Regulation of Catecholamine Enzyme Gene Expression and Identification of Enhancer-Like Elements in the Rat TH Gene Joanne M. Carroll1, Marian J. Evinger1, Howard M. Goodman2, Tong H. Joh1 1Cornell University Medical College, New York, NY, and Tyrosine hydroxylase (TH), the first and rate-limiting enzyme in catecholamine biosynthesis, is highly regulated by a variety of physiological factors. Our goal is to study that part of the regulation occurring at the level of gene transcription. Primary bovine chromaffin cell cultures have been utilized as a model system in which to identify potential transcriptional modulators. The steady state level of TH mRNA in chromaffin cells following treatment with activators of adenylate cyclase, i.e., forskolin or cyclic AMP analogs (e.g., 8-bromo cyclic AMP), increases five- to tenfold. Examination of the 5' flanking region of the rat TH gene revealed a 9 bp sequence at -45, sharing homology with cyclic AMP regulated elements (CRE) described in other genes. A plasmid containing 151 bp of upstream TH sequence linked to bacterial CAT was transfected into mammalian cells. The sequences in this region were sufficient to drive CAT transcription and responded to elevated intracellular cyclic AMP. These experiments suggest that the element at -45 in the TH gene is a functional enhancer. We are currently evaluating other potential transcriptional regulators using the bovine chromaffin cell culture system and mapping the cis-acting elements in the TH gene. Expression of the mRNA Encoding Rat Cellular Retinol Binding Protein (CRBP): Mapping By In Situ Hybridization Analyses Robert J. D'Amato, Brian L. Largent, and Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Cellular retinol binding protein (CRBP) is an intracellular protein thought to be directly involved in retinol's action on cellular growth and differentiation. It has been suggested that CRBP transports retinol into the nucleus where regulation of gene expression could occur. Additionally, CRBP is essential in transporting retinol transcellularly across the retina/blood barrier (produced by the retinal pigment epithelium) to the photoreceptors. We have localized the mRNA for CRBP in various tissues by in situ hybridization using a novel technique which allows production of oligonucleotide probes of defined specific activity and length. Probes complementary to CRBP cDNA were synthesized as 58mer oligonucleotides containing 46 nucleotides of a target specific sequence followed by an additional 12 nucleotides (CAR-common annealing region). A second oligonucleotide (22mer), consisting of T10 followed by the sequence complementary to the CAR region, was annealed to the 58mer oligo and extended with alpha 35S-dATP and Klenow fragment of DNA polymerase. mRNA encoding CRBP is found to be highly abundant in liver, kidney, and retina. Presently, we are examining the cellular localization of the mRNA and its regulation. Effect of Cysteamine Administration on the In Vivo Biosynthesis of Somatostatin, Oxytocin, and Vasopressin in Rat Hypothalamus J.D. Fernstrom, R.P.S. Kwok, and J.L. Cameron Departments of Psychiatry and Behavioral Neuroscience, and Center for Neuroscience, University of Pittsburgh, Pittsburgh, PA 15213 Cysteamine (CSH) injection causes a rapid (1-4 hr), longlasting (ô72 hr) depletion of immunoreactive (IR) somatostatin levels in the rat hypothalamus. IR-Vasopressin (AVP) and IR-oxytocin (OXT) levels are unaffected. We previously showed that (35S) cysteine incorporation into somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28) also falls within 1-4 hr of CSH injection, but is normal 1 week later (Cameron and Fernstrom, Endocrinology 119:1292, 1986). Such results suggest CSH injection inhibits SRIF synthesis but do not define the role of synthesis inhibition in the prolonged depression of hypothalamic IR-SRIF levels and their eventual recovery. We have, therefore, performed a more thorough temporal analysis of CSH's effects on the in vivo synthesis and IR levels of SRIF (and AVP and OXT) in the rat hypothalamus. Male rats received CSH (300 mg/kg, sc) 1, 2, 4, 8, 10, 12 and 24 hr before the injection of 50 microcuries (35S) cysteine into the third ventricle under urethane anesthesia. Four hours later, hypothalami were removed and prepared for HPLC, to separate labeled SRIF-14, SRIF28, OXT, and AVP. Other rats received CSH, but not label, and were killed at intervals ranging from 15 min to 120 hr later for measurements of IR-SRIF, IR-AVP, and IR-OXT in hypothalamus and posterior pituitary. As previously observed, hypothalamic IR-SRIF levels fell within 2-4 hr to values 50-60 percent below normal, and returned to normal by 72 hr. In contrast, hypothalamic and posterior pituitary IR-AVP and