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Acute and chronic exposure to ethanol produces specific changes in several signal transduction cascades. Such alterations in signaling are thought to be a crucial aspect of the central nervous system's adaptive response, which occurs with chronic exposure to ethanol. We have recently identified and isolated several genes whose expression is specifically induced by ethanol in neural cell cultures. The product of one of these genes has extensive sequence homology to phosducin, a phosphoprotein expressed in retina and pineal gland that modulates trimeric guanine nucleotide-binding protein (G protein) function by binding to G-protein fly subunits. We identified from a rat brain cDNA library an isolate encoding the phosducin-like protein (PhLP), which has 41% identity and 65% amino acid homology to phosducin. PhLP cDNA is expressed in all tissues screened by RNA blot-hybridization analysis and shows marked evolutionary conservation on Southern, hybridization. We have identified four forms ofPhLP cDNA varying only in their 5' ends, probably due to alternative splicing. This 5'-end variation generates two predicted forms of PhLP protein that differ by 79 aa at the NH2 terminus.

The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus regulates synthesis of its photosystem in response to two environmental stimuli, oxygen tension and light intensity. Here we describe the identification and characterization of the trans-acting regulatory gene hvrA, which we show is involved in differentially controlling reaction center and light-harvesting gene expression in response to alterations in light intensity. An hvrA mutant strain is shown to lack the capability to trans-activate light-harvesting-I and reaction center gene expression but retain normal light-harvesting-II and photopigment regulation, in response to a reduction in light intensity. As a consequence of altered expression, hvrA mutant strains exhibit reduced photosynthetic growth capabilities under dim-light conditions. The results of this study and additional studies indicate that regulated synthesis of the photosystem involves complex sets of overlapping regulatory circuits that differentially control photosystem gene expression in response to environmental stimuli such as oxygen tension and light intensity.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) play a major role in transmembrane signaling processes by transducing extracellular signals from the superfamily of heptahelical cell surface receptors to their appropriate intracellular effectors (1, 2). In its trimeric form, G␣␤␥ is inactive, and the G␣ subunit binds a molecule of GDP. Upon ligand binding, the receptor catalyzes the exchange of GDP for GTP on G␣ that causes its activation and dissociation from the tightly bound G␤␥ complex.1 Inactivation and reassociation of the heterotrimer is initiated by the hydrolysis of bound GTP into GDP by an intrinsic GTPase activity of the G␣ subunit. It is now known that both the free GTP-bound G␣ and the G␤␥ dimer can bind and regulate downstream effectors including adenylyl cyclases, phospholipases, and ion channels, and thereby modulate second messenger levels and ion flux (3).The discovery of several specific G␤␥ binding proteins has recently shed light on new roles for G␤␥ in the propagation and termination of cellular signaling. The dimer has been shown to recruit ␤-adrenergic receptor kinase (␤ARK) 2 to its membraneassociated receptor substrate and thus initiate receptor desensitization (4, 5). This process occurs via direct binding of G␤␥ to the C terminus of a putative pleckstrin homology domain on ␤ARK (6). Furthermore, the responsiveness of G protein-regulated signaling systems may be directly modulated through the interaction of G␤␥ subunits with intracellular regulatory proteins. For instance, phosducin, a phosphoprotein mainly expressed in the retina and pineal gland, inhibits the phototransduction cascade by scavenging ␤␥ subunits of the G protein transducin (G t ), thus preventing their reassociation with the G t ␣ subunit (7,8). Because phosducin has a higher affinity for G t ␤␥ than does G t ␣, it has been suggested that the formation of the phosducin/G t ␤␥ complex is a major factor regulating photoreceptor responsiveness (9). From in vitro binding and cotransfection assays, it was proposed that phosducin may also compete with other targets for G␤␥ binding, such as ␤ARK and phospholipase C type ␤2 (10, 11).We recently isolated a rat brain cDNA encoding a phosducinlike protein (PhLP), which has 65% amino acid homology to phosducin (12). We also described several 5Ј-end splice variants that generate two predicted isoforms of the protein: PhLP long (PhLP) of 301 amino acids containing the entire coding sequence and PhLP short (PhLP S ) of 218 amino acids missing the first 83 N-terminal residues of PhLP (12, 13). Based on sequence homology with phosducin, we have suggested that PhLP proteins regulate G␤␥ signaling in nonretinal tissues. In favor of this hypothesis, a recent report showed that recombinant PhLP S inhibits several G␤␥ functions in vitro (14). Interestingly, these authors suggested that unlike phosducin (11,15), the N terminus of PhLP was unlikely to contain a G␤␥-binding domain.To more directly characterize the interaction of PhLP with G␤␥, we studied PhLP binding to G␤␥ both...

