Control of gene expression is important to gene therapy for purposes of both dosing and safety. In vivo regulation of gene expression was demonstrated following co-injection of two separate recombinant adeno-associated virus vectors, one encoding an inducible murine erythropoietin transgene and the other a transcriptional activator, directly into the skeletal muscle of adult immunocompetent mice. Transcription was controlled by systemic administration or withdrawal of tetracycline over an 18 week period, demonstrating that the two vectors were capable of transducing the same cell. Cellular or humoral immune responses against the transactivator protein were not detected.

Prader-Willi syndrome (PWS), a human neuroendocrine disorder, is associated with deficiencies of paternal chromosome 15q12. Small nuclear ribonucleoprotein polypeptide N (SNRPN) is the first expressed gene identified in the PWS critically deleted region. Following our demonstration that the murine homologue of SNRPN is imprinted, we have characterized a sequence polymorphism within expressed portions of human SNRPN and show that human SNRPN is monoallelically expressed in fetal brain and heart and in adult brain. Analysis of maternal DNA and SNRPN cDNA confirmed that the maternal allele of SNRPN is not expressed in fetal brain and heart. Maternal imprinting of SNRPN supports the hypothesis that paternal absence of SNRPN is responsible for the PWS phenotype.

Transcripts of the rat calcitonin/calcitonin gene-related peptide (CGRP) gene are alternatively spliced in a tissue-specific manner resulting in the production of calcitonin mRNA and peptide in thyroid C cells and CGRP mRNA and peptide in neurons. Transfection studies using calcitonin and chimaeric human beta-globin/calcitonin exon minigene constructs showed that the splice acceptor and exon specific to calcitonin mRNA are spliced much less efficiently in CGRP-producing cells (F9 teratocarcinomas) than in cells that preferentially make calcitonin (HeLa cells). In vitro splicing of chimaeric human beta-globin/calcitonin transcripts in HeLa nuclear extracts were inhibited by the addition of nuclear extract from CGRP-favoring cells or tissues such as rat brain. This inhibition was specific as splicing of human beta-globin first intron transcripts was not affected by comparable amounts of rat brain extract. Fractionation of rat brain nuclear extracts allowed the partial purification of two brain-specific polypeptides of apparent molecular mass of 43 and 41 kDa which preferentially bind RNA containing the calcitonin-specific splice acceptor. Since these polypeptides cofractionate with the calcitonin mRNA-specific splicing inhibition activity, we suggest that they may mediate the inhibition of splicing observed in vitro and underlie, in part, the inefficient calcitonin mRNA production observed in CGRP-favoring cells in vivo.

The best examples of imprinting in humans are provided by the Angelman and Prader-Willi syndromes (AS and PWS) which are associated with maternal and paternal 15q11-13 deletions, respectively, and also with paternal and maternal disomy 15. The region of the deletions has homology with a central part of mouse chromosome 7, incompletely tested for imprinting effects. Here, we report that maternal duplication for this region causes a murine imprinting effect which may correspond to PWS. Paternal duplication was not associated with any detectable effect that might correspond with AS. Gene expression studies established that Snrpn is not expressed in mice with the maternal duplication and suggest that the closely-linked Gabrb-3 locus is not subject to imprinting. Finally, an additional new imprinting effect is described.

Prader-Willi syndrome (PWS) is associated with paternal gene deficiencies in human chromosome 15q11-13, suggesting that PWS is caused by a deficiency in one or more maternally imprinted genes. We have now mapped a gene, Snrpn, encoding a brain-enriched small nuclear ribonucleoprotein (snRNP)-associated polypeptide SmN, to mouse chromosome 7 in a region of homology with human chromosome 15q11-13 and demonstrated that Snrpn is a maternally imprinted gene in mouse. These studies, in combination with the accompanying human mapping studies showing that SNRPN maps in the Prader-Willi critical region, identify SNRPN as a candidate gene involved in PWS and suggest that PWS may be caused, in part, by defects in mRNA processing.

The calcitonin gene related peptide (CGRP) gene is a complex transcription unit that is expressed in a highly restricted pattern in both the nervous system, particularly in sensory ganglia and brainstem, and in the thyroid C cells of the endocrine system, with tissue-specific alternative RNA processing events generating transcripts encoding either the hormone, calcitonin, or the neuropeptide, CGRP. This pattern of expression in neural and endocrine tissues raises the question whether similar or distinct genomic elements are responsible for activation in both neural and endocrine cell types. We have identified a complex enhancer element, located more than 1 kilobase 5' of the transcription initiation site of the calcitonin/CGRP gene that functions in cells of neuronal or C cell origin, but not in any other cell type tested. At least two complementary regulatory sequences are required for the function of the cell-specific enhancer.

This chapter focuses on alternative RNA processing events operate in a tissue-specific, developmentally regulated fashion in the neuroendocrine system to produce alternative, discrete polypeptide products serving to further increase the diversity required to subserve the biological demands of complex organisms. The similarity of the alternative RNA processing events in genes of the neuroendocrine system with those of other families of eukaryotic genes suggest that common underlying biochemical mechanisms may operate in this form of regulated expression of many eukaryotic transcription units. Using an antisera generated against a synthetic peptide corresponding to the 14 C1-terminal amino acids of Calcitonin Gene-Related Product (CGRP), immunoreactive CGRP was identified in a unique distribution in a large number of cell groups and pathways in the central nervous system distinct from that of any known neuropeptide. The possible existence of other gene products related to CGRP was investigated by screening libraries of chimaeric plasmids containing inserts complementary to mRNAs from rat medullary thyroid carcinomas with a clonal alpha-CGRP cDNA probe. Sequence- or site-specific poly(A) site selection, or choice of splice acceptor were considered as the alternative regulated event that accounts for the developmentally-regulated RNA processing events resulting in CGRP production.

The interactions of dopaminergic agonists and antagonists with 3H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [3H]dopamine and [3H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed "D3" binding site. Antagonist/[3H]dopamine competition curves are of uniformly steep slope (nH = 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [3H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [3H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [3H]dopamine binding are also correlated with estimates of these agonists' affinities for the high affinity binding component of agonist/[3H]flupentixol competition curves. Both D3 specific [3H] dopamine binding and the high affinity agonist-binding component of dopamine/[3H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor.

Many researchers now believe that neuroleptic drugs, such as the phenothiazines, exert their actions in the brains of schizophrenics at sites that are involved with specific schizophrenic disturbance itself. Thus, it has been hypothesized that if one understood the mechanism of action of these drugs, such as their neuronal recognition sites and the physiological and cellular responses with which they interfere, one might gain insight into the organic abnormalities responsible for the disease process. The neuronal recognition sites to which neuroleptics bind appear to be receptors for the neurotransmitter dopamine, and enhanced dopaminergic transmission has long been hypothesized to be etiologic in some forms of schizophrenia. This chapter focuses on neuronal binding sites for neuroleptics. It also discusses these sites with respect to their association with dopamine receptors. It also describes the methods employed to study separately the neuronal localization, relative levels, and pharmacological and biochemical characteristics of each dopamine receptor subtype.

The pharmacological classification of dopamine receptors is controversial. While as many as five subclasses of dopamine receptors have been proposed, in this review we attempt a synthesis which suggests that the preponderance of data can be encompassed by a model proposing only two major classes of dopamine receptors. Hopefully, this review will provide the reader with a useful conceptual framework within which to evaluate future research on dopaminergic pharmacology.