium, retinal cell-types emerged and acquired a laminar organization resembling that in vivo. Characterization of rod photoreceptor cells at both the light and electron microscopic level showed that these cells exhibit differentiated structural features including inner segments, connecting cilia and membranous expansions suggestive of forming outer segments. Immunofluorescent labeling with an antibody to a synaptic vesicle protein, and electron microscopic identification of synaptic elements showed formation of synapses by the photoreceptor cells within the explant. Neurites extending from the explants exhibited growth on laminin, fibronectin and collagen substrates. Since the neurites immunolabeled with antibodies to the 140 kDa subunit of neurofilament and with antibodies to Thy-1, they could be identified as axons of ganglion cells. Antibodies to a variety of cell-type specific antigens showed that the cells expressed molecules associated with the fully differentiated cell. Furthermore, since our approach has been to explant embryonic retina at an age when the antigens are not yet expressed in vivo, the appearance of the antigens in culture represented de novo expression. In contrast, neural retinal cells in dissociated cultures did not exhibit de novo expression of differentiated molecular properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