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J Nathans

Publications and source records attributed to J Nathans.

At least 73 records · Page 4Linked to original sources
PubMed1994

Autosomal dominant retinitis pigmentosa in a large family: a clinical and molecular genetic study.

PURPOSE: To characterize the pedigree, visual function phenotype, and responsible mutation in a large family with autosomal dominant retinitis pigmentosa. METHODS: Pedigree data were obtained by personal interviews and corroborated with community records. One hundred twenty-eight members of the family were examined clinically, and a subset of 12 affected subjects was further studied with dark- and light-adapted static perimetry and electroretinography. The coding region of the rhodopsin gene was polymerase chain reaction (PCR) amplified and resolved by denaturing gradient gel electrophoresis. Genomic DNA samples from nine affected and five unaffected family members were analyzed by PCR amplification and restriction enzyme digestion. RESULTS: A 14-generation pedigree was identified in which retinitis pigmentosa (RP) was inherited in an autosomal dominant fashion. Affected individuals reported early night blindness and showed vessel attenuation and bone spicule-like pigmentary changes. In these individuals, the rod electroretinogram (ERG) was not detectable, and the cone ERG was reduced in amplitude and delayed in timing. With dark-adapted perimetry, rod function could be detected in only one young patient, and it was markedly abnormal. Light-adapted perimetry indicated that cone sensitivity could be relatively well preserved in the central field, but it was diminished in the periphery even in the most mildly affected subjects. A valine345-to-leucine mutation was identified in the rhodopsin gene and shown to cosegregate in the heterozygous condition with the disease. CONCLUSIONS: The natural history of RP in this family begins with a loss of rod function, progresses to involve the cone system, and leads eventually to a severe loss of visual function. The invariance of valine345 in all functional vertebrate visual pigments sequenced to date, and the unusually conservative nature of the valine345-to-leucine mutation suggests that the carboxy terminus of rhodopsin is involved in a highly specific interaction with one or more rod proteins.

Adolescent↗
PubMed1994

Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa.

PURPOSE: To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein. METHODS: Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels. RESULTS: There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes. CONCLUSIONS: There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Adolescent↗
PubMed1994

Blue cones and cone bipolar cells share transcriptional specificity as determined by expression of human blue visual pigment-derived transgenes.

Sequences 5' of the human blue visual pigment gene have been assayed in transgenic mice for their ability to direct cell-type-specific expression of linked beta-galactosidase (lacZ) and placental alkaline phosphatase (P ALP1) reporters. Constructs containing either 5.4 kilobases (kb) or 0.47 kb of 5' flanking DNA direct expression exclusively to the retina. Within the retina, transgene expression is confined to blue cones and cone bipolar cells, as determined, respectively, by double labeling with anti-cone pigment antibodies and by morphologic analyses. These results imply that blue cones and cone bipolar cells have partially overlapping transcriptional specificities.

Animals↗
PubMed1993

Rhodopsin activation: effects on the metarhodopsin I-metarhodopsin II equilibrium of neutralization or introduction of charged amino acids within putative transmembrane segments.

We have studied the metarhodopsin I (M I)-metarhodopsin II (M II) equilibria of expressed wild-type and mutant rhodopsins. We studied two classes of mutants with amino acid substitutions in or near the putative transmembrane segments: those in which a charged residue was replaced by a neutral residue (or in one case another charged residue) and those in which a neutral residue likely (or postulated) to be in proximity to the retinylidene Schiff's base was replaced by a charged residue. In the first class, we found mutants that abnormally favored M II (replacements of Asp-83, Glu-134, or Arg-135) as well as one that abnormally favored M I (replacement of Glu-122). In the second class, we found several mutants that abnormally favored M I, the most extreme being those in which glutamate replaced His-211 or Ala-292. These studies suggest that electrostatic forces play a major role in the energetics of the M 1-to-M II transition, and they indicate that electrostatic perturbation in the vicinity of the protonated retinylidene Schiff's base is a plausible mechanism for the change in its pKa that is associated with the M I-M II transition. They further suggest that the highly conserved pair of charged residues homologous to Glu-134 and Arg-135 may play a general role in agonist-dependent conformational changes in G-protein-coupled receptors.

Animals↗
PubMed1993

Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain.

Over 40 mutations in the rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa. Twenty-one of these mutations have been introduced into a human rhodopsin cDNA by site-directed mutagenesis, and the encoded proteins have been produced by transfection of a human embryonic kidney cell line (293S). Three of the mutant proteins (G51V, V345M, and P347S) resemble the wild type in yield, regenerability with 11-cis-retinal, and accumulation in the plasma membrane (class I). The remaining 18 mutant proteins are produced at lower levels, regenerate variably or not at all with 11-cis-retinal, and accumulate partially or predominantly in the endoplasmic reticulum (class II). Together with an earlier analysis of 13 mutant rhodopsins (Sung, C.-H., Schneider, B., Agarwal, N., Papermaster, D.S., and Nathans, J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8840-8844), these experiments define distinct classes of biochemical defects in human rhodopsin and further show that amino acid substitutions in class II reside within the transmembrane and extracellular domains, whereas class I mutants cluster in the first transmembrane domain and at the extreme carboxyl terminus.

