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MAGIC, an in vivo genetic method for the rapid construction of recombinant DNA molecules: Nature Genetics, Vol. 37, No. 3. (30 January 2005), pp. 311-319.Mamie Li, Stephen Elledge

Source: Nature Genetics, Vol. 37, No. 3. (30 January 2005), pp. 311-319.
PCR-suppressio n effect: Kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples: Journal of Biotechnology, Vol. In Press, Corrected ProofIn the present study, we analyzed the kinetics of the PCR-suppressio n effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10 kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switc hing full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcrip t enriched cDNA library construction, especially when only a small amount of total RNA is available.Zhon g-Min Dai, Xiao-Jing Zhu, Qi Chen, Wei-Jun Yang

Source: Journal of Biotechnology, Vol. In Press, Corrected Proof
Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC: Nature Methods, Vol. 4, No. 3. (11 February 2007), pp. 251-256.Mamie Li, Stephen Elledge

Source: Nature Methods, Vol. 4, No. 3. (11 February 2007), pp. 251-256.
Generation and Characterizati on of SCARs by Cloning and Sequencing of RAPD Products: A Strategy for Species-specif ic Marker Development in Bamboo: Annals of Botany, Vol. 95, No. 5. (16 April 2005), pp. 835-841.MALAY Malay, Bhattacharya Samik, AMITA Amita

Source: Annals of Botany, Vol. 95, No. 5. (16 April 2005), pp. 835-841.
Structure of a cannabinoid receptor and functional expression of the cloned cDNA.: Nature, Vol. 346, No. 6284. (9 August 1990), pp. 561-564.Mariju ana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-in duced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecificall y disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-couple d receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent , stereoselectiv e and pertussis toxin-sensitiv e manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoacti ve cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-couple d receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-in duced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.LA Matsuda, SJ Lolait, MJ Brownstein, AC Young, TI Bonner

Source: Nature, Vol. 346, No. 6284. (9 August 1990), pp. 561-564.
Human cloning action stalled: Current Biology, Vol. 13, No. 24. (16 December 2003), pp. 937-939.M Gross

Source: Current Biology, Vol. 13, No. 24. (16 December 2003), pp. 937-939.
The gene controlling marijuana psychoactivity : molecular cloning and heterologous expression of Delta1-tetrahy drocannabinoli c acid synthase from Cannabis sativa L.: The Journal of biological chemistry, Vol. 279, No. 38. (17 September 2004), pp. 39767-39774.De lta(1)-tetrahy drocannabinoli c acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of Delta(1)-tetra hydrocannabino l. We cloned a novel cDNA (GenBank trade mark accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotid e open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-in sect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristic s of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterizati on of an enzyme specific to cannabinoid biosynthesis.S Sirikantaramas , S Morimoto, Y Shoyama, Y Ishikawa, Y Wada, Y Shoyama, F Taura

Source: The Journal of biological chemistry, Vol. 279, No. 38. (17 September 2004), pp. 39767-39774.
Cloning and Characterizati on of a Gene Encoding the Major Surface Protein of the Bacterial Endosymbiont Wolbachia pipientis: J. Bