Ethanol affects many functions of the brain and peripheral organs. Here we show that ethanol opens G-protein-activated, inwardly rectifying K + (GIRK) channels, which has important implications for inhibitory regulation of neuronal excitability and heart rate. At pharmacologically relevant concentrations, ethanol activated both brain-type GIRK1/2 and cardiac-type GIRK1/4 channels without interaction with G proteins or second messengers. Moreover, weaver mutant mice, which have a missense mutation in the GIRK2 channel, showed a loss of ethanol-induced analgesia. These results suggest that the GIRK channels in the brain and heart are important target sites for ethanol.
Neuregulins (also known as ARIA, NDF, heregulin, GGF) are a family of widely expressed growth and differentiation factors. Neuregulins secreted from motor neurons accumulate at maturing neuromuscular junctions, where they stimulate transcription of genes encoding specific acetylcholine receptors. How these factors function at central synapses, however, is unknown. In the maturing cerebellum, neuregulins are concentrated in glutamatergic mossy fibres that innervate granule cells in the internal granule-cell layer. We have analysed the effects of neuregulins on the expression of genes encoding NMDA (N-methyl-D-aspartate) receptors in the cerebellum, because receptor composition changes dramatically as expression of the receptor NR2C subunit is specifically induced in neurons in the internal granule-cell layer during synaptogenesis. Here we report that addition of a neuregulin-beta isoform to cultured cerebellar slices specifically increases the expression of NR2C messenger RNAs by at least 100-fold; effects are only minor with a neuregulin-alpha isoform. This stimulation of NR2C expression requires synaptic activity by NMDA receptors, as well as neuregulin-beta. Addition of the NMDA-receptor-channel blocker AP-5 prevents upregulation of the NR2C subunit by neuregulin, whereas an AMPA/kainate-receptor antagonist does not. Consistent with these effects of neuregulin, we find that granule cells express its receptors ErbB2 and ErbB4 before the NR2C subunit of the NMDA receptor. Our results indicate that neuregulins regulate the composition of neurotransmitter receptors in maturing synapses in the brain, in a manner analogous to the neuromuscular junction.
The 35S rRNA gene of the yeast Saccharomyces cerevisiae was fused to the GAL7 promoter. This hybrid gene, when present on a multicopy plasmid and induced by galactose, suppressed the growth defects of a temperaturesensitive RNA polymerase I (pol I) mutant and those of a mutant in which the gene for the second largest subunit of pol I was deleted. Analysis of pulse-labeled RNA directly demonstrated that rRNA synthesis in this deletion mutant is from the GAL7 promoter. These experiments show that the sole essential function of pol I is the transcription ofthe rRNA genes, that pol I is not absolutely required for the synthesis of rRNA and ribosomes or cell growth if 35S rRNA synthesis is achieved by some other means, and that the tandemly repeated structure of the chromosomal rRNA genes is also not absolutely required for the synthesis of rRNA and ribosomes. pCl/l carrying GAL7-35SrDNA (see Fig. 1) pNOY102 pAA7 carrying GAL7-35SrDNA (see Fig. 1) *NOY401 was constructed by crossing NOY265 (10) and W303-la (7) and then screening meitotic segregants for a temperature-sensitive (rpal90-3) and strongly galactose-positive phenotype.tNOY408-la, -lb, -lc, and -ld are four haploid segregants derived from a single ascospore produced by diploid strain NOY408 on galactose plates.
We have previously isolated mutants ofSaccharomyces cerevisiae that are primarily defective in transcription of 35S rRNA genes by RNA polymerase I and have identified genes (RRN1 to RRN9) involved in this process. We have now cloned the RRN4 gene by complementation of the temperature-sensitive phenotype of the rrn4-1 mutant and have determined its complete nucleotide sequence. The following results demonstrate that the RRN4 gene encodes the A12.2 subunit of RNA polymerase I. First, RRN4 protein expressed in Escherichia coli reacted with a specific antiserum against A12.2. Second, amino acid sequences of three tryptic peptides obtained from A12.2 were determined, and these sequences are found in the deduced amino acid sequence of the RRN4 protein. The amino acid sequence of the RRN4 protein (A12.2) is similar to that of the RPB9 (B12.6) subunit of yeast RNA polymerase II; the similarity includes the presence of two putative zinc-binding domains.Thus, A12.2 is a homolog of B12.6. We propose to rename the RRN4 gene RPA12. Deletion ofRPA12 produces cells that are heat but not cold sensitive for growth. We have found that in such null mutants growing at permissive temperatures, the cellular concentration ofA190, the largest subunit of RNA polymerase I, is lower than in the wild type. In addition, the temperature-sensitive phenotype of the rpa12 null mutants can be partially suppressed by RPA190 (the gene for A190) on multicopy plasmids. These results suggest that A12.2 plays a role in the assembly of A190 into a stable polymerase I structure.In eukaryotic cells, rRNA synthesis is carried out by a unique RNA polymerase, RNA polymerase I (Pol I), in the nucleolus with the aid of various transcription factors and perhaps appropriate nucleolar structures (for recent reviews, see references 22 and 28). Although studies of transcription factors and other possible components involved in rRNA transcription are only at the beginning stage, characterization of Pol I itself, specifically with respect to its subunit composition, has been carried out fairly extensively, mostly by using Pol I from the yeast Saccharomyces cerevisiae (for reviews, see references 24 and 31). Pol I from S. cerevisiae consists of 14 polypeptide subunits. Five of them (ABC27, ABC23, ABC14.5, ABC1Oa, and ABC10B) are shared by all three nuclear RNA polymerases, two of them (AC40 and AC19) are shared by Pol I and RNA polymerase III (Pol III), and the remaining seven (A190, A135, A49, A43, A34.5, A14, and A12.2) are subunits unique to Pol I. Genes for all subunits of Pol I except A14 and A12.2 have been cloned, and their nucleotide sequences have been determined (3,13,15,18,32,37,38; unpublished work cited in reference 31). The two largest subunits, A190 and A135, like the two largest subunits of Pol II and Pol III, are homologs of the ,B' and a subunits of Escherichia coli RNA polymerase, while AC40 and AC19 (and the B44 subunit of Pol II) appear to be homologs of the E. coli a subunit (3,11,12,16). Thus, these four subunits may constitute a core enzyme co...
SRPI, a suppressor of certain temperaturesensitive mutations in RNA polymerase I in Saccharomyces cerevisuae, encodes a protein that is associated with nuclear pores. By using a system of conditional SRPI expression and by isolating temperature-sensitive srpl mutants, we have demonstrated that Srplp is essential for maintenance of the crescent-shaped nucleolar structure, RNA transcription, and the proper functions of microtubules as inferred from analysis of nuclear division/segregation and immunofluorescence microscopy of microtubules. Different mutant alleles showed significantly different phenotypes in relation to these apparently multiple functional roles of the protein. We have also found that eight imperfect 42-amino-acid tandem repeats present in Srplp are similar to the 42-amino-acid repeats in armadillo/plakoglobin/jJcatenin proteins present in adhesive junction complexes of higher eukaryotes. We discuss this similarity in connection with the observed pleiotropic effects of srpl mutations.The SRPJ gene in Saccharomyces cerevisiae was originally identified as a specific suppressor (SRPI-1) of temperaturesensitive (ts) mutations in the zinc-binding domain of the A190 subunit of RNA polymerase I (Pol I) (1). The suppressor, SRPJ-1, was also found to suppress ts mutations in the zinc-binding domain of the A135 subunit of Pol I but not other ts mutations in both A190 and A135. There is evidence to suggest that the zinc-binding domains of the two polymerase subunits, one close to the N terminus of A190 and the other close to the C terminus of A135, are in physical proximity and interact with each other (2). It was therefore originally inferred that the SRPI gene product interacts directly or indirectly with this region of Pol I (1). The SRPI gene was cloned and sequenced, and the encoded protein (Srplp) was localized, by immunofluorescence microscopy as well as biochemical fractionation, to the periphery of the nucleus, in the vicinity of the nuclear pores (1). This localization, which was subsequently confirmed by additional experiments (mentioned below), was surprising in view of the specific genetic interaction between SRPI and the Pol I subunit genes mentioned above. In this paper, we first describe the results of mutational analysis of SRPI as well as Srplp depletion experiments. We then describe our finding of structural similarity of Srplp to a family of proteins including the vertebrate adhesive junction proteins f3-catenin and plakoglobin (3) and the Drosophila segment polarity-determining protein armadillo (4, 5) and discuss possible functions of Srplp in relation to the observed pleiotropic effects caused by mutations in this protein or by its depletion. MATERIALS AND METHODSSynthetic glucose medium is 2% glucose, 0.67% Bacto yeast nitrogen base (Difco), and 0.5% Casamino acids (Difco), which was supplemented with tryptophan and required bases, as described (6).-Synthetic galactose medium is the same as above, except that 2% galactose is substituted for glucose.Yeast strains NOY477 and NOY4...
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