We succeeded in cloning the rhesus monkey nociceptin/orphanin FQ peptide (NOP) receptor. The nucleotide sequence and amino acid sequence of the rhesus monkey NOP receptor were 95.9% and 97.8%, respectively, identical to the human NOP receptor. There was no significant difference between the rhesus monkey NOP receptor and the human NOP receptor in the binding affinity of [125I] [Thy14]nociceptin and the binding of [35S]guanosine 5'-O-(γ thio)triphospate ([35S]GTPγS) stimulated by nociceptin/orphanin FQ (N/OFQ). A selective NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one ((+)-J-113397) inhibited the [35S]GTPγS binding activated by N/OFQ using the membrane of the rhesus monkey NOP receptor. The antagonistic activity of (+)-J-113397 to the rhesus monkey NOP receptor was comparable to that to the human NOP receptor. Thus, N/OFQ acts via activation of the NOP receptor in both human and rhesus monkeys without significant species differences.
Intracisternal A-particle retrotransposons (IAPs) are known, moveable, retrovirus-like elements and are defective in envelope protein synthesis in the mouse genome. Insertion of IAP elements can either interupt or enhance gene function or expression. Using a mouse model called lethal wasting (lew), we recently identified the insertion of an IAP sequence in a gene, 9630033F20Rik, that contains domains involved in glycolysis. The expression pattern of the 9630033F20Rik gene between various normal and diseased tissues was determined by semi-quantitative RT-PCR. The effect of the insertion mutation in 9630033F20Rik on glycolysis in heart, muscle, and brain tissues was further investigated using oligonuleotide microarray analysis. Results indicated that the expression of 9630033F20Rik is ubiquitous and its signal is relatively higher in heart and brain tissues. The insertion caused the deletion of exon 5 and decreased expression of this gene in all the tissues studied in the lew mice. Changes in the expression levels of glycolytic genes mainly occured in muscle tissue, raising a possibility that 9630033F20Rik may function as one of the transcriptional regulators of glycolytic genes in skeletal muscle. However, considering the fact that a single nucleotide mutation in vesicle-associated membrane protein 1 (VAMP1) has been reported as the causal gene for the lew mouse, how much of an impact the IAP insertion in the lew mouse phenotype has on glycolytic genes compared to the effect from the VAMP1 mutation responsible for the lew mouse phenotype should be further investigated.
Activin type II receptor (ActRII) is crucial for the assembly of the ligand/receptor complex and the activation of downstream cascades in activin signaling pathway. In this study, we identified six variants of grass carp ActRIIA which can be generated through three alternative splicing events (defined as AS1, AS2 and AS3). AS1 induces a spliced segment encoding 14 amino acids located in the external juxtamembrane region of the receptor. However, both AS2 and AS3 occur at the same cleavage site of kinase domain and induce a premature termination codon. Indeed, AS2 inserts a fragment of 79 bp while AS3 generates a new 3’ terminal of cDNA with poly(A) signals. The full length cDNA of the shortest variant was shown to be 2001 bp encoding 514 amino acids with sequence identity of 79–95% to counterparts in other species. Homology modeling studies showed grass carp ActRIIA exhibits a characteristic three-finger toxin fold in the extracellular domain and a conserved bilobal architecture in the intracellular kinase domain. Semi-quantitative RT-PCR analysis revealed that six variants showed different expression patterns in selected tissues of grass carp. This study will be helpful for a better understanding of the physiological role of activin signaling in lower vertebrates.
DNA-based transposable elements are present in the genomes of various organisms, and generally occur in autonomous and nonautonomous forms, with a good correspondence to complete and defective copies, respectively. In vertebrates, however, the vast majority of DNA-based elements occur only in the nonautonomous form. Until now, the only clear exception known has been the Tol2 element of the medaka fish, which still causes mutations in genes of the host species. Here, we report another exception: the Tol1 element of the same species. This element was thought likely to be a "dead" element like the vast majority of vertebrate elements, but recent identification of an autonomous Tol1 copy in a laboratory medaka strain gave rise to the possibility that the element is still "alive" in medaka natural populations. We examined variation in the structure of Tol1 copies through genomic Southern blot analysis, and revealed that 10 of the 32 fish samples examined contained full-length Tol1 copies in their genomes. The frequency at which these copies occur among Tol1 copies is at most 0.5%, yet some of them still have the ability to produce a functional transposase. The medaka fish thus harbors two active DNA-based elements in its genome, and is in this respect unique among vertebrates.
