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J Nathans

Publications and source records attributed to J Nathans.

At least 19 recordsLinked to original sources
PubMed1992

Absorption spectra of the hybrid pigments responsible for anomalous color vision.

Unequal homologous recombination events between green and red cone pigment genes produce the red-green or green-red hybrid pigment genes found in many individuals with variant color vision. Photobleaching difference absorption spectroscopy of hybrid pigments produced in cultured cells shows that the spectral sensitivity of each hybrid pigment is intermediate between the parental green and red pigment sensitivities. Amino acids encoded by exons 2, 3, 4, and 5 produce spectral shifts at the wavelength of maximal absorbance of 0 to 4, 0 to 4, 3 to 4, and 15 to 21 nanometers, respectively, the exact value depending on the identities of amino acids elsewhere in the hybrid.

Color Perception↗
PubMed1992

A visual pigment from chicken that resembles rhodopsin: amino acid sequence, gene structure, and functional expression.

The amino acid sequence of a rhodopsin-like visual pigment from chickens has been determined by isolating and sequencing its gene. The predicted sequence is between 70% and 80% identical to bovine, human, and chicken rhodopsins and between 40% and 50% identical to human blue, green, and red cone pigments, the chicken red cone pigment, and cavefish long-wave cone pigments. The encoded pigment, produced by transfection of cDNA into cultured cells, absorbs maximally at 495 nm as determined from photobleaching difference spectra and reacts at 20 degrees C with 50 mM hydroxylamine with a half-time of 16 min. These properties, together with a high pI predicted from the amino acid sequence, suggest that this cloned gene encodes the chicken green pigment previously identified by biochemical and spectroscopic studies. This sequence defines a new branch of the visual pigment gene family.

Amino Acid Sequence↗
PubMed1992

Absorption spectra of human cone pigments.

Human colour vision is mediated by three light-sensitive pigments, each found in a different cone-cell type. The absorption spectra of the human cone pigments have been sought for over a century using techniques such as psychophysical colour matching, reflection densitometry, electroretinography, single-cell action spectra and, most directly, microspectrophotometry. We report here a direct determination of the human cone pigment photobleaching difference absorption spectra after the production of each cone pigment apoprotein in tissue culture cells transfected with the corresponding complementary DNA clones. The mean values for the wavelength of maximal absorption are 426 nm for the blue pigment, 530 nm for the green pigment, and 552 nm and 557 nm for two polymorphic variants of the red pigment.

Amino Acid Sequence↗
PubMed1992

Abnormal rod dark adaptation in autosomal dominant retinitis pigmentosa with proline-23-histidine rhodopsin mutation.

We studied rod and cone function in 13 patients from four families with autosomal dominant retinitis pigmentosa and the proline-23-histidine rhodopsin mutation. In patients with early stages of this disease, rod sensitivity was mildly abnormal throughout the retina and cone sensitivity was normal. In more severely affected patients, sensitivity loss varied with retinal region, some regions showing mild rod loss only and other regions having pronounced rod and cone dysfunction. Rhodopsin levels were decreased below normal by amounts that indicated the rod sensitivity loss was determined by the reduced ability to absorb light. The most characteristic abnormality of this genotype was a slowed rod branch of dark adaptation, which was present regardless of the extent or severity of disease. The time required for recovery of rod sensitivity was more than twice the normal time. These findings with dark-adapted perimetry, fundus reflectometry, and dark adaptometry showed intrafamilial and interfamilial consistency.

Adolescent↗
PubMed1992

A locus control region adjacent to the human red and green visual pigment genes.

Deletion of sequences 5' of the human red and green pigment gene array results in blue cone monochromacy, a disorder in which both red and green cone function are absent. To test whether these sequences are required for transcription of the adjacent visual pigment genes in cone photoreceptors, we produced transgenic mice carrying sequences upstream of the red and green pigment genes fused to a beta-galactosidase reporter. The patterns of transgene expression indicate that the human sequences direct expression to both long and short wave-sensitive cones in the mouse retina and that a region between 3.1 kb and 3.7 kb 5' of the red pigment gene transcription initiation site is essential for expression. Sequences within this region are highly conserved among humans, mice, and cattle, even though the latter two species have only a single visual pigment gene at this locus. These experiments suggest a model in which an interaction between the conserved 5' region and either the red or the green pigment gene promoter determines which of the two genes a given cone expresses.

Animals↗
PubMed1992

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.

Amino Acid Sequence↗
PubMed1992

Photobleaching difference absorption spectra of human cone pigments: quantitative analysis and comparison to other methods.

Four human cone pigment apoproteins were expressed by transfection of human tissue culture cells with the corresponding complementary DNA clones. Following reconstitution of the cone pigments by incubation with 11-cis retinal, photobleaching difference absorption spectra were obtained for the blue pigment, the green pigment, and two polymorphic variants of the red pigment. These spectra were analyzed to determine the wavelengths of maximal absorbance and the bandwidths. The recombinant cone pigment spectra were compared to human cone spectral sensitivities and cone pigment absorption spectra determined by microspectrophotometry, single-cell electrophysiology, reflection densitometry, electroretinography, and psychophysical color and brightness matching.

Cloning, Molecular↗
PubMed1992

Human rod photoreceptor cGMP-gated channel: amino acid sequence, gene structure, and functional expression.

