Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

Using data derived from peptide sequencing of p68/70, a protein doublet induced during optic nerve regeneration in goldfish, we have isolated cDNAs that encode RICH (regeneration-induced CNPase homolog) from a goldfish regenerating retina cDNA library. The predicted RICH protein comprises 411 amino acids, possesses a pI of 4.48, and shows significant homology to the mammalian myelin marker enzyme 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase; EC 3.1.4.37). The mRNA encoding RICH was demonstrated, by both Northern blot analysis and RNase protection assays, to be induced as much as 8-fold in regenerating goldfish retinas at 20 days after nerve crush. Analysis of total RNA samples from various tissues showed a broad distribution of RICH mRNA, with the highest levels observed in gravid ovary. The data obtained strongly suggest that RICH is identical or very similar to p68/70. The molecular cloning of RICH provides the means for a more detailed analysis of its function in nerve regeneration. Additionally, the homology of RICH and CNPase suggests that further investigation may provide additional insight into the role of these proteins in the nervous system.

Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.

A channeled tRNA cycle during mammalian protein synthesis.

In earlier studies it was shown that the mammalian translation system is highly organized in vivo and that the intermediates in the process, aminoacyl-tRNAs, are channeled--i.e., they are directly transferred from the aminoacyl-tRNA synthetases to the elongation factor to the ribosomes without dissociating into the cellular fluid. Here, we examine whether spent tRNAs leaving the ribosome enter the fluid phase or are transferred directly to their cognate aminoacyl-tRNA synthetases to complete a channeled tRNA cycle. Using a permeabilized CHO cell system that closely mimics living cells, we find that there is no leakage of endogenous tRNA during many cycles of translation, and protein synthesis remains linear during this period, even though free aminoacyl-tRNA is known to rapidly equilibrate between the inside and outside of these cells. We also find that exogenous tRNA and periodate-oxidized tRNA have no effect on protein synthesis in this system, indicating that they do not enter the translation machinery, despite the fact that exogenous tRNA rapidly distributes throughout the cells. Furthermore, most of the cellular aminoacyl-tRNA synthetases function only with endogenous tRNAs, although a portion can use exogenous tRNA molecules. However, aminoacylation of these exogenous tRNAs is strongly inhibited by oxidized tRNA; this inhibitor has no effect on endogenous aminoacylation. On the basis of these and the earlier observations, we conclude that endogenous tRNA is never free of the protein synthetic machinery at any stage of the translation process and, consequently, that there is a channeled tRNA cycle during protein synthesis in mammalian cells.

Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.

For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC 3.1.8.1) has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.

Stringent chemical and thermal regulation of recombinant gene expression by vaccinia virus vectors in mammalian cells.

We developed a stringently regulated expression system for mammalian cells that uses (i) the RNA polymerase, phi 10 promoter, and T phi transcriptional terminator of bacteriophage T7; (ii) the lac repressor, lac operator, rho-independent transcriptional terminators and the gpt gene of Escherichia coli; (iii) the RNA translational enhancer of encephalomyocarditis virus; and (iv) the genetic background of vaccinia virus. In cells infected with the recombinant vaccinia virus, reporter beta-galactosidase synthesis was not detected in the absence of inducer. An induction of at least 10,000- to 20,000-fold occurred upon addition of isopropyl beta-D-thiogalactopyranoside or by temperature elevation from 30 to 37 degrees C using a temperature-sensitive lac repressor. Regulated synthesis of the secreted and highly glycosylated human immunodeficiency virus 1 envelope protein gp120 was also demonstrated. Yields of both proteins were approximately 2 mg per 10(8) cells in 24 hr. Plasmid transfer vectors for cloning and expression of complete or incomplete open reading frames in recombinant vaccinia viruses are described.

Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase.

