Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two methods are described for identifying transplanted photoreceptors in a foreign host retina. One involves the use of [3H]thymidine to label the nuclei of photoreceptors which are dividing for 1 week after birth in myomorphic retina. These photoreceptors can be identified by autoradiography. The second involves the use of a transgenic mouse carrying a bovine rhodopsin promoter in tandem with the bacterial LacZ gene. These mice express beta-galactosidase in their rods. X-gal reaction allows these rods to be identified by routine light and electron microscopy. These methods have been used to follow photoreceptor transplants in adult Royal College of Surgeons strain rat and C3H mouse mutants which have lost virtually all their photoreceptors. Dissociated photoreceptors transplanted to the subretinal space of these animals survive for at least 3 months. The inner segment, cell body, and synaptic terminal of these transplanted photoreceptors remain morphologically normal; the outer segment, however, becomes rudimentary.
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PMID:Monitoring photoreceptor transplants with nuclear and cytoplasmic markers. 172 78

To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli beta-galactosidase gene (lacZ). Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 bp direct photoreceptor-specific expression. All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5' of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.
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PMID:Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. 189 80

Rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and N-acetylglucosamine. In the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. Treatment of bovine rod outer segments (ROS) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. Rats injected intravitreally with [3H]galactose and [14C]leucine and maintained in darkness were killed 1 hr, 6 hr, 1, 3 or 5 days following the injection. Retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by ConA sepharose chromatography and SDS-PAGE. During the first 6 hr, galactose selectively labeled rhodopsin in the Golgi-enriched fraction resulting in increased [3H]/[14C] ratios in both Golgi and ROS. The data suggested that trimming was occurring at the transition from Golgi to ROS. Furthermore, a decrease in isotope ratio in the ROS between 6 hr and 1 day suggested further trimming of rhodopsin after membrane assembly in the ROS. Additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. Rats injected intravitreally with [35S]methionine were dark-adapted for 2 hr. Following subcellular fractionation of retinas, ConA purified rhodopsin from ROS was applied to one of two additional lectin columns: Ricinus communis agglutinin (RCA) or Griffonia simplicifolia I (GSA). Eight to nine percent of the labeled rhodopsin was bound to and eluted from RCA, whereas none bound to GSA, indicating the presence of a beta-galactoside. The RCA agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. Galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. Binding of rhodopsin's oligosaccharide to RCA was abolished by pre-treatment with beta-galactosidase. Decreased binding of rhodopsin to RCA was observed following intravitreal injection of castanospermine but not swainsonine. Of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. The association of galactose with rat rhodopsin appeared to be a transient one. At 2 hr, 8-9% of rhodopsin contained galactose, at 6 hr only 2.2% had galactose and by 24 hr less than 1% did. The galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. Separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that 75% of the galactose-containing rhodopsin was in the plasma membrane and only 25% was in the discs. These findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed.
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PMID:Transient hyperglycosylation of rhodopsin with galactose. 193 88

We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells. DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression. Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression. Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs. We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112. We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC). By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli.
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PMID:Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster. 297 15

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.
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PMID:Structure and expression of the cAMP cell-surface receptor. 324 22

Rhodopsin gene expression has been used as a model system to study the mechanisms regulating photoreceptor gene expression. Previous transgenic experiments using rhodopsin promoter/lacZ fusion constructs identified some of the cis-acting DNA elements responsible for photoreceptor cell-specific expression. However, the issue of rod specificity vs. photoreceptor (rod and cone) specificity of the elements was not resolved. To address this issue, the specificity of reporter gene expression in the retinas of transgenic mice carrying bovine rhodopsin promoter/lacZ (beta-galactosidase) fusion genes was assessed using X-gal staining and electron microscopy. Two independent transgenic lines, one carrying a rhodopsin promoter fragment extending from -2174 to +70 base pairs (bp) relative to the messenger RNA start site and another line carrying a fragment from -222 to +70 bp, both showed reporter gene expression in cones as well as rods, although the level of staining appeared to be less in the cones than in the rods. These results demonstrate that the -2174 to +70 bp and -222 to +70 bp bovine rhodopsin promoter fragments are not rod-specific in transgenic mice and indicate that the existence of rod promoter mediated-expression in cones must be considered when interpreting results from transgenic experiments utilizing the rhodopsin promoter.
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PMID:Reporter gene expression in cones in transgenic mice carrying bovine rhodopsin promoter/lacZ transgenes. 784 Nov 29

This study was undertaken to determine whether there are age-related changes in the specific activities of several glycosidases in fresh retinal pigment epithelial cells (RPE) isolated from the posterior pole of human donor eyes. One hundred and twenty-one pairs of eyes from human donors, between the ages of 43 and 95 years, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and the Cleveland Ohio Eye Bank within 18 to 24 h of death. None had histories of diabetes, hepatitis, HIV infection, intraocular surgery, or documented age-related macular degeneration, although several older donors with evidence of drusen were included in the study. RPE cells were isolated from the posterior third of the retina using the conventional rush method and homogenized with a glass, Broeck tissue grinder. All post-nuclear supernatants were analyzed for glycosidase activity; a smaller number of nuclear pellets were assayed to verify that the majority of the enzyme activity was associated with the post-nuclear sypernatants. Glycosidase activity was quantitated fluorometrically by measuring the enzymatic release of umbelliferone from synthetic substrate preparations, specific for each enzyme. Total protein was determined by a micro BCA protein assay. Regression analysis revealed statistically significant age-related decreases for the specific activities of alpha-mannosidase (p = 0.0001), beta-galactosidase (p = 0.0001), N-acetyl-beta-glucosaminidase (p = 0.0001), and N-acetyl beta galactosaminidase (p = 0.0001) in fresh human donor RPE cells taken from the region of the posterior third of the retina that included the macula. Mannose and N-acetyl-glucosamine are major carbohydrate monomers of the oligosaccaride chains of human rhodopsin, and a relatively high percentage of the oligosaccharide chains are galactosylated. Defects in their degradation may lead to the accumulation of undigested residual material in the RPE.
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PMID:Age-related changes of glycosidases in human retinal pigment epithelium. 867 Jul 43

We have produced transgenic mice (rdta mice) that express the gene for an attenuated diphtheria toxin under the control of a portion of the rhodopsin promotor. Morphologically, expression of this transgene results in the elimination of the majority of cell bodies in the outer nuclear layer (ONL) of the retina. This cell loss is evident as early as postnatal day 7 (P7), which corresponds closely to the onset of expression of rhodopsin in the mouse retina that occurs about P5. Reverse transcription-PCR (RT-PCR) analysis of mRNA from the retinae of rdta mice shows that the level of rhodopsin mRNA is reduced by 50% as early as P14 and by P28, has declined to approximately 15% of that in the retinae of control mice. Electroretinographic recordings from the dark-adapted rdta mice at P17 reveal that their retinae do not generate any rod-mediated signals. The majority of the cell bodies that persist in the ONL of the rdta retinae have the morphological features of cone photoreceptors, although these cells never develop normal inner and outer segments. To confirm that the surviving cells are cones we crossed the rdta mice to a different line of transgenic mice that express the E. coli beta-galactosidase (lacZ positive) reporter gene in all cone photoreceptors. In retinae from mice that inherit both transgenes, nearly every cell that remains in the ONL expresses lacZ and, thus, is a cone. This finding also is consistent with RT-PCR analyses, which show that cone opsin mRNAs persist in the retinae of our rdta mice at ages when rhodopsin mRNA is significantly reduced. Electroretinograms can be obtained from the rdta mice under conditions that saturate the rod response and, thus, providing evidence that the cones that remain are functional, even though they lack inner and outer segments. Finally, we have examined the inner nuclear layer for changes that result from rod photorecptors ablation. We show that, while the elimination of the rod photoreceptors has little or no effect on the morphology of the post-synaptic neurons, this deletion does alter their laminar position.
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PMID:Morphological and physiological consequences of the selective elimination of rod photoreceptors in transgenic mice. 898 62

Crx, an Otx-like homeobox gene, is expressed primarily in the photoreceptors of the retina and in the pinealocytes of the pineal gland. The CRX homeodomain protein is a transactivator of many photoreceptor/pineal-specific genes in vivo, such as rhodopsin and the cone opsins. Mutations in Crx are associated with the retinal diseases, cone-rod dystrophy-2, retinitis pigmentosa, and Leber's congenital amaurosis, which lead to loss of vision. We have generated transgenic mice, using 5'- and/or 3'-flanking sequences from the mouse Crx homeobox gene fused to the beta-galactosidase (lacZ) reporter gene, and we have investigated the promoter function of the cell-specific and developmentally regulated expression of Crx. All of the independent transgenic lines commonly showed lacZ expression in the photoreceptor cells of the retina and in the pinealocytes of the pineal gland. We characterized the transgenic lines in detail for cell-specific lacZ expression patterns by 5-bromo-4-chloro-3-indolyl beta-D-galactoside staining and lacZ immunostaining. The lacZ expression was observed in developing and developed photoreceptor cells. This observation was confirmed by coimmunostaining of dissociated retinal cells with the lacZ and opsin antibodies. The ontogeny analysis indicated that the lacZ expression completely agrees with a temporal expression pattern of Crx during retinal development. This study demonstrates that the mouse Crx 5'-upstream genomic sequence is capable of directing a cell-specific and developmentally regulated expression of Crx in photoreceptor cells.
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PMID:The mouse Crx 5'-upstream transgene sequence directs cell-specific and developmentally regulated expression in retinal photoreceptor cells. 1188 Apr 94

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