Phototransduction in retinal rods involves a G-protein-mediated signaling cascade that leads to cGMP hydrolysis and the closure of a cGMP-gated channel. This channel has recently been purified from bovine retina and molecularly cloned (Kaupp et al., 1989). We report here the cloning of cDNA and genomic DNA encoding the human rod cGMP-gated channel, based upon its homology to the bovine counterpart. The human mRNA structure differs from the bovine in containing an Alu repetitive element spliced into the 5' untranslated region. The human cGMP-gated channel gene (CNCG) is located on chromosome 4 and contains at least 10 exons. One large exon encodes the carboxy-terminal two-thirds of the protein, whereas seven small exons encode the amino-terminal one-third of the protein. Alternative splicing removes one of the small exons in a subset of transcripts in the human retina, producing an internal in-frame deletion of 36 codons. When expressed in a human embryonic kidney cell line (293S), the full-length cDNA clone, but not the differentially spliced variant, produced functional ion channels broadly similar to the native channels in vertebrate rods.

Amino Acid Sequence↗
PubMed1992

Human tritanopia associated with two amino acid substitutions in the blue-sensitive opsin.

Tritanopia is an autosomal dominant genetic disorder of human vision characterize by a selective deficiency of blue spectral sensitivity. The defect is manifested within the retina and could be caused by a deficiency in function or numbers (or both) of blue-sensitive cone photoreceptors. We have used PCR, denaturing gradient gel electrophoresis, and DNA sequencing of amplified exons to detect in four of nine unrelated tritanopic subjects two different point mutations in the gene encoding the blue-sensitive opsin, each leading to an amino acid substitution. Segregation analysis within pedigrees and hybridization of oligonucleotides specific for each allele to DNA samples from control subjects support the hypothesis that these mutations cause tritanopia. These results complete the genetic evidence for the trichromatic theory of human color vision.

Arginine↗
PubMed1991

Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.

Blotting, Western↗
PubMed1991

Retinal function and rhodopsin levels in autosomal dominant retinitis pigmentosa with rhodopsin mutations.

We studied rod and cone function in 20 patients from six families with autosomal dominant retinitis pigmentosa, who represented five different point mutations in the gene encoding rhodopsin. In a family with a stop codon mutation at the carboxyl end of the molecule (glutamine-344), young members with the mutation were asymptomatic and clinically unaffected but showed about 1 log unit of rod sensitivity loss across the visual field and decreased rhodopsin levels; at this stage, cone function was essentially normal. In three families with mutations at the border of a transmembrane segment (arginine-135-leucine and arginine-135-tryptophan), there was neither detectable rod function nor measurable rhodopsin; cone function was variably impaired. Two families carrying different mutations (threonine-17-methionine and threonine-58-arginine) had altitudinal visual field defects with less impaired rod and cone function in the inferior than in the superior field. Rod adaptation was abnormal in both families, but the time course of adaptation differed between patients with the two mutations. Differences in the pattern of retinal dysfunction were therefore discernible in patients with different rhodopsin mutations.

Adolescent↗
PubMed1991

Rhodopsin mutations in autosomal dominant retinitis pigmentosa.

DNA samples from 161 unrelated patients with autosomal dominant retinitis pigmentosa were screened for point mutations in the rhodopsin gene by using the polymerase chain reaction and denaturing gradient gel electrophoresis. Thirty-nine patients were found to carry 1 of 13 different point mutations at 12 amino acid positions. The presence or absence of the mutations correlated with the presence or absence of retinitis pigmentosa in 174 out of 179 individuals tested in 17 families. The mutations were absent from 118 control subjects with normal vision.

Base Sequence↗
PubMed1991

Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas.

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.

Aging↗
PubMed1990

Determinants of visual pigment absorbance: identification of the retinylidene Schiff's base counterion in bovine rhodopsin.

The role of negatively charged residues in tuning the absorbance spectrum of bovine rhodopsin has been tested by mutating each aspartate and glutamate to asparagine and glutamine, respectively. Previous work demonstrated that aspartate83, glutamate122, and glutamate134 can be replaced by neutral residues with little or no effect on the absorbance spectrum of the resulting pigment [Nathans, J. (1990) Biochemistry 29, 937-942]. With one exception, mutations at the remaining 19 aspartate and glutamate residues result in very nearly wild-type absorbance spectra. The exception is glutamate113: mutation to glutamine causes the pigment to absorb at 380 nm, reflecting deprotonation of the retinylidene Schiff's base. Upon addition of either chloride, bromide, or iodide, the absorbance rapidly shifts to 495, 498, or 504.5 nm, respectively, reflecting protonation of the Schiff's base. The progressive red shift observed upon addition of halides with larger atomic radii strongly suggests that halides are serving as the Schiff's base counterion. Halides have no effect on the absorbance spectrum of wild-type rhodopsin. I infer, therefore, that glutamate113 is the retinylidene Schiff's base counterion in wild-type rhodopsin. Sakmar et al. [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8309-8313] and Zhukovsky and Oprian [(1989) Science 246, 928-930] have arrived at the same conclusion based upon a related series of experiments. These data support a model in which spectral tuning in bovine rhodopsin results from interactions between the polyene chain of 11-cis-retinal and uncharged amino acids in the binding pocket.

Animals↗