Candidate genes for alcohol preference identified by expression profiling in alcohol-preferring and -nonpreferring reciprocal congenic rats

Background

Selectively bred alcohol-preferring (P) and alcohol-nonpreferring (NP) rats differ greatly in alcohol preference, in part due to a highly significant quantitative trait locus (QTL) on chromosome 4. Alcohol consumption scores of reciprocal chromosome 4 congenic strains NP.P and P.NP correlated with the introgressed interval. The goal of this study was to identify candidate genes that may influence alcohol consumption by comparing gene expression in five brain regions of alcohol-naïve inbred alcohol-preferring and P.NP congenic rats: amygdala, nucleus accumbens, hippocampus, caudate putamen, and frontal cortex.

Results

Within the QTL region, 104 cis-regulated probe sets were differentially expressed in more than one region, and an additional 53 were differentially expressed in a single region. Fewer trans-regulated probe sets were detected, and most differed in only one region. Analysis of the average expression values across the 5 brain regions yielded 141 differentially expressed cis-regulated probe sets and 206 trans-regulated probe sets. Comparing the present results from inbred alcohol-preferring vs. congenic P.NP rats to earlier results from the reciprocal congenic NP.P vs. inbred alcohol-nonpreferring rats demonstrated that 74 cis-regulated probe sets were differentially expressed in the same direction and with a consistent magnitude of difference in at least one brain region.

Conclusions

Cis-regulated candidate genes for alcohol consumption that lie within the chromosome 4 QTL were identified and confirmed by consistent results in two independent experiments with reciprocal congenic rats. These genes are strong candidates for affecting alcohol preference in the inbred alcohol-preferring and inbred alcohol-nonpreferring rats.

     

Ancora: a web resource for exploring highly conserved noncoding elements and their association with developmental regulatory genes

Metazoan genomes contain arrays of highly conserved noncoding elements (HCNEs) that span developmental regulatory genes and define regulatory domains. We describe Ancora , a web resource that provides data and tools for exploring genomic organization of HCNEs for multiple genomes. Ancora includes a genome browser that shows HCNE locations and features novel HCNE density plots as a powerful tool to discover developmental regulatory genes and distinguish their regulatory elements and domains.     

In silico identification of regulatory elements of GRIN1 genes

The ionotropic receptor of glutamate activated by N-methyl-D-aspartate (iGluR-NMDA) is a multiheteromeric complex constituted by at least three different types of subunits, encoded by seven different genes. The subunits of iGluR-NMDA have a complex system of regulation of their gene expression. Their expression is specific for each type of neural cell, as well as for the age of the organism. Moreover, there are reports that iGluR-NMDA expression is species-specific. Even though this macromolecular complex is very important in physiology and pathology of the central nervous system, knowledge to date about the regulatory elements controlling expression is scarce. We present the results of an in silico prediction of potential regulatory elements, some of which coincide with the few known experimentally. We also present the important differences regarding the presence and the localization of the regulatory elements among human, rat, and mouse species.     

Comparative functional analysis of the cow and mouse myostatin genes reveals novel regulatory elements in their upstream promoter regions

Myostatin is a paracrine/autocrine factor that inhibits muscle growth, and mutations that affect myostatin activity or expression produce dramatic increases in muscle mass in several species. However, at present it is less clear whether differences in myostatin expression or activity exist between species with differing body sizes. Here we demonstrate that mouse muscle expresses far greater levels of myostatin mRNA than cow. In addition, activity of a 1200 bp mouse myostatin promoter construct was significantly greater than that of a 1200 bp cow myostatin promoter construct in C₂C₁₂ myotubes. In contrast, activity of reporter constructs flanked by one or both untranslated regions (UTRs) was not significantly different between the two species. Sequence analysis identified a number of promoter regions which differed between larger species (cow, pig, goat, sheep, human) and smaller (mouse, rat), including a TATA-box sequence, a CACCC box, two AT-rich regions (AT1 and AT2), and a palindromic sequence (PAL). We therefore used mutagenesis to alter the mouse sequence for each of these elements to that of the cow. Mutagenesis of the TATA, CACC, and AT1 sequences of the mouse to those of the cow significantly decreased activity of the mouse myostatin promoter compared to the wild type mouse promoter, while mutation of the AT2 and PAL sequences tended to increase promoter activity. Finally, the cow myostatin promoter was less responsive to FoxO signaling than the mouse myostatin promoter. Together these data support the hypothesis that differences in promoter activity between mouse and cow may contribute to differences in expression of the myostatin gene between these species.     

Candidate genes for drought tolerance and improved productivity in rice (Oryza sativa L.)

Candidate genes are sequenced genes of known biological action involved in the development or physiology of a trait. Twenty-one putative candidate genes were designed after an exhaustive search in the public databases along with an elaborate literature survey for candidate gene products and/or regulatory sequences associated with enhanced drought resistance. The downloaded sequences were then used to design primers considering the flanking sequences as well. Polymerase chain reaction (PCR) performed on 10 diverse cultivars that involvedJaponica, Indica and local accessions, revealed 12 polymorphic candidate genes. Seven polymorphic candidate genes were then utilized to genotype 148 individuals of CT9993 × IR62266 doubled haploid (DH) mapping population. The segregation data were tested for deviation from the expected Mendelian ratio (1:1) using a Chi-square test (<1%). Based on this, four candidate genes were assessed to be significant and the remaining three, as non-significant. All the significant candidate genes were biased towards CT9993, the female parent in the DH mapping population. Single-marker analysis strongly associated (<1%) them to different traits under both well-watered and low-moisture stress conditions. Two candidate genes,EXP15 andEXP13, were found to be associated with root number and silicon content in the stem respectively, under both well-watered and low-moisture stress conditions     

Complexity in evolved regulatory variation for alcohol dehydrogenase genes in HawaiianDrosophila

Summary The alcohol dehydrogenase gene (Adh gene) ofDrosophila affinidisjuncta is expressed at a higher level in the larval midgut and Malpighian tubules than the homologous gene fromDrosophila hawaiiensis. This study analyzed thecis-acting sequences responsible for these regulatory differences in larval tissues ofDrosophila melanogaster transformants. A series of 10 chimeric and deletedAdh genes was introduced into the germ line ofD. melanogaster, and tissue-specific expression levels were quantified by gel electrophoresis of tissue extracts. Sequences in the upstream region of the two genes had the strongest influence on enzyme production in the midgut and Malpighian tubules. Other sequence elements also showed effects, some of which were tissue specific. Most gene fragments displayed context-dependent effects, thus supporting the proposed model of polygenic regulation ofAdh gene expression.     

Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies

Most classical integrases of prokaryotic genetic elements specify integration into tRNA or tmRNA genes. Sequences shared between element and host integration sites suggest that crossover can occur at any of three sublocations within a tRNA gene, two with flanking symmetry (anticodon-loop and T-loop tDNA) and the third at the asymmetric 3' end of the gene. Integrase phylogeny matches this classification: integrase subfamilies use exclusively either the symmetric sublocations or the asymmetric sublocation, although tRNA genes of several different aminoacylation identities may be used within any subfamily. These two familial sublocation preferences imply two modes by which new integration site usage evolves. The tmRNA gene has been adopted as an integration site in both modes, and its distinctive structure imposes some constraints on proposed evolutionary mechanisms.     

Identification and properties of imprinted genes and their control elements

Imprinted genes have the unusual characteristic that the copy from one parent is destined to remain inactive. Though few in number they nonetheless constitute a functionally important part of the mammalian genome. With their memory of parental origin, imprinted genes represent an important model for the epigenetic regulation of gene function and will provide invaluable paradigms to test whether we can predict epigenetic state from DNA sequence. Since their first discovery, systematic screens and some good fortune have led to identification of over seventy imprinted genes in the mouse and human: recent microarray analysis may reveal many more. With a significant number of imprinted genes now identified and completion of key mammalian genome sequences, we are able systematically to examine the organization of imprinted loci, properties of their control elements and begin to recognize common themes in imprinted gene regulation.     

Pleiotropic control of five eucaryotic genes by multiple regulatory elements.

We have previously shown that allophanate acts as an inducer for five structural genes whose products participate in the degradation of allantoin by Saccharomyces cerevisiae. This observation led us to hypothesize that these genes might be controlled in common and to test the hypothesis by searching for mutants unable to induce production of the allantoin-degrading enzymes. Such mutants have been found. These strains grew poorly when provided with any of the allantoin pathway intermediates, but used other nitrogen sources normally. The mutations carried in these strains were recessive to wild-type alleles and complemented mutations in all known loci associated with the allantoin pathway. The locus containing the most thoroughly studied mutation (dal81-1) was not fund to be tightly linked to any of the allantoin pathway structural genes. The low basal levels of allantoin pathway enzymes observed in Dal81- strains remained the same whether or not the inducer was present in the growth medium. However, the levels of enzyme increased moderately when mutants were grown on poor nitrogen sources. From these observations, we conclude that dal81 mutant strains possess a defect in the induction of enzyme synthesis; enzyme production due to relief of nitrogen catabolite repression, however, appears normal. The observed epistatic relationships of mutations in the DAL80 and DAL81 loci suggest that their products may possess a reasonable degree of functional independence.     

Synergism of closely adjacent estrogen-responsive elements increases their regulatory potential

Recent gene transfer experiments have shown that an estrogen-responsive DNA element (ERE) GGTCANNNTGACC mediates the estrogen inducibility of the Xenopus laevis vitellogenin A1 and A2 genes as well as the chicken vitellogenin II gene. We report here on experiments that explain the estrogen regulation of the Xenopus vitellogenin B1 and B2 genes. In these genes, two ERE homologues, which have only low, if any, regulatory capacity on their own, act synergistically to achieve high estrogen inducibility. Furthermore we show that synergism of EREs is most efficient, when the two elements are closely adjacent and that it is lost when the synergistic elements are separated by 125 basepairs. In-vitro estrogen receptor binding experiments indicate that co-operative binding of estrogen receptors to closely adjacent EREs is not essential for synergism of ERE homologues that have no intrinsic regulatory capacity. Functional synergism of EREs is observed in the human estrogen-responsive MCF-7 cell line as well as in mouse fibroblasts (Ltk-) cotransfected with estrogen receptor expression vectors. Even expression of a truncated receptor protein lacking 178 amino acid residues of the amino-terminal end allows synergism, suggesting that the amino-terminal end preceding the DNA-binding domain of the estrogen receptor is not required.