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W Baehr

Publications and source records attributed to W Baehr.

At least 73 records · Page 4Linked to original sources
PubMed1991

Chlamydia trachomatis developmentally regulated protein is homologous to eukaryotic histone H1.

Chlamydiae are prokaryotic obligate intracellular parasites that undergo a biphasic life cycle involving an infectious, extracellular form known as elementary bodies and an intracellular, replicating form termed reticulate bodies. We have purified from Chlamydia trachomatis a very basic elementary body-specific protein with an apparent molecular mass of 18 kDa, determined its N-terminal amino acid sequence, and cloned the encoding gene. Sequence analysis of the cloned gene revealed some remarkable properties for its expressed product, including a high lysine content (29%), a correspondingly high pI, and significant homology to the H1 class of eukaryotic histones. Furthermore, a monoclonal antibody to this chlamydial histone analog, termed Hc1, displayed immunoblot and antinuclear specificity suggestive of cross-reactivity to H1 histones. The gene was expressed only during the late stages of the chlamydial life cycle concomitant with the reorganization of chlamydial reticulate bodies into elementary bodies, suggesting that the Hc1 protein plays a role in the condensation of chlamydial chromatin during intracellular differentiation.

Amino Acid Sequence↗
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↗
PubMed1991

Interphotoreceptor retinoid-binding protein: biochemistry and molecular biology.

Interphotoreceptor retinoid-binding protein (IRBP) is a recent and still somewhat enigmatic newcomer in the family of photoreceptor-specific proteins involved in vision. It has been isolated, characterized, cloned and sequenced from several species in less than a decade. Its extracellular localization and the increased amount of bound all-trans retinol following illumination are consistent with the proposed function of IRBP in extracellular transport or buffering of retinol and retinal isomers in the IPM. Its capacity to bind a great many other hydrophobic ligands, however, may indicate a multi-purpose function. Many questions concerning biosynthesis and processing, secretion, tertiary structure and ligand binding remain to be answered. Perhaps most interesting, from a molecular biology point of view, will be the regulation of transcription and tissue-specific expression of this photoreceptor protein, and possible involvement of its gene in human retinal degenerations. Constructs consisting of IRBP regulatory sequences and foreign antigens incorporated into the germ line of transgenic mice will help in the characterization of promoters and enhancer elements, while suppression of IRBP gene expression and its consequences may give us a more precise answer about IRBP function.

Amino Acid Sequence↗
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

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.

Animals↗
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↗
PubMed1989

Human interstitial retinoid-binding protein. Gene structure and primary structure.

Interstitial retinoid-binding protein (IRBP) is synthesized and secreted by rod photoreceptor cells into the interphotoreceptor matrix and is known to bind retinoids and fatty acids. We have used cDNA clones encoding human IRBP to isolate a 15-kilobase genomic fragment that encompasses the complete human IRBP gene. The IRBP gene spans more than 11 kilobases and is interrupted by three introns, all of which are positioned near the 3'-end of the coding sequence. The 3741-base pair coding region of IRBP appears to have been generated by quadruplication of an approximately 900 base pair long ancestral gene. The deduced amino acid sequence predicts a mature protein of 1,230 residues (calculated molecular weight 133,000). The protein sequence can be aligned into four homologous segments, each consisting of about 300 residues. Sequence similarity between segments is as high as 60% when conservative substitutions are taken into account. Two putative N-linked glycosylation sites are located in highly conserved domains in the center of the first and second segment of IRBP. A domain consisting of 41 residues at the COOH-terminal end of the third segment has 15 matching residues (38%) with an intradiscal loop of rhodopsin, a retinal-binding protein in rod photoreceptors.

Amino Acid Sequence↗
PubMed1989

The virulence-associated gonococcal H.8 gene encodes 14 tandemly repeated pentapeptides.

H.8 is a virulence-associated, surface-exposed immunogenic macromolecule composed of lipid and protein, common to Neisseria gonorrhoeae and Neisseria meningitidis. The H.8 DNA sequence predicted a 6.9 kD peptide comprising 14 tandemly repeated pentameric sequences. Ten were identical: Pro, Ala, Ala, Glu, Ala. Also predicted was a lipoprotein leader consensus sequence which probably specified acylation since the Escherichia coli-expressed protein was tightly associated with lipid. Lipid appeared to contribute significantly to H.8 antigen's electrophoretic mobility. This is the first description of a prokaryotic outer membrane protein composed solely of tandem repeats. Furthermore, DNA encoding this repeat appears to have been duplicated and translocated into another neisserial gene encoding an azurin.

Amino Acid Sequence↗
PubMed1989

Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains.

We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.

