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Biomedical subjects

J Nathans

Publications and source records attributed to J Nathans.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1990

Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments.

I have investigated the effect on bovine rhodopsin's absorbance spectrum of charged amino acid changes in the putative membrane-spanning regions. A total of 14 site-directed mutants were constructed at 6 amino acid positions: 83, 86, 122, 134, 135, and 211. Two of these positions are occupied by charged amino acids that are conserved in all four human visual pigments (positions 134 and 135). In the four variable positions, single and double mutants were constructed to reproduce the intramembrane distribution of charged amino acids predicted for each human cone pigment. Following solubilization in digitonin and reconstitution with 11-cis-retinal, the photobleaching difference spectrum of each pigment was determined in the presence of hydroxylamine. The absorbance spectra of the mutant pigments are all surprisingly close to that of native bovine rhodopsin (lambda max = 498 nm), ruling out a significant role for these residues in spectral tuning.

Amino Acid Sequence↗
PubMed1989

Molecular genetics of human blue cone monochromacy.

Blue cone monochromacy is a rare X-linked disorder of color vision characterized by the absence of both red and green cone sensitivities. In 12 of 12 families carrying this trait, alterations are observed in the red and green visual pigment gene cluster. The alterations fall into two classes. One class arose from the wild type by a two-step pathway consisting of unequal homologous recombination and point mutation. The second class arose by nonhomologous deletion of genomic DNA adjacent to the red and green pigment gene cluster. These deletions define a 579-base pair region that is located 4 kilobases upstream of the red pigment gene and 43 kilobases upstream of the nearest green pigment gene; this 579-base pair region is essential for the activity of both pigment genes.

Adolescent↗
PubMed1989

Production of bovine rhodopsin by mammalian cell lines expressing cloned cDNA: spectrophotometry and subcellular localization.

Cloned cDNA encoding bovine rhodopsin has been recombined into an expression vector and cotransfected with an antibiotic resistance plasmid into cultured human embryonic kidney cells. The resulting cell lines produce 100-200 micrograms of bovine opsin per liter of saturated tissue culture medium (10(9) cells). Incubation in vitro with 11-cis retinal produces a photolabile pigment the absorbance spectrum of which is indistinguishable from that of bona fide bovine rhodopsin. Expressed rhodopsin accumulates in the plasma membrane as determined by immunoelectron microscopy.

Animals↗
PubMed1989

The genes for color vision.

Three centuries of experimentation in optics, psychophysics and biochemistry established the dependence of color vision on three kinds of light-absorbing molecules, or pigments, in the cone cells of the retina. The author has isolated the genes that encode the pigments and has identified genetic anomalies that lead to color blindness.

Animals↗
PubMed1988

Tandem array of human visual pigment genes at Xq28.

Unequal crossing-over within a head-to-tail tandem array of the homologous red and green visual pigment genes has been proposed to explain the observed variation in green-pigment gene number among individuals and the prevalence of red-green fusion genes among color-blind subjects. This model was tested by probing the structure of the red and green pigment loci with long-range physical mapping techniques. The loci were found to constitute a gene array with an approximately 39-kilobase repeat length. The position of the red pigment gene at the 5' edge of the array explains its lack of variation in copy number. Restriction maps of the array in four individuals who differ in gene number are consistent with a head-to-tail configuration of the genes. These results provide physical evidence in support of the model and help to explain the high incidence of color blindness in the human population.

Color Vision Defects↗