IR-OXT levels remained normal throughout. Label incorporation into HPLC-isolated SRIF-14, SRIF28, AVP, and OXT in hypothalamus was negligible 1 hr after CSH injection, remained low at 4 hr, but was essentially normal 8-10 hr post-CSH administration. The data suggest that SRIF synthesis returns to normal much more rapidly than total endogenous stores. The rapid restoration of SRIF synthesis may account for the longer-term replenishment of peptide pools, though other mechanisms may also contribute. (Supported by a grant from the NIH [NS 20014]). Localization Of Increased C-Fos mRNA Content in Rat CNS Following Recurrent Seizures Christine M. Gall and Jeffrey D. White University of California, Irvine, CA, and SUNY, Stony Brook, NY The product of the cellular proto-onogene c-fos has been suggested to be a regulatory factor in gene transcription. Recently, several groups, including ourselves, have demonstrated that c-fos protein and/or mRNA content in the CNS is increased in response to seizures. We previously demonstrated that recurrent seizures induce an increase in preproenkephalin A mRNA content in a number of limbic and olfactory forebrain regions. In the present study, we have employed in situ hybridization autoradiography to evaluate the correspondence between changes in the abundance of mRNAs for c-fos and preproenkephalin A following seizure activity. Recurrent seizures were induced by unilateral electrolytic lesion of the dentate gyrus hilus in anesthetized male rats. In this paradigm, limbic seizures begin approximately 1.5 hrs postlesion and recur for 8 to 10 hrs thereafter. At 3 and 6 hrs postlesion, experimental animals and paired controls were sacrificed by perfusion with 4% paraformaldehyde, and free-floating vibratome sections through the forebrain were processed for the localization of c-fos mRNA using a 35S-labeled cRNA probe and both film and emulsion autoradiographic techniques. At both time points, the hilus lesion-induced seizures led to increases in hybridization to c-fos mRNA, which were greatest in dentate gyrus stratum granulosum and layer II of piriform and entorhinal cortices. Modest increases in hybridization were also evident throughout the pyramidal cells of hippocampus, olfactory tubercle, and anterior olfactory nucleus. The areas of increased c-fos mRNA include most, but not all, areas in which preproenkephalin A mRNA becomes elevated in the same paradigm. The data are consistent with an activation of c-fos gene in response to increased neuronal activity induced by seizures, but further work, including examination of additional time points, is needed to determine if c-fos transcription is elevated in all areas which exhibit seizure-induced increases in enkephalin synthesis. Restriction of T-Cell Receptor Variable Region Genes In Experimental Autoimmune Encephalitis C. Gomez, J. Urban, V. Kumar, D. Kono, and L. Hood Division of Biology, 147-75, California Institute of Technology, To investigate the molecular biology of T-cell antigen receptors relevant in experimental allergic encephalomyelitis (EAE), helper T cells (TH) were raised to an N-terminal nonapeptide of myelin basic protein (MBP) known to induce experimental autoimmune encephalomyelitis (EAE) in PL/J (H-2d) mice. Variable gene segment usage was identified by nucleotide sequence analysis for seven T-cell receptor (Tcr) a chain cDNAs and six ẞ chain cDNAs. An additional 29 helper T cells were analyzed by Northern and Southern blot for Tcr gene usage. The results show a striking restriction in both variable (V) and joining (J) gene segment use in both the a and ß chains of Tcr responding to this encephalitogenic peptide. The Va2 gene segment was expressed in 9 out of 14 TH analyzed (64%) and the remainder (36%) were found to express Va4. Sequence analysis demonstrated six of six Vɑ2 genes to be exactly identical, utilizing a newly described member of the Va2 gene subfamily (Va2.3) joined to a newly described Ja gene segment (Ja51). Ja51 was also used by the other sequenced a chain gene expressing Va4.1. Almost all TH expressed the same Vẞ gene consisting of Vẞ8.2 joined to Jẞ2.6 (29 out of 33 TH, 88%). At least two of the remaining four TH expressed Vẞ13. Junctional diversity was completely absent from the Va2 genes. It was present in four out of five sequenced Vẞ8 genes, but the translated amino acid sequences of these genes were almost completely identical. These results represent the first detailed study of Ter important in the pathogenesis of autoimmunity. The fact that such Tcr appear to bear remarkable similarities in their V genes suggests that antibodies directed against specific Ter V domains may prove useful both diagnostically and therapeutically in the treatment of autoimmune disease. |