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PubMed1987

The halo-opsin gene. I. Identification and isolation.

Halorhodopsin (HR), the light-driven chloride pump in halobacteria, was purified in the denatured as well as in the native state and chemically cleaved into peptide fragments. Isolation of peptide and liquid phase sequencing yielded approximately 20% of the halo-opsin (HO) structure in non-overlapping peptides. Chemically synthesized oligodeoxynucleotides corresponding to a peptide sequence obtained from both HR preparations were used to screen a cosmid gene bank of Halobacterium halobium strain L-33. A positive clone contained cosmid pAB H47 which by subcloning and nucleotide sequencing was shown to encode at least part of the HO gene.

Blotting, Southern↗
PubMed2004

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice.

BACKGROUND: The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. METHODS: The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). RESULTS: The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. CONCLUSIONS: The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.

Animals↗
PubMed1996

Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.

ATP-Binding Cassette Transporters↗
PubMed1991

An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker.

1. The spectral response of the circadian pacemaker of the eye of the mollusk Bulla gouldiana was examined in two ways: by using the latency of the first light-evoked compound action potential (CAP) as an acute photoresponse of the putative pacemaker cells of the eye, the basal retinal neurons (BRNs), and by measuring the effectiveness of monochromatic light pulses at resetting the pacemaker. 2. Through measurements of the spectral sensitivity of the acute response of the BRNs, a photopigment absorbing maximally near 490 nm (lambda max) was described. Action spectra of the acute response following isolation of the BRNs, by surgical removal of the distal photoreceptor layer or the use of low Ca2+ media to block chemical synapses on the BRNs, further suggested that a 490 nm lambda max photopigment is used in generating the acute light response. The spectral sensitivity of eyes adapted to a dim background illumination also agreed with the expected absorption of a 490 lambda max rhodopsin. 3. The effectiveness of monochromatic light pulses at shifting the phase of the circadian rhythm in CAP frequency suggested that the photopigment used in the entrainment of the pacemaker is the opsin based molecule identified through acute response measurements.

Animals↗
PubMed1994

The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae.

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium. This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

Alcohol Dehydrogenase↗
PubMed1996

Effect of transmembrane helix packing on tryptophan and tyrosine environments in detergent-solubilized bacterio-opsin.

Bacterio-opsin (bO) is folded in a nearly native conformation in mixed micelles of dimyristoyl phosphatidyl choline (DMPC) and 3-[(3-cholamidopropyl)-dimehtylamonio]-1-propane sulfonic acid (CHAPS), but bO is partially unfolded in sodium dodecyl sulfate (SDS). UV difference spectroscopy was used to study the changes in environment of bO aromatic amino acid side chains that occur upon partial unfolding. The UV difference spectra of peptides in CHAPS/DMPC minus peptides in SDS were measured for bO and the following subfragments of bO: C1 (residues 72-248), C2 (1-71), V1 (1-166), V2 (167-248), CB7 (119-145), CB9 (164-209), and CB10 (72-118). The spectra show that, in partially unfolded bO in SDS, the Tyr and Trp absorbance is blue-shifted. The difference spectra were compared to solvent perturbation difference spectra of N-acetyl-L-tyrosine ethyl ester and N-acetyl-L-tryptophanamide. The exposure change calculated from the difference spectra was found to correlate with the change in the number of van der Waals contacting atoms upon partial unfolding, and also with the number of transmembrane helical segments. This result suggests a simple experimental method of testing helix packing arrangements derived from hydropathy plots and model building.

Amino Acid Sequence↗
PubMed1984

The structure of mammalian rod opsins.

Ovine rhodopsin is organised in disc membranes as a monomer. The determination of its amino acid sequence has permitted the utilisation of structure prediction programmes which indicate the probable disposition of the polypeptide chain in the bilayer. This putative model is consistent with labelling data using the chemical probes, [14C]succinic anhydride, [125I]diazodiido sulphanilic acid and [125I]iodophenyl azide, and with the cleavage points for several proteases. More surprisingly the predicted structure points to the occurrence of breaks/distortions in the transmembrane helical segments. These distorted regions may be of primary functional importance to the protein and at least one is associated with the attachment point of the chromophore. This particular part of the structure is also identified as a "mutational hot spot", for bovine, equine, ovine and porcine opsins exhibit different sequences (but conserved molecular volumes) in the four residues following the retinyllysine. In an otherwise highly conserved protein with no obvious functional differences between the four species, the high substitution rate in this region is unexplained.

Amino Acid Sequence↗
PubMed1993

Homologous bacterio-opsin-encoding gene expression via site-specific vector integration.

Homologous recombination in the archaebacterium Halobacterium halobium has been investigated and exploited for the wild-type (wt) level of expression of the bacterio-opsin-encoding gene (bop). The Haloferax volcanii-Escherichia coli shuttle vector, pWL102, was used to construct a shuttle-mutagenesis vector, pEF191, bearing bop and short flanking sequences. Transformation of a bacteriorhodopsin (BR)-negative H. halobium strain with pEF191 resulted in plasmid integration at the homologous bop locus. A model for this site-specific vector integration is presented which has been confirmed by determining the arrangement of the repeated homologous sequences on the chromosome. Two different configurations are obtained after integrative transformation due to the presence of an insertion element in the genomic copy of bop. In one configuration, the functional bop cluster containing the regulatory bat and brp genes was in wt arrangement. In the second configuration, the bop cluster is interrupted by 10 kb of plasmid vector sequences, and the upstream region required for bop expression was limited to 400 bp. The BR production for both configurations was determined and found to be at wt level. These results suggest that the function of the putative bop promoter does not depend on the defined upstream positions of bat and brp. The system presented here can be easily exploited for structure-function studies on BR and introduces homologous gene targeting as a powerful tool in the study of halobacterial genetics.

Bacteriorhodopsins↗
PubMed2003

Downstream coding region determinants of bacterio-opsin, muscarinic acetylcholine receptor and adrenergic receptor expression in Halobacterium salinarum.

The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M(1)) and adrenergic (a2b) receptors by fusing the coding region of the m1 and a2b genes to nucleotide sequences known to direct bacterio-opsin (bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification. bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both. m1 and a2b mRNA levels were low, and accumulation was sensitive to both the extent of the bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in H. salinarum.

Amino Acid Sequence↗
PubMed2001

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants.

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless. under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.

Bacteriorhodopsins↗
PubMed2003

Opsin activation as a cause of congenital night blindness.

Three different mutations of rhodopsin are known to cause autosomal dominant congenital night blindness in humans. Although the mutations have been studied for 10 years, the molecular mechanism of the disease is still a subject of controversy. We show here, using a transgenic Xenopus laevis model, that the photoreceptor cell desensitization that is a hallmark of the disease results from persistent signaling by constitutively active mutant opsins.

Animals↗
PubMed1968

Studies on vision. The nature of the retinal-opsin linkage.

1. Convenient methods for the preparation of tritiated 11-cis-retinol, 11-cis-retinal and rhodopsin are described. Irradiation of labelled rhodopsin in the presence of sodium borohydride resulted in the irreversible binding of the retinyl moiety to the active site. Degradative studies established that the retinyl moiety in this reduced derivative of rhodopsin was attached to the in-amino group of lysine. In connexion with this investigation the synthesis of a number of N-retinylidene- and N-retinyl-amino acids was achieved. Derivatives of lysine with the retinyl moiety attached either to the alpha-amino group or to the in-amino group were also synthesized and characterized. 2. It is suggested that the involvement of a charge-transfer interaction between the retinylidene chromophore and a suitable group -X or -X.H on the opsin best explains the spectroscopic properties of rhodopsin and other visual proteins.

Amino Acids↗
PubMed2002

Mechanism of cell death and disease resistance induction by transgenic expression of bacterio-opsin.

One of the earliest signal transduction events that trigger the hypersensitive response (HR) of plants against pathogen attack is thought to be an alteration of proton flux across the plasma membrane (PM). However, no direct genetic evidence for the involvement of PM-localised proton channels or pumps in the induction of this response has been reported. We previously showed that expression of the bacterial proton pump bacterio-opsin (bO) in transgenic plants resulted in the spontaneous activation of the HR. Here we show that the bO protein is likely localised to the PM in transgenic tobacco plants. Furthermore, mutational analysis shows that induction of the HR by bO expression is dependent upon the capability of bO to translocate protons. Although bO functions as a light-driven proton pump in Halobacteria when assembled with retinal, we also show by mutational analysis that this chromophore binding is unnecessary for its in planta activity. Taken together, our results suggest that expression of bO in plants leads to the insertion of a passive proton channel into the PM. The activity of this channel in the PM results in spontaneous activation of cell death and HR-associated phenotypes including enhanced resistance to a broad spectrum of plant pathogens. Our work provides direct molecular evidence to support a working model in which alterations in ionic homeostasis at the level of the PM may work as one of the critical steps in the signalling pathway for the activation of the HR.

Bacteriorhodopsins↗
PubMed1983

High-frequency spontaneous mutation in the bacterio-opsin gene in Halobacterium halobium is mediated by transposable elements.

We have recently characterized a transposable element, ISH1, which inactivates the bacterio-opsin (BO) gene in two purple membrane-deficient (Pum-) mutants of Halobacterium halobium. Examination of nine additional Pum- mutants now shows that in all of these the BO gene has been inactivated by insertion of one of two types of transposable elements. Four Pum- strains contain ISH1 within the BO gene, probably at the same site that we have previously characterized. A second element, ISH2, which is present in four more strains, inserts at multiple sites within the BO coding sequence. Significantly, another Pum- strain contains the ISH2 element 102 nucleotides upstream from the initiator codon for BO. ISH2, which is 520 nucleotides long, is the smallest insertion sequence known. Its sequence has been determined: it is A + T-rich (53%), contains a 19-base-pair inverted repeat at its termini, and, interestingly, duplicates either 10 or 20 base pairs at the target site during insertion. ISH2 is present in multiple copy numbers in the genome and contains several relatively short open reading frames.

Apoproteins↗
PubMed1990

Expression of the archaebacterial bacterio-opsin gene with and without signal sequences in Escherichia coli: the expressed proteins are located in the membrane but bind retinal poorly.

In a further effort to obtain functional expression of the bacterio-opsin gene (bop) in Escherichia coli, the bop gene with E. coli signal sequences as well as the bop gene with the native presequence were expressed in E. coli. The location of the expressed products in the E. coli cell and their processing and folding to a structure that binds retinal as in Halobacterium halobium were investigated. All the expressed proteins were in the membrane. The proteins were largely unprocessed, and they were distributed between the outer and the inner membrane. The processed fractions, which were minor, were exclusively in the inner membrane. The processed proteins bound exogenously added all-trans-retinal but only partially, indicating that these proteins were present in at least two folded states.

Amino Acid Sequence↗
PubMed1984

Characterization of insertions affecting the expression of the bacterio-opsin gene in Halobacterium halobium.

We have determined the sequence of the inverted repeats and duplicated target DNA of the halobacterial insertion elements ISH2 (520 bp), ISH23 (900 bp) and ISH24 (3000 bp) associated with bacterio-opsin (bop) mutants. ISH2 has a perfect 19 bp inverted repeat (3,5), while both ISH23 and ISH24 have imperfect inverted repeats of 29 bp and 14 bp respectively. ISH23 was shown to be highly homologous to ISH50 (6). Variable lengths of duplicated target DNA are found when ISH2 and ISH23 (ISH50) transpose into different sites. A 550 bp DNA insert ("ISH25") reverts the Bop mutation caused by ISH24. "ISH25" lacks typical structural features of a transposable element. "ISH25" and ISH24 are found adjacent to each other upstream of the bop gene. An identical arrangement of "ISH25" and ISH24 is found in the cccDNA of H. halobium NRC817. Comparative sequence analysis of both areas suggests that the translocation of "ISH25" to the bop gene region occurred by a recombination event.

Bacteriorhodopsins↗
PubMed2000

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR.

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.

Amino Acid Sequence↗
PubMed1997

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Bacteriorhodopsins↗