DNA, Complementary↗
PubMed1993

Cloning and expression of goldfish opsin sequences.

Five opsin cDNA clones were isolated from a goldfish retina cDNA library and sequenced. On the basis of homology to previously characterized visual pigments, one clone was identified as goldfish rod opsin and a second as a goldfish red cone opsin. Two rhodopsin-like clones were found to be similar to the chicken green opsin, a pigment which shares properties with both rod and cone pigments. A fifth clone was equally homologous to human blue cone opsin and human rod opsin. In order to characterize the spectral properties of the encoded pigments, the five clones were expressed in tissue culture cells and the apoproteins reconstituted with 11-cis-retinal. The wavelength of maximal absorption for goldfish rhodopsin is 492 nm and for the fifth pigment, identified as the goldfish blue pigment, 441 nm. Pigments encoded by the two rhodopsin-like clones absorb at 505 and 511 nm and are likely to correspond to the goldfish green pigment previously characterized by microspectrophotometry. The putative red cone opsin cDNA may encode a pigment that is a polymorphic variant of goldfish red since it absorbs maximally at 525 nm.

Amino Acid Sequence↗
PubMed1993

Bovine pancreatic trypsin inhibitor-trypsin complex as a detection system for recombinant proteins.

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10(-14) and 1.1 x 10(-13) M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.

Animals↗
PubMed1993

Brn-3b: a POU domain gene expressed in a subset of retinal ganglion cells.

A search for POU domain transcription factors in human retina cDNA has led to the identification of Brn-3b, a class IV POU domain protein. Immunohistochemical experiments show that chicken, mouse, rabbit, monkey, and human retinas contain Brn-3b exclusively within a subpopulation of ganglion cells. In the adult mouse brain, Brn-3b is found only within cells in the deep layers of the superior colliculus, in the dorsal periaqueductal gray, and in a small cluster of cells in the brain stem near the area postrema. During the immediate postnatal period, cells containing Brn-3b are distributed in a number of regions within the brain stem and cerebellum. These data suggest that Brn-3b plays a role in determining and/or maintaining the identities of a small number of neurons, including a subset of visual system neurons.

Amino Acid Sequence↗
PubMed1993

Role of hydroxyl-bearing amino acids in differentially tuning the absorption spectra of the human red and green cone pigments.

The human red and green cone pigments differ at either 15 or 16 amino acids, depending upon which polymorphic variants are compared. Seven of these amino acid differences involve the introduction or removal of a hydroxyl group. One of these differences, a substitution of alanine for serine at position 180, was found previously to produce a 5 nm blue shift. To determine the role of the remaining six hydroxyl group differences in tuning the absorption spectra of the human red and green pigments, we have studied six site-directed mutants in which single amino acids from the green pigment have been substituted for the corresponding residues in the red pigment. Blue shifts of 7 and 14 nm were observed upon substitution of phenylalanine for tyrosine at position 277 and alanine for threonine at position 285, respectively. Single substitutions at positions 65, 230, 233, and 309 produced spectral shifts of 1 nm or less. These data are in good agreement with a model based upon sequence comparisons among primate pigments and with the properties of site-directed mutants of bovine rhodopsin. Nonadditive effects observed in comparing the absorption spectra of red-green hybrid pigments remain to be explained.

Cell Line↗
PubMed1993

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.

Base Sequence↗
PubMed1993

Genetic heterogeneity among blue-cone monochromats.

Thirty-three unrelated subjects with blue-cone monochromacy or closely related variants of blue-cone monochromacy were examined for rearrangements in the tandem array of genes encoding the red- and green-cone pigments. In 24 subjects, eight genotypes were found that would be predicted to eliminate the function of all of the genes within the array. As observed in an earlier study, the rearrangements involve either deletion of a locus control region adjacent to the gene array or loss of function via homologous recombination and point mutation. One inactivating mutation, Cys203-to-Arg, was found in 15 probands who carry single genes and in both visual pigment genes in one subject whose array has two genes. This mutation was also found in at least one of the visual pigment genes in 1 subject whose array has multiple genes and in 2 of 321 control subjects, suggesting that preexisting Cys203-to-Arg mutations constitute a reservoir of chromosomes that are predisposed to generate blue-cone-monochromat genotypes by unequal homologous recombination and/or gene conversion. Two other point mutations were identified: (a) Arg247-to-Ter in one subject with a single red-pigment gene and (b) Pro307-to-Leu in one subject with a single 5' red-3' green hybrid gene. The observed heterogeneity of genotypes points to the existence of multiple one- and two-step mutational pathways to blue-cone monochromacy.

Base Sequence↗
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↗