Amino Acid Sequence↗
PubMed1988

Isolation and analysis of the mouse opsin gene.

We have identified three overlapping 5'-truncated mouse opsin cDNA clones by immunologically screening a lambda gt11 retina expression library. Using one of the cDNA clones as a probe, we isolated a 5 kb genomic fragment that encompassed the complete coding sequence for mouse opsin. The coding region for opsin was interrupted by four introns positioned precisely as those previously described for other mammalian opsins. In contrast to the single major opsin mRNA in the bovine and human retina, Northern analysis of mouse retina RNA demonstrated the presence of at least five distinct species of polyadenylated opsin mRNAs. Their sizes ranged from 1.7 kb to 5.1 kb.

Amino Acid Sequence↗
PubMed1988

Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes.

Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells. Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants. To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs). Predominantly conserved regions of the genes of both serogroups are interspersed with four short variable domains (I-IV). Recombinant phage clones expressing specific MOMP antigenic determinants revealed that protective serotype-specific recognized epitopes in variable domains I and II. Protective subspecies and serogroup-specific mAbs recognized overlapping determinants in variable domain IV near the C terminus. A nonprotective species-specific mAb mapped to an invariant peptide of nine residues contained within variable domain IV. In the intact chlamydial organism of serovar B, variable domains II and IV were susceptible to proteolytic digestion, whereas both N and C termini were protected. These results suggest an arrangement of MOMP in the outer membrane in which three of the four variable domains are exposed to the outside and in which both N and C termini are presumably oriented toward the periplasmic space. This molecular analysis of MOMP antigenic determinants and their surface topology on intact chlamydiae will be useful toward the development of a recombinant subunit or synthetic chlamydial vaccine.

Amino Acid Sequence↗
PubMed1986

Visual pigment homologies revealed by DNA hybridization.

A bovine rhodopsin complementary DNA probe was used to detect homologous visual pigment genes in a variety of species. Under stringent DNA hybridization conditions, genomic DNA from most vertebrate species carried a single homologous fragment. Additional homologies were detected in some vertebrates by reducing the hybridization stringency. Homologous fragments were also detected in DNA isolated from invertebrate species, a unicellular alga, and an archaebacterium; many of these fragments were homologous to a Drosophila opsin probe. These results suggest that photosensory pigments in a wide variety of species arose from a common precursor.

Animals↗
PubMed1986

Light-dependent binding of G-protein to outer segment membranes of toad photoreceptors.

Light-dependent changes in the binding of G-protein were analyzed in outer segment disk membranes obtained from photoreceptors of the toad (Bufo marinus) retina. Isolated, intact retinas, incubated in oxygenated Ringer's solution at 23 +/- 1 degree C, were subjected to various conditions of illumination and then incubated in darkness for specified periods. The retinas were then chilled (0-4 degrees C) and the receptor outer segments (ROS) were isolated. Binding of the alpha- and beta-subunits of G-protein to the ROS membranes was analyzed by quantitating G alpha and G beta extracted from the membranes with hypotonic medium lacking GTP vs. hypotonic medium containing GTP (H and HG extracts, respectively). For retinas illuminated and then immediately chilled for analysis, the extent of G binding (relative abundance of G alpha, beta in the HG extract) increased with the extent of bleaching of the visual pigment. Near-maximal binding was observed after bleaches of greater than or equal to 30%. With an increasing period of incubation in darkness after approximately 70% bleaching, the extent of binding declined gradually to low levels characteristic of unbleached retinas. The period required for half-completion of the decline was approximately 10(3) s. A gradual decline in G binding, from a rapidly developing peak value, was also observed with an increasing period of exposure to intense light. Viewed in the context of previous electrophysiological data, our results indicate that sustained bleaching desensitization of the rods does not depend upon a persisting state of "tight binding" (immobilization) of G-protein by bleached visual pigment.

Adaptation, Physiological↗
PubMed1985

The Drosophila ninaE gene encodes an opsin.

The Drosophila ninaE gene was isolated by a multistep protocol on the basis of its homology to bovine opsin cDNA. The gene encodes the major visual pigment protein (opsin) contained in Drosophila photoreceptor cells R1-R6. The coding sequence is interrupted by four short introns. The positions of three introns are conserved with respect to positions in mammalian opsin genes. The nucleotide sequence has intermittent regions of homology to bovine opsin coding sequences. The deduced amino acid sequence reveals significant homology to vertebrate opsins; there is strong conservation of the retinal binding site and two other regions. The predicted protein secondary structure strikingly resembles that of mammalian opsins. We conclude the Drosophila and vertebrate opsin genes are derived from a common ancestor.

Amino Acid Sequence↗