Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in alpha -gustducin.

Biochemical and genetic studies have implicated alpha-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, alpha-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of alpha-gustducin and expressed it as a transgene from the alpha-gustducin promoter in both wild-type and alpha-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of alpha-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type alpha-gustducin as a transgene in alpha-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the alpha-gustducin gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous alpha-gustducin's interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the alpha-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the alpha-gustducin lineage of taste cells mediate these responses.

An In vitro assay useful to determine the potency of several bitter compounds.

Gustducin and transducin are guanine nucleotide binding regulatory proteins (G proteins) expressed in taste receptor cells and implicated in transducing taste cell responses to certain compounds that humans consider bitter or sweet. These G proteins can be activated in vitro by taste receptor-containing membranes plus any of several bitter compounds. This activation can be monitored using limited trypsin digestion, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Scanning of the autoradiograms enables one to quantitate the level of activation (defined as an activation index), obtain dose-response profiles and estimate the potency of the tastant. This assay may provide a useful substitute for, or adjunct to, the time-consuming human psychophysical analysis and costly animal studies typically used in taste sensory analysis. It may be used to identify and determine the concentration-response function of many bitter components of oral pharmaceuticals and food ingredients. A potential limitation of the assay is that only about half of all bitter compounds tested demonstrated in vitro activity, perhaps due to the presence of multiple transduction pathways. Nevertheless, the rapid throughput and microsample handling capability of this assay make it an ideal method to screen for high-potency bitterness inhibitors.

Directing gene expression to gustducin-positive taste receptor cells.

We have demonstrated that an 8.4 kb segment (GUS(8.4)) from the upstream region of the mouse alpha-gustducin gene acts as a fully functional promoter to target lacZ transgene expression to the gustducin-positive subset of taste receptor cells (TRCs). The GUS(8. 4) promoter drove TRC expression of the beta-galactosidase marker at high levels and in a developmentally appropriate pattern. The gustducin minimal 1.4 kb promoter (GUS(1.4)) by itself was insufficient to specify TRC expression. We also identified an upstream enhancer from the distal portion of the murine gustducin gene that, in combination with the minimal promoter, specified TRC expression of transgenes. Expression of the lacZ transgene from the GUS(8.4) promoter and of endogenous gustducin was coordinately lost after nerve section and simultaneously recovered after reinnervation, confirming the functionality of this promoter. Transgenic expression of rat alpha-gustducin restored responsiveness of gustducin null mice to both bitter and sweet compounds, demonstrating the utility of the gustducin promoter.

Characterization and solubilization of bitter-responsive receptors that couple to gustducin.

The tastes of many bitter and sweet compounds are thought to be transduced via guanine nucleotide binding protein (G-protein)-coupled receptors, although the biochemical nature of these receptors is poorly understood at present. Gustducin, a taste-specific G-protein closely related to the transducins, is a key component in transducing the responses to compounds that humans equate with bitter and sweet. Rod transducin, which is also expressed in taste receptor cells, can be activated by the bitter compound denatonium in the presence of bovine taste membranes. In this paper, we show that gustducin is expressed in bovine taste tissue and that both gustducin and transducin, in the presence of bovine taste membranes, can be activated specifically by several bitter compounds, including denatonium, quinine, and strychnine. We also demonstrate that the activation in response to denatonium of gustducin by presumptive bitter-responsive receptors present in taste membranes depends on an interaction with the C terminus of gustducin and requires G-protein betagamma subunits to provide the receptor-interacting heterotrimer. The taste receptor-gustducin interaction can be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin. Finally, as the initial step toward purifying taste receptors, we have solubilized this bitter-responsive taste receptor and maintained its biological activity.

A brain-derived neurotrophic factor (BDNF) related system is involved in the maintenance of the polyinnervate Torpedo electric organ.

Target-derived molecules are essential for the maintenance of neuron survival. In the present work, we introduce the electric organ of Torpedo marmorata as a tool for the study of trophic interactions in a polyinervate system. This electric organ maintains a large number of cholinergic terminals on the postsynaptic cell surface. We have observed that a soluble extract derived from the electric organ induces the maturation of Xenopus oocytes injected with presynaptic plasma membranes (PSPM), indicating that a trophic system may exist. Moreover, we have detected a p75NGFR related protein in PSPM by Western blot analysis. These results suggest the presence of a neurotrophin-related system maintaining the polyinnervate electric organ. Furthermore, molecular experiments showed that the brain-derived neurotrophic factor (BDNF) is the neurotrophin operating in our model. Using degenerate oligonucleotides which comprise a conserved fragment of all neurotrophins, we have only amplified by polymerase chain reaction a BDNF fragment. In a similar way, we have amplified and cloned a fragment of the TrkB/C high affinity BDNF receptor. The fact that degenerate oligonucleotides only amplify BDNF allows us to conclude that the polyinnervation is maintained by this neurotrophin either alone or in combination with other trophic factors.

The eukaryotic translation initiation factor 5, eIF-5, a protein from Zea mays, containing a zinc-finger structure, binds nucleic acids in a zinc-dependent manner.

A maize cDNA encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from an 8-day-old seedling cDNA library. The 1975 bp cDNA encodes a protein of 451 amino acids, with a predicted molecular weight of 49.04 kDa, and hybridizes to a single sequence in the maize genome. The deduced sequence contains motifs characteristic of proteins belonging to the GPTase superfamily, a zinc finger well conserved in all the protein sequences for eIF-5 reported so far, and a fragment also present in prokaryotic and chloroplast L11 ribosomal protein. Polymer-binding assays have been used to assess the predicted RNA binding property of the protein and to characterize its function. It is shown that the eIF-5-encoded protein binds to single-stranded DNA and to polyuridylic acid and that the binding is dependent on the presence of Zn2+ ions. These results suggest that the zinc-finger structure is involved in the binding of the eIF-5 protein to RNA.

Functional reconstitution of KCl-evoked, Ca(2+)-dependent acetylcholine release system in Xenopus oocytes microinjected with presynaptic plasma membranes and synaptic vesicles.

We have developed a new method for the generation of functionally active presynaptic chimeras in Xenopus laevis oocytes. Frog oocytes injected with presynaptic subcellular fractions extracted from the electric organ of Torpedo marmorata release acetylcholine in a calcium-dependent manner upon chemical stimulation. Neither oocytes injected without presynaptic plasma membranes nor oocytes injected with ghost erythrocyte plasma membrane instead of presynaptic plasma membrane release acetylcholine. This suggests that specific presynaptic components necessary for KCl-evoked, Ca(2+)-dependent acetylcholine release become functionally integrated in the Xenopus laevis oocytes. Moreover, rhodaminated presynaptic plasma membranes and the synaptic vesicle protein synaptophysin are detected on the oocyte surface by fluorescence or immunofluorescence, respectively, showing that the injected presynaptic components are incorporated into the membrane of the frog oocyte. Furthermore, Botulinum neurotoxin type A, a specific blocker of acetylcholine release in the neuromuscular junction, inhibits the neurotransmitter release from the chimerical oocytes. This suggests that targets for toxin action are also functionally incorporated in the oocyte upon injection of membranous presynaptic components. Our results show that oocytes injected with presynaptic components behave as cholinergic nerve ending chimeras, at least in terms of neurotransmitter release and toxin targets. The system bypasses some problems associated with messenger RNA expression because not only proteins, but native presynaptic components are incorporated. This new technique may provide a useful approach for electrophysiological and pharmacological studies in order to characterize the synaptic transmission.

Coupling of bitter receptor to phosphodiesterase through transducin in taste receptor cells.

The rod and cone transducins are specific G proteins originally thought to be present only in photoreceptor cells of the vertebrate retina. Transducins convert light stimulation of photoreceptor opsins into activation of cyclic GMP phosphodiesterase (reviewed in refs. 5-7). A transducin-like G protein, gustducin, has been identified and cloned from rat taste cells. We report here that rod transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue. Furthermore, the bitter compound denatonium in the presence of taste-cell membranes activates transducin but not Gi. A peptide that competitively inhibits rhodopsin activation of transducin also blocks taste-cell membrane activation of transducin, arguing for the involvement of a seven-transmembrane-helix G-protein-coupled receptor. These results suggest that rod transducin transduces bitter taste by coupling taste receptor(s) to taste-cell phosphodiesterase. Phosphodieterase-mediated degradation of cyclic nucleotides may lead to taste-cell depolarization through the recently identified cyclic-nucleotide-suppressible conductance.

Cloning characterization and expression of the cDNA encoding a neuron-specific alpha-tubulin isoform highly represented in the electric lobe of Torpedo marmorata.

A cDNA (alpha T6) encoding an alpha-tubulin from Torpedo marmorata (Tm) was isolated and sequenced. The deduced 451-amino-acid (aa) sequence codes for an alpha-tubulin of 50,161 Da. The aa sequence of alpha T6 of Tm showed a 70-99.6% identity to the other alpha-tubulins previously described. Moreover, the alpha T6 aa sequence was 95-99.6% identical to neural-specific tubulins of mouse, rat, human and siberian salmon. The corresponding mRNA is highly represented in the giant motoneurons of the electric lobe. All neuronal populations of the Tm brain exhibit variable levels of alpha T6 expression, with the highest levels in the long-axon-projecting neurons. These results suggest that this alpha-tubulin isoform may play an important role in the maintenance and/or remodeling of the neuronal cytoskeleton.

Characterization of a rabbit serum raised against a botulinum toxin type A binding protein from presynaptic plasma membranes from Torpedo electric organ.

Botulinum neurotoxin type A blocks acetylcholine release from the peripheral nervous system. We have previously described a putative botulinum neurotoxin type A receptor of presynaptic plasma membranes from Torpedo. The electric organ of Torpedo, which is largely enriched in cholinergic nerve endings, is homologous to the neuromuscular junction, allowing us to isolate large scale of presynaptic components. In order to characterize this protein we have raised a polyclonal antibody (a-P140) against this receptor. The antiserum a-P140 recognizes a 140,000 mol. wt band in non-reducing conditions and an 80,000 band in reducing conditions. The immunohistochemistry assay reveals the P140 protein on the ventral face of the electrocytes where the nerve terminals are localized. Moreover, a-P140 antiserum recognizes the P140-BoNT/A complex after binding and cross-linking experiments. In addition, we have immunoprecipitated an in vitro translated product which is closely coincident in mol. wt to the 80,000 band of the receptor.

Role of structural domains for maize gamma-zein retention in Xenopus oocytes.

In order to examine the role of cysteine (Cys)-rich domains in the accumulation of maize (Zea mays L.) gamma-zein within the endoplasmic-reticulum-derived protein bodies, we studied the localization of gamma-zein and of two truncated forms of gamma-zein in Xenopus laevis oocytes. The two derivatives were constructed from a DNA encoding the gamma-zein: one by deletion of the Pro-X linker region (21 amino acids) and the other by deletion of the Cys-rich domain (94 amino acids). In-vitro-synthesized transcripts were injected into oocytes and the distribution of the translation products was then analyzed. The entire gamma-zein and both truncated forms of the gamma-zein had accumulated efficiently in microsomes and no traces of secretion were observed. We suggest that neither C-terminal Cys-rich nor Pro-X domains are essential for gamma-zein retention in oocyte vesicles. Therefore, structural features derived from disulphide bonds are not necessary for gamma-zein targeting on the endoplasmic reticulum.

Regulation of the maize HRGP gene expression by ethylene and wounding. mRNA accumulation and qualitative expression analysis of the promoter by microprojectile bombardment.

The expression of the maize gene coding for a hydroxyproline-rich glycoprotein (HRGP) has been studied by measuring the mRNA accumulation after wounding or ethylene treatment. RNA blot and in situ hybridization techniques have been used. The temporal and tissue-specific expression has been observed: the cells related to the vascular system show the more intense HRGP mRNA accumulation. Transcriptional constructions of the maize HRGP promoter have been tested on different maize tissues by microbombarding. A 582 bp promoter is able to direct the expression of the gus gene on calli and young leaves. Constructions having shorter promoter sequences lose this ability. The 582 bp construction retains the general specificity of expression observed for the HRGP gene.

A maize embryo-specific gene encodes a proline-rich and hydrophobic protein.

A gene from maize that encodes a hybrid proline-rich protein (HyPRP) formed by two well-defined domains, proline-rich and hydrophobic, respectively, has been characterized at the level of its structure and expression. The proline-rich domain is composed of elements PPYV and PPTPRPS, similar to those found in PRP proteins from soybean. The hydrophobic domain is rich in cysteine and is similar to seed proteins, mainly to a soybean hydrophobic seed protein. In maize, HyPRP is encoded by a single gene, and its mRNA accumulates in immature maize zygotic embryos, with a maximum accumulation between 12 and 18 days after pollination. The HyPRP mRNA can also be detected in ovary prior to pollination. In situ hybridization experiments on embryo sections show an expression of the gene in scutellum and in nonvascular cells from the embryo axis. Functional hypotheses related to HyPRP are discussed.

Accumulation of cell wall hydroxyproline-rich glycoprotein mRNA is an early event in maize embryo cell differentiation.

The accumulation of the mRNA coding for a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the wall from the cells of vegetative tissues, has been observed in maize embryo by in situ hybridization. The HRGP mRNA accumulates in the embryo axis and not in the scutellum and preferentially in dividing and provascular cells. The histone H4 mRNA is distributed in similar tissues but is restricted to defined groups of cells, indicating that these two gene products have a different steady-state level of accumulation during the cell cycle. The HRGP mRNA appears to be a useful marker for early formation of the vascular systems. The mRNA accumulation correlates in space and time with cells having a low content of cellulose in their walls, suggesting that the mRNA is produced in the early stages of cell wall formation before complete deposition of cellulose.

Spatial and temporal expression of a maize lipid transfer protein gene.

We studied the temporal and spatial pattern of lipid transfer protein (LTP) gene expression, as well as the localization of this protein, in maize. Using an LTP gene, we observed an accumulation of LTP mRNA in embryos and endosperms during seed maturation. LTP gene expression was also investigated in young seedlings. After germination, the level of LTP mRNA in the coleoptile increased, with a maximum at 7 days, whereas LTP mRNA levels were low in the scutellum and negligible in roots. The high levels of LTP mRNA found in coleoptiles and embryos were confirmed by in situ hybridization. Moreover, LTP gene expression appeared to be localized in the external cellular layers and around the leaf veins. Using immunogold methods, we also observed that LTP was distributed heterogeneously in the different cells of coleoptiles and leaves. The highest concentrations of LTP were found in the outer epidermis of the coleoptiles as well as the leaf veins. Together, our observations indicate that LTP gene expression is not only organ specific and time specific but also cell specific.

Differential expression of a hydroxyproline-rich cell-wall protein gene in embryonic tissues of Zea mays L.

A hydroxyproline-rich glycoprotein (HRGP) component of the maize cell wall was shown to be present in different organs of the plant by extraction of cell wall proteins and detection by Western blotting and immunocytochemistry. Antibodies raised against the protein or against synthetic peptides designed from the protein sequence immunoprecipitated a proline-rich polypeptide which was synthesized in-vitro from poly(A) (+) RNA extracted from different tissues of the plant and from the complete in-vitro-transcribed mRNA. A very low amount of the protein was found in immature embryos. In particular, the protein could not be detected in the scutellum either by Western blotting or by immunocytochemistry. In agreement with this finding, HRGP mRNA was barely detected in the scutellum, in contrast to its accumulation in the embryo axis. Our results indicate the existence of a unique cell wall structure in embryonic tissues from maize as well as a tissuespecific component of the control of maize HRGP gene expression, distinct to others already described such as cell division.

Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation.

The spatial pattern of expression for a maize gene encoding a hydroxyproline-rich glycoprotein (HRGP) was determined by in situ hybridization. During normal development of roots and leaves, the expression of the gene was transient and particularly high in regions initiating vascular elements and associated sclerenchyma. Its expression was also associated with the differentiation of vascular elements in a variety of other tissues. The gene encoded an HRGP that had been extracted from the cell walls of maize suspension culture cells and several other embryonic and post-embryonic tissues. The gene was present in one or two copies in different varieties of maize and in the related monocots teosinte and sorghum. A single gene was cloned from maize using a previously characterized HRGP cDNA clone [Stiefel et al. (1988). Plant Mol. Biol. 11, 483-493]. In addition to the coding sequences for the HRGP and an N-terminal signal sequence, the gene contained a single intron in the nontranslated 3' end.

Expression of genes for cell-wall proteins in dividing and wounded tissues ofZea mays L.

Hydroxyproline-rich glycoproteins (HRGPs) fromZea mays have been immunolocalized in the cell wall of root tip cells using ultrathin sections and antibodies ellicited against the purified protein. The accumulation of mRNA corresponding to this protein was studied using the cDNA probe. Maximum accumulation of the mRNA was found in tissues with a high proportion of dividing cells such as those in the root tip of young maize seedlings and a close relationship with cellular division was also observed in in-vitro cultures. However, the level of the mRNA in elongating tissues was minimal, as shown by studies carried out on the elongation zones of root tips and coleoptiles. The mRNA was induced by stress conditions, particularly by wounding young leaves and coleoptiles. It is concluded that in maize this group of proline-rich cell-wall proteins accumulates during cell division and not during cell elongation or differentiation, and participates in the stress-response mechanisms of the plant.

Molecular cloning of cDNAs encoding a putative cell wall protein from Zea mays and immunological identification of related polypeptides.

Copy DNAs corresponding to a highly repetitive, proline-rich protein from maize have been cloned by differential screening of a coleoptile cDNA library. The deduced amino acid sequence contains a single repetitive element of carrot extensin (Ser-Pro-Pro-Pro-Pro). The related mRNAs have a defined distribution in tissues of the plant and are accumulated mainly in the coleoptile node and root tip. A peptide that corresponds to one of the repetitive elements of the protein has been synthesized and antisera have been obtained in rabbits. These antibodies react against crude preparations of coleoptile cell wall and against polypeptides extracted following the protocols described for the extraction of extensin. From these data it is concluded that the cDNAs correspond to a family of cell wall glycoproteins from maize.