We have developed a strategy to generate mutant genes in mammalian cells in a conditional manner by employing a fusion protein, Cre-ER, consisting of the loxP site-specific Cre recombinase linked to the ligand-binding domain of the human estrogen receptor. We have established homozygous retinoid X receptor alpha-negative (RXR alpha-/-) F9 embryonal carcinoma cells constitutively expressing Cre-ER and have shown that estradiol or the estrogen agonist/antagonist 4-hydroxytamoxifen efficiently induced the recombinase activity, whereas no activity was detected in the absence of ligand or in the presence of the antiestrogen ICI 164,384. Furthermore, using a targeting vector containing a selection marker flanked by loxP sites, we have inactivated one retinoic acid receptor alpha allele in such a line, demonstrating that the presence of the recombinase does not inhibit homologous recombination. Combining this conditional site-specific recombination system with tissue-specific expression of Cre-ER may allow modification of the mammalian genome in vivo in a spatiotemporally regulated manner.

Transformation of mammalian cells by overexpressing H2O2-generating peroxisomal fatty acyl-CoA oxidase.

Peroxisome proliferators induce qualitatively predictable pleiotropic responses, including development of hepatocellular carcinomas in rats and mice despite the inability of these compounds to interact with and damage DNA directly. In view of the nongenotoxic nature of peroxisome proliferators, it has been postulated that hepatocarcinogenesis by this class of chemicals is due to a receptor-mediated process leading to transcriptional activation of H2O2-generating peroxisomal fatty acyl-CoA oxidase (ACOX) in liver. To test this hypothesis, we overexpressed rat ACOX in African green monkey kidney cells (CV-1 cells) under control of the cytomegalovirus promoter. A stably transfected CV-1 cell line overexpressing rat ACOX, designated CV-ACOX4, when exposed to a fatty acid substrate (150 microM linoleic acid) for 2-6 weeks, formed transformed foci, grew efficiently in soft agar, and developed adenocarcinomas when transplanted into nude mice. These findings indicate that sustained overexpression of H2O2-generating ACOX causes cell transformation and provide further support for the role of peroxisome proliferation in hepatocarcinogenesis induced by peroxisome proliferators.

Use of an oocyte expression assay to reconstitute inductive signaling.

We have developed a paracrine signaling assay capable of mimicking inductive events in the early vertebrate embryo. RNA encoding one or more secreted proteins is microinjected into a Xenopus laevis oocyte. After a brief incubation to allow translation, a piece of embryonic tissue competent to respond to the signaling protein is grafted onto the oocyte. The secreted protein's effect on the grafted explant is then scored by assaying expression of tissue-specific markers. Explants of ectodermal tissue from blastula or gastrula stage embryos were grafted onto oocytes that had been injected with RNA encoding activin or noggin. We found that the paracrine assay faithfully reconstitutes mesoderm induction by activin and neural induction by noggin. Blastula-stage explants grafted onto activin-expressing oocytes expressed the mesodermal marker genes brachyury, goosecoid, and muscle actin. Gastrula-stage explants grafted onto noggin-expressing oocytes expressed neural cell adhesion molecule (NCAM) and formed cement gland. By injecting pools of RNA synthesized from a cDNA expression library into the oocyte, we also used the assay to screen for secreted neural-inducing proteins. We assayed 20,000 independent transformants of a library constructed from LiCl-dorsalized Xenopus laevis embryos, and we identified two cDNAs that induced neural tissue in ectodermal explants from gastrula-stage embryos. Both cDNAs encode noggin. These results suggest that the paracrine assay will be useful for the cloning of novel signaling proteins as well as for the analysis of known factors.

Mammalian karyopherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins.

Although only 44% identical to human karyopherin alpha 1, human karyopherin alpha 2 (Rch1 protein) substituted for human karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and karyopherin beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, karyopherin beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous karyopherin beta. Recombinant karyopherin beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant karyopherin alpha 1 or alpha 2 did not bind unless karyopherin beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (karyopherin alpha 1 or alpha 2, karyopherin beta, Ran, and p10) import depended on karyopherin beta. Localization of the exogenously added transport factors after a 30-min import reaction showed karyopherin beta at the nuclear envelope and karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled karyopherin beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.

cAMP-dependent, long-lasting inhibition of a K+ current in mammalian neurons.

We report the long-term modulation of K+ channels by cAMP in cultured murine colliculi neurons. A short (1-2 s) application of 8-Br-cAMP induced a long-lasting broadening of the action potential, a loss of after-hyperpolarization, and a reduction in spike accommodation. In agreement with these changes, 8-Br-cAMP produced a long-lasting (2 hr) inhibition of a K+ current. These effects were also observed after a short activation of the pituitary adenylyl cyclase-activating polypeptide, beta-adrenergic, and 5-hydroxytryptamine type 4 (5-HT4) receptors, all known to increase cAMP. A transient activation of the cAMP-dependent protein kinase and a long-lasting inhibition of phosphatases (up to 2 hr) were detected. The blockade of the K+ current resulting from a brief application of 8-Br-cAMP or 5-hydroxytryptamine was prolonged from 2 to 4 hr when protein-serine/threonine phosphatases 1 and 2A were inhibited with 10 nM okadaic acid. The critical steps following the cAMP-dependent protein kinase activation and resulting in a long-term blockade of phosphatases are discussed in this report.

Molecular coevolution of mammalian ribosomal gene terminator sequences and the transcription termination factor TTF-I.

Both the DNA elements and the nuclear factors that direct termination of ribosomal gene transcription exhibit species-specific differences. Even between mammals--e.g., human and mouse--the termination signals are not identical and the respective transcription termination factors (TTFs) which bind to the terminator sequence are not fully interchangeable. To elucidate the molecular basis for this species-specificity, we have cloned TTF-I from human and mouse cells and compared their structural and functional properties. Recombinant TTF-I exhibits species-specific DNA binding and terminates transcription both in cell-free transcription assays and in transfection experiments. Chimeric constructs of mouse TTF-I and human TTF-I reveal that the major determinant for species-specific DNA binding resides within the C terminus of TTF-I. Replacing 31 C-terminal amino acids of mouse TTF-I with the homologous human sequences relaxes the DNA-binding specificity and, as a consequence, allows the chimeric factor to bind the human terminator sequence and to specifically stop rDNA transcription.

Meso1, a basic-helix-loop-helix protein involved in mammalian presomitic mesoderm development.

To identify genes involved in the regulation of early mammalian development, we have developed a dominant-negative mutant basic-helix-loop-helix (bHLH) protein probe for interaction cloning and have isolated a member of the bHLH family of transcription factors, Meso1. Meso1-E2A heterodimers are capable of binding to oligonucleotide probes that contain a bHLH DNA recognition motif. In mouse embryos, Meso1 is expressed prior to MyoD1 family members. Meso1 expression is first detected at the neural plate stage of development in the paraxial mesoderm of the head and in presomitic mesodermal cells prior to their condensation into somites. Our findings suggest that Meso1 may be a key regulatory gene involved in the early events of vertebrate mesoderm differentiation.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.

Activation of mammalian retinoid X receptors by the insect growth regulator methoprene.

We report that methoprene and its derivatives can stimulate gene transcription in vertebrates by acting through the retinoic acid-responsive transcription factors, the retinoid X receptors (RXRs). Methoprene is an insect growth regulator in domestic and agricultural use as a pesticide. At least one metabolite of methoprene, methoprene acid, directly binds to RXR and is a transcriptional activator in both insect and mammalian cells. Unlike the endogenous RXR ligand, 9-cis-retinoic acid, this activity is RXR-specific; the methoprene derivatives do not activate the retinoic acid receptor pathway. Methoprene is a juvenile hormone analog that acts to retain juvenile characteristics during insect growth, preventing metamorphosis into an adult, and it has been shown to have ovicidal properties in some insects. Thus, a pesticide that mimics the action of juvenile hormone in insects can also activate a mammalian retinoid-responsive pathway. This finding provides a basis through which the potential bioactivity of substances exposed to the environment may be reexamined and points the way for discovery of new receptor ligands in both insects and vertebrates.