Speciation genes are responsible for genetic incompatibilities in hybrids of incipient species and therefore participate in reproductive isolation leading to complete speciation. Hybrid males between Drosophila melanogaster females and D. simulans males die at late larval or prepupal stages due to a failure in chromosome condensation during mitosis. However a mutant male of D. simulans, named Lethal hybrid rescue (Lhr), produces viable hybrid males when crossed to females of D. melanogaster. Recently the Lhr gene has been proposed as corresponding to the CG18468 gene in D. melanogaster. However this identification relied on sequence characteristics more than on a precise mapping and the use of the GAL4/UAS system to drive the transgene in D. melanogaster might have increased the complexity of interaction. Thus here we propose an independent identification of the Lhr gene based on a more precise mapping and transgenic experiments in D. simulans. We have mapped the Lhr gene by using Single Nucleotide Polymorphisms (SNPs) and identified within the candidate region the gene homologous to CG18468 as the Lhr gene as it was previously reported. Transgenic experiments in D. simulans with the native promoter of CG18468 prove that it is the Lhr gene of D. simulans by inducing the lethality of the hybrid males.
In order to analyze mutations induced by gamma irradiation in higher plants, we irradiated rice with gamma rays and screened for mutations expressing phenotypes of glutinous endosperm (wx), chlorophyll b deficiency, endosperm protein deficiency, gibberellin-related dwarfism, and shortened plastochron—in order to clarify types of mutations. Nucleotide sequence analysis showed that the most frequent mutation induced by gamma rays was deletion, particularly small deletion. Of the 24 mutations, 15 were small deletions (1–16 bp), four were large deletions (9.4–129.7 kbp), three were single-base substitutions, and two were inversions. Deletions 100 bp–8 kbp in length were not found, suggesting that gamma irradiation is unlikely to induce deletions of 100 bp to 8 kbp but is more likely to induce deletions between 1 and several ten bp or those of around 10 kbp or more. Based on the results, reverse genetics applications may be possible for gamma irradiation-induced deletions in rice by mismatch cleavage analysis used in Targeting Induced Local Lesions IN Genomes (TILLING) to detect small deletions and base substitutions or by using array comparative genomic hybridization (aCGH) to detect large deletions.
A total of 476 accessions of seven cultivated and 32 wild potato species previously characterized by nuclear DNA (nDNA) and chloroplast DNA (ctDNA) marker analyses were employed to the mitochondrial DNA (mtDNA) marker analysis. Fourteen simple sequence repeat (SSR) markers with mononucleotide repeat regions were developed from the potato mtDNA, although their variability was extremely low. Six mtDNA markers including three developed SSR markers disclosed 40 banding patterns that discriminated 63 different mtDNAs. For the same set of samples, 72 ctDNA banding patterns discriminated 129 different ctDNAs. Consequently, 164 haplotypes were distinguished. The correlation between ctDNA and mtDNA differentiation was positive (r = 0.226), but poor when compared with that between ctDNA and nDNA (r = 0.415), which likely lowered the utility of mtDNA polymorphisms in evaluating relationships among these species. Nevertheless, a finding of a unique mtDNA type in all T-type ctDNA holders (S. tuberosum and S. tarijense) strongly supports S. tarijense functioned as a maternal ancestor of potato.
The PpCCA1a and PpCCA1b genes of the moss Physcomitrella patens are functional homologs of the Arabidopsis thaliana circadian clock genes CCA1/LHY. We made use of disruptant lines for PpCCA1a and/or PpCCA1b to elucidate the physiological significance of these genes in the growth of moss protonemal tissue under alternating day/night cycles. Protonemal cells of the double disruptant line, carrying neither of the two genes, grew faster than those of the wild-type plant (WT) in long days (LD), whereas no difference in the growth rate was detected between them in short days (SD). The double disruptant line also showed day length-dependent phenotypic changes in the PpCCA1b promoter activity: the diurnal profile of bioluminescence from the PCCA1b::LUC+ reporter strain was more significantly affected in LD than in SD. These observations are the first demonstration of a physiological function of the circadian clock in non-angiosperm land plants, and are consistent with recent findings that the clock controls hypocotyl elongation of A. thaliana in a photoperiod-dependent manner.
In recent years, interest has been growing in the study of complex networks. Since Erdös and Rényi (1960) proposed their random graph model about 50 years ago, many researchers have investigated and shaped this field. Many indicators have been proposed to assess the global features of networks. Recently, an active research area has developed in studying local features named motifs as the building blocks of networks. Unfortunately, network motif discovery is a computationally hard problem and finding rather large motifs (larger than 8 nodes) by means of current algorithms is impractical as it demands too much computational effort. In this paper, we present a new algorithm (MODA) that incorporates techniques such as a pattern growth approach for extracting larger motifs efficiently. We have tested our algorithm and found it able to identify larger motifs with more than 8 nodes more efficiently than most of the current state-of-the-art motif discovery algorithms. While most of the algorithms rely on induced subgraphs as motifs of the networks, MODA is able to extract both induced and non-induced subgraphs simultaneously. The MODA source code is freely available at: http://LBB.ut.ac.ir/Download/LBBsoft/MODA/