The genetic locus ahcY, encoding the enzyme S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) from the bacterium Rhodobacter capsulates, has been mapped by mutational analysis to within a cluster of genes involved in regulating the induction and maintenance of the bacterial photosynthetic apparatus. Sequence analysis demonstrates that ahcY encodes a 51-kDa polypeptide that displays 64% sequence identity to its human homolog. Insertion mutants in ahcY lack detectable S-adenosyl-L-homocysteine hydrolase activity and, as a consequence, S-adenosyl-L-homocysteine accumulates in the cells, resulting in a 16-fold decrease in the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine as compared to wild-type cells. The ahcY disrupted strain fails to grow in minimal medinm; however, growth is restored in minimal medium supplemented with methionine or homocysteine or in a complex medium, thereby indicating that the hydrolysis of S-adenosyl-L-homocysteine plays a key role in the metabolism of sulfur-containing amino acids. The ahcY mutant, when grown in supplemented medium, synthesizes significantly reduced levels of bacteriochlorophyll, indicating that modulation of the intracellular ratio of S-adenosyl-L-methionine to S-adenosyl-L-homocysteine may be an important factor in regulating bacteriochlorophyll biosynthesis.S-Adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) was first described in rat liver extracts as the activity responsible for the reversible hydrolysis of S-adenosyl-Lhomocysteine (AdoHcy) to adenosine and homocysteine ( Fig. 1) (1). AdoHcy is formed as a direct product of transmethylation reactions involving S-adenosyl-L-methionine (AdoMet) (2) and is known to be a potent inhibitor of most AdoMet-mediated methyl-transfer reactions.AdoHcyase has been found in all cells that have been tested, with the exception of Escherichia coli and certain related bacteria (3-5). In the latter, the hydrolysis ofAdoHcy to adenine and homocysteine requires two enzymatic steps catalyzed by a specific AdoHcy nucleosidase (6) and S-ribosyl-L-homocysteine hydrolase (7). AdoHcyase isolated from various sources always consists of a number of identical subunits, each containing 1 mol of tightly bound NAD+ (8).The amino acid sequences of the rat liver, human placenta, and Dictyostelium enzymes have been deduced from cDNA sequences and show a high degree of sequence conservation (9-11). Site-directed mutational analyses coupled with in vitro biochemistry have demonstrated the presence of a nucleotide-binding domain (12, 13), but further conclusions regarding the protein structure have been hindered by lack of significant sequence diversity and a convenient system for genetic analysis.

The c-fos immediate-early gene is induced by morphine and other drugs of abuse in the nucleus accumbens (NAc), a mesolimbic region implicated in drug abuse and reward. This study examined the role of c-fos in the acquisition and expression of the conditioned place paradigm (CPP) in the rat by suppressing Fos protein expression with c-fos antisense oligodeoxynucleotides (ODNs). CPP was completely prevented by c-fos antisense ODN infused bilaterally into the NAc prior to each systemic morphine injection, whereas sense and missense NAc injections had no effect on CPP. NAc administration of c-fos antisense ODN after the last systemic morphine conditioning session did not affect the expression of morphine-CPP. These results suggest that c-fos expression in NAc is necessary for the acquisition but not expression of morphine-CPP, and they have important implications for understanding conditioned behaviours and drug craving and addiction.

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Here we present the nucleotide sequence and characterization of two genes, hvrB and oriS, that are located in the regulatory gene cluster from Rhodobacter capsulatus. The hvrB gene, which encodes a protein with a predicted molecular mass of 32 kDa, is shown to be highly homologous to genes encoding members of the LysR famil of bacterial transcriptional regulators. A chromosomal disruption of hvrB is shown to result in the failure to regulate expression from the nearby ahcY and orfS genes in response to alterations in light intensity. We show by primer extension mapping that the 5' end of ahcY-specific mRNA defines a promoter region exhibiting sequence similarity to known R. capsulatus promoter elements. Our mutational analysis further demonstrates that hvrB autoregulates its own expression in vivo.S-Adenosyl-L-homocysteine hydrolase (AdoHcyase) is an enzyme responsible for the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine and homocysteine (8). AdoHcy is formed as a direct product of transmethylation reactions involving S-adenosyl-L-methionine (AdoMet) (25) and is known to be a potent inhibitor of most AdoMetmediated methyl group transfer reactions. Previously, Sganga et al. (29) cloned and sequenced the gene encoding the AdoHcyase enzyme, ahcY, from the purple, nonsulfur photosynthetic bacterium Rhodobacter capsulatus. The results of their analysis demonstrated that the AdoHcyase primary structure was highly conserved, exhibiting >60% sequence identity between the eubacterial enzyme and its homologs from such evolutionarily divergent species as Caenorhabditis elegans, Dictyostelium discoideum, rat, and human (7,19,24). A mutant strain of R. capsulatus containing a disruption of the ahcYgene (SLB1) was shown to be incapable of growth on minimal medium but viable on complex medium or on minimal medium containing exogenously added methionine or homocysteine (29). The viability of this strain on complex medium was somewhat surprising in light of the greatly elevated intracellular AdoHcy level that the cells exhibited as a consequence of the absence of AdoHcyase activity (29). The elevated AdoHcy/ AdoMet ratio should be toxic to many methyl group transfer reactions that occur within the cell.In photosynthetic bacteria, methylation of the sixth propyl group of Mg-protoporphyrin IX during the synthesis of bacteriochlorophyll is catalyzed by the enzyme S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase (MPMT) (18). The reaction is accompanied by a methyl group transfer from AdoMet to Mg-protoporphyrin IX, resulting in the formation of Mg-protoporphyrin monomethylester. Mg-protoporphyrin monomethylester is the first stable intermediate which is unique to the bacteriochlorophyll biosynthetic path-* Corresponding author. Mailing address: