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S J Pittler

Publications and source records attributed to S J Pittler.

35 records · Page 2Linked to original sources
PubMed1993

PCR analysis of DNA from 70-year-old sections of rodless retina demonstrates identity with the mouse rd defect.

Rodless retina (gene symbol, r) was discovered in mice by Keeler 70 years ago and was first described in this journal as an autosomal recessive mutation leading to "the absence of the visual cells (rods), the external nuclear layer, and the external molecular layer" [Keeler, C. E. (1924) Proc. Natl. Acad. Sci. USA 10, 329-333]. The mutation was studied by Keeler and others in the United States and Europe over the next decade, but Keeler's stock was destroyed in 1939, and mice definitively related to his by pedigree and progeny tests also appeared to have been lost by the end of World War II. In the early 1950s Brückner in Basel recognized mice with a similar retinal phenotype. Investigators in London and Strasbourg analyzed descendants of Brückner's mice and concluded, on the basis of different pathogenesis from r, that they carried a new mutation, which came later to be called retinal degeneration, rd. The relationship of r and rd has been unsettled ever since. Now that the rd phenotype is known to be due to a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene, we hoped to settle the question by direct analysis of r DNA. DNA was liberated from 70-year-old histological sections of +/r and r/r eyes, the only extant r DNA, and the regions encompassing the nonsense mutation amplified by the polymerase chain reaction (PCR). Sequence analysis of the PCR products revealed the presence of the same nonsense mutation and two intron polymorphisms in r DNA. PCR and direct sequence analysis of 11 strains of mice known to carry rd (or a similar allele) also revealed the presence of the nonsense mutation and the same intron polymorphisms. The fact that all r and rd mice contain an identical defect and intron polymorphisms in the phosphodiesterase beta-subunit gene settles beyond reasonable doubt that a single mutation arising > 70 years ago is now widely distributed through inbred mouse strains. Because of the extensive use of the name in publications of the past 40 years, we propose that the gene continue to be designated retinal degeneration, rd.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Primary structure of frog rhodopsin.

Amphibians have been employed extensively to study the anatomy, physiology, biochemistry, and cell biology of the visual system for decades, yet there have been no reports concerning the primary structure of amphibian visual transduction components. Thus, we have determined the entire nucleotide sequence of frog (Rana pipiens) rhodopsin cDNA, including a putative transcription start point and poly A tail, by sequence analysis of PCR products and mRNA. The open reading frame predicts an opsin of 354 residues, six residues longer than the mammalian rod opsins, containing 11 potential phosphorylation sites in the C-terminal domain. RNA blot analysis revealed two transcripts of ca. 1.7 and 3.1 kb. Frog rhodopsin exhibits approximately 85% identity to mammalian rhodopsin at the amino acid level. Sequence analysis of additional components will produce the framework from which a more detailed understanding of amphibian phototransduction can emerge.

Amino Acid Sequence↗
PubMed1992

In vitro isoprenylation and membrane association of mouse rod photoreceptor cGMP phosphodiesterase alpha and beta subunits expressed in bacteria.

We investigated the specificity of CAAX box-related isoprenylation of rod photoreceptor cGMP phosphodiesterase (PDE) subunits expressed in bacteria and the consequences of this modification on rod disk membrane association. Full-length cDNA sequences of the alpha and beta subunits of mouse PDE, inserted into bacterial pET expression vectors, were overexpressed as fusion proteins containing 28 (bMP-alpha) and 26 (bMP-beta) additional amino acid residues at their N termini. Both fusion proteins were overexpressed and stored in inclusion bodies. Purified bMP-alpha and bMP-beta were recognized by bovine PDE-specific polyclonal antibodies, but did not associate with depleted rod disk membranes and were catalytically inactive. Using bovine brain or retina extracts as sources of protein prenyltransferases and tritiated farnesyl- or geranylgeranylpyrophosphate as donors, bMP-alpha (CAAX sequence CCIQ) was exclusively farnesylated, and bMP-beta (CAAX sequence CCIL) was exclusively geranylgeranylated. After isoprenylation, bMP-alpha and bMP-beta each associated with rod photoreceptor outer segment disk membranes under isotonic, but not under hypotonic, conditions. The results indicate that isoprenylated bMP-alpha and bMP-beta each interact independently with membranes and that isoprenylation is the key modification that facilitates membrane association.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Primary structure and chromosomal localization of human and mouse rod photoreceptor cGMP-gated cation channel.

We have determined the primary structures of the human and mouse retinal rod cGMP-gated cation channel by analysis of cDNA clones and amplified DNA. The open reading frames predicted polypeptides of 690 and 683 residues exhibiting 88% sequence similarity. Sequence comparison indicated that the rod channels consist of a variable 90-residue N-terminal region, a short highly charged segment rich in lysine and glutamate, and a 540-residue C-terminal portion that is well conserved in three mammalian species. Significant sequence similarity (59%) of the visual cGMP-gated channel to the olfactory cAMP-gated channel established the existence of a family of cyclic nucleotide-gated ion channel genes. RNA blot analysis revealed transcripts of 3.2 kilobases (kb) in human, mouse, and dog, 3.2, 4.6, and 5.2 kb in bovine, and 3.6 kb in fish. The human channel gene was mapped by polymerase chain reaction of somatic cell hybrid DNAs to chromosome 4 (p14-q13) near the centromere. The mouse channel gene locus (Cncg) was mapped by interspecific backcross haplotype analysis 0.9 centimorgan proximal of the Kit locus on chromosome 5.

Amino Acid Sequence↗
PubMed1992

Chromosome mapping of the rod photoreceptor cGMP phosphodiesterase beta-subunit gene in mouse and human: tight linkage to the Huntington disease region (4p16.3).

The retinal degeneration mouse (gene symbol, rd) is an animal model for certain forms of human hereditary retinopathies. Recent findings of a nonsense mutation in the rd mouse PDE beta-subunit gene (Pdeb) prompted us to investigate the chromosome locations of the mouse and human genes. We have utilized backcross analysis in mice to verify and define more precisely the location of the Pdeb locus 6.1 +/- 2.3 cM distal of Mgsa on mouse chromosome 5. We have determined that the human gene (PDEB) maps to 4p16.3, very close to the Huntington disease (HD) region. Analysis of the comparative map for mice and humans shows that the mouse homologue of the HD gene will reside on chromosome 5. Linkage of the mouse Pdeb locus with other homologues in the human 4p16.3 region is maintained but gene order is not, suggesting at least three possible sites for the corresponding mouse HD gene.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1992

Syntenic assignments of visual transduction genes in cattle.

To establish syntenic relationships of phototransduction genes, we have mapped the genes encoding the alpha-, beta-, and gamma-subunits of rod cGMP phosphodiesterase (PDE) (PDEA, PDEB, PDEG), the alpha'-subunit of cone PDE (PDEA2), and the rod cGMP-gated channel (CNCG) to bovine syntenic groups. The rod cGMP PDE alpha-, beta-, and gamma-subunit genes map to bovine syntenic groups U22, U15 (chromosome 6), and U21 (chromosome 19), respectively. The rod cGMP-gated channel gene also maps to syntenic group U15, and the bovine cone alpha'-subunit gene maps to U26 (chromosome 26). With the exception of the cone PDE alpha'-subunit gene, which has not been mapped in other mammals, all of these genes have been assigned to conserved chromosomal regions shared among bovine, human, and mouse. A compilation of currently known syntenic assignments and predictions regarding future assignments of phototransduction genes in human, mouse, and cattle is presented.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse.

Retinal degeneration in the mouse mutant, rd, was previously shown to be a disorder of cyclic nucleotide metabolism involving a deficiency in the activity of the rod photoreceptor cGMP phosphodiesterase (PDE). We have characterized the normal and rd PDE beta-subunit gene, and their respective transcripts, by PCR and direct sequence analysis. We show that the gene consists of at least 22 exons ranging in size from 48 base pairs to several hundred base pairs, covering greater than 25 kilobases. Within a 67-base-pair exon of the rd PDE beta-subunit gene, we identified a nonsense ochre mutation (a C----A transversion in codon 347) that truncates the normal gene product, eliminating more than one-half of the peptide chain, including the putative catalytic domain. The consequences of the truncation are consistent with the observed phenotypes in rd mice heterozygous and homozygous for the disorder. The nonsense mutation was also found in another related and in six unrelated strains displaying the rd phenotype, indicating that the rd allele arose from a single genetic event. The results strongly argue for the nonsense mutation being responsible for retinal degeneration in the rd mouse.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

Complete cDNA sequences of mouse rod photoreceptor cGMP phosphodiesterase alpha- and beta-subunits, and identification of beta'-, a putative beta-subunit isozyme produced by alternative splicing of the beta-subunit gene.

We have characterized overlapping cDNA clones encoding cGMP phosphodiesterase (PDE) alpha- and beta-subunits of mouse retinal rod photoreceptors. The open reading frames predict an alpha-subunit of 100 kDa (856 residues), and a beta-subunit of 99 kDa (853 residues). Sequence analysis of two of twelve beta-subunit clones predicts the presence in the retina of an additional PDE, termed beta', which is generated by alternative splicing of the beta-subunit gene. beta' differs from beta only at the C-terminus being 55 residues shorter and lacking the Caax motif found at the C-termini of both the alpha- and beta-subunits. A 300 residue segment thought to contain the active site is present in the C-terminal half of alpha, beta and beta'.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1991

HSV antigens and HSV DNA in avascular and vascularized lesions of human herpes simplex keratitis.

Fifty-one corneal buttons obtained by penetrating keratoplasty from patients with a preoperative clinical diagnosis of nonulcerative herpetic keratitis and/or disciform stromal scarring (44) as well as ulcerative necrotizing stromal keratitis (7) were processed for herpes simplex virus (HSV) antigens using an immunoperoxidase technique and for HSV DNA by the polymerase chain reaction (PCR). HSV antigens were detected significantly more often (p less than 0.025) in specimens with avascular nonulcerative keratitis than in those with vascularization. In contrast to HSV antigens, HSV DNA was identified at equal proportions in avascular and vascularized lesions. Both HSV antigens and HSV DNA were detected in all specimens from patients with ulcerative necrotizing stromal keratitis. The implications of these findings with regard to possible mechanisms underlying herpetic keratitis in man are discussed.

Adolescent↗
PubMed1991

The molecular genetics of retinal photoreceptor proteins involved in cGMP metabolism.

Metabolism of cGMP is critically important for the functioning of phototransduction in the mammalian retina. In rod and cone photoreceptors, two types of antagonistic enzymes, guanylate cyclases and cGMP phosphodiesterases, carefully balance the available amount of the intracellular messenger. Guanylate cyclase produces cGMP and phosphodiesterase rapidly hydrolyzes cGMP upon bleaching of the photopigment. Regulation of their activity in light and dark, influence of Ca++, and feed-back mechanisms are currently under intense investigation. A molecular analysis on both the gene and protein levels will contribute significantly to our understanding of their respective roles in phototransduction. The two types of enzymes have been characterized molecularly to a very different extent. Rod phosphodiesterase was purified to homogeneity almost fifteen years ago, but photoreceptor guanylate cyclase has evaded all attempts for molecular characterization. Characterization of retinal guanylate cyclase cDNA(s), however, will most likely be achieved in the near future. Cone PDE was shown to be a distinct enzyme, different from, but related to, the rod enzyme. Molecular cloning has provided sequence information of two of the three subunits of rod PDE; the small inhibitory subunit has been expressed in bacterial expression vectors, giving us an elegant tool for exploring mechanisms of activation and inhibition. The gene encoding the alpha subunit was shown to be a member of a large gene family of cyclic nucleotide phosphodiesterases, present in many eucaryotes ranging from unicellular organisms (yeast) to mammals. While much has been achieved, many questions remain to be answered. The beta subunit of rod phosphodiesterase has evaded complete molecular characterization, and its origin (one gene and posttranslational modification of the gene product generating alpha and beta, alternative splicing, or two separate genes with distinct gene products) has not been elucidated. Mechanisms of interaction of subunits, activation and inhibition, the active site(s) of the enzyme are undefined. Virtually nothing is known about the molecular organization of the photoreceptor guanylate cyclase(s). Recent cloning of two apparently unrelated mammalian guanylate cyclases, however, containing a common homologous domain signals increasingly rapid progress in this field.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Mouse opsin. Gene structure and molecular basis of multiple transcripts.

The single copy mouse opsin gene produces five major transcripts, varying in size from 1.7 to 5.1 kilobases. The mRNAs are present at levels that vary over 2 orders of magnitude and can be detected as early as postnatal day 1. Each of the transcripts is polyadenylated and can be identified in polysome-bound RNA, suggesting that each is translated in vivo. To elucidate the molecular basis of this complex transcription pattern, we have characterized genomic fragments covering the entire mouse opsin gene, including several kilobases of 5'- and 3'-flanking regions. Transcription initiates at a single site 97 base pairs upstream of the translation start codon. Northern hybridization with exon- and intron-specific probes demonstrated that the various transcripts are not generated by partial or alternative splicing. Sequence analysis of the 3' end of the gene showed the presence of multiple polyadenylation signals. Analysis by polymerase chain reaction of the 3' end of opsin cDNA demonstrated that the complex transcription pattern originated from the selective use of these polyadenylation sites, generating transcripts that differ only in the length of the 3'-untranslated region. Transcript heterogeneity similar to that observed in mouse was also found in rat and, to a lesser degree, in human and frog opsin mRNAs.

Amino Acid Sequence↗
PubMed1990

Molecular characterization of human and bovine rod photoreceptor cGMP phosphodiesterase alpha-subunit and chromosomal localization of the human gene.

Defects in proteins that function in photoreceptor signal transduction are prime suspects as causes of some human hereditary retinal degenerations. We have characterized cDNA clones encoding the alpha-subunit of human and bovine rod cell cGMP phosphodiesterase a key phototransduction enzyme. Clones from both species contain an open reading frame capable of coding for an approximately 100-kDa polypeptide of 859 amino acids, 94% of which are identical. Two or more transcripts were detected in both human and bovine retinal poly(A)+ RNA preparations, although the human transcripts ranging from 5.3 to 4.9 kb are significantly larger than the two bovine transcripts of 4.6 and 4.0 kb. The bovine and human genes appear to exist in single copy, with the bovine gene spanning more than 140 kb of genomic DNA. Somatic cell hybrids were used to map the human gene to the long arm of chromosome 5 (5q31.2----q34). Finally, the use of the candidate gene approach in the study of hereditary retinal dystrophies is discussed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

An interchromosomal gene conversion of the Drosophila dunce locus identified with restriction site polymorphisms: a potential involvement of transposable elements in gene conversion.

Females heterozygous for the two alleles dnc2 and dncM14 of the X-linked gene dunce (dnc), and carrying a copy of dnc+ on the second chromosome, have produced a cluster of six dnc+ progeny X-chromosomes from recombination experiments. Restriction site polymorphisms have been used as genetic markers to follow the parentage of dnc locus segments in these chromosomes. All six chromosomes are identical with respect to the spectrum of restriction site markers they carry in the dnc+ chromosomal region. In the progeny chromosomes, this region is comprised of sequences like the dncM14 X-chromosome and the translocation copy of dnc+. Sequences flanking the dnc gene in the progeny chromosomes are like the dncM14 chromosome. Internal to the gene but near the 5' end, is a segment from the dnc+ translocation which has apparently originated from an interchromosomal and premeiotic gene conversion event. In addition, two transposable elements have inserted into the progeny chromosomes, one towards the 5' end of dnc and the other near the 3' end. The insertion of these elements occurred premeiotically since all six chromosomes are structurally identical. The data are interpreted with respect to a potential role of transposable element transposition in the process of gene conversion.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

A new family of the poly-deoxyadenylated class of Drosophila transposable elements identified by a representative member at the dunce locus.

Two transposable elements have been identified at the dunce locus in chromosomes recovered after a premeiotic and interchromosomal conversion event occurred at this gene. One is approximately 8.2 kb and is inserted near the 5' end of the gene. This element was identified by sequence analysis as a member of the B104 (roo) family of copia-like transposable elements. The second resides near the 3' end of the gene and represents a new family of the class of poly-deoxyadenylated [poly(dA)] transposable elements. It is 0.38 kb in length and has one terminus consisting of a stretch of 29 deoxyadenosine residues with a polyadenylation site like those found in mRNA molecules, located about 20 bp away from the poly(dA) stretch. Fourteen base pairs of genome DNA is duplicated at the target site of this element.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

In vitro synthesis of rat brain hexokinase.

Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) has been synthesized in the rabbit reticulocyte lysate system directed by poly(A)+ mRNA isolated from rat brain. Identification of the in vitro synthesis product as hexokinase was based on its immunoprecipitation with anti-hexokinase serum as well as the generation of identical peptide maps after partial cleavage of the in vitro product and authentic hexokinase with Staphylococcus aureus V8 proteinase or chymotrypsin. The in vitro product and authentic hexokinase were indistinguishable in molecular weight (SDS-gel electrophoresis); thus, despite the fact that, in situ, much of the hexokinase in brain is found in association with mitochondria, it is not synthesized in the form of a higher molecular weight precursor as is characteristic of other mitochondrial proteins. This is in accord with the view that hexokinase is best considered as a classical 'soluble' enzyme which is capable of exhibiting reversible association with mitochondria. The in vitro product cochromatographs (during anion-exchange HPLC) with authentic hexokinase previously shown to have a blocked (presumably acetylated) N-terminus; this procedure is capable of resolving the N-terminally blocked form of the enzyme from a partially proteolyzed form having a free N-terminal amino group. Thus the in vitro product is apparently N-acetylated by an enzyme system previously shown to be present in reticulocyte lysates. A significant fraction of the in vitro synthesized hexokinase attained a conformation characteristic of the native enzyme as judged by the observations that it could be immunoprecipitated by monoclonal antibodies recognizing the native enzyme but not by antibodies recognizing denatured hexokinase, and limited tryptic cleavage of the in vitro product gave fragments identical to those seen with the native enzyme and thought to reflect the organization of structural domains in that enzyme. However, based on these same criteria, the majority of the hexokinase synthesized in vitro appears to exist in a folding state that is not identical to that of either the fully denatured or native enzyme.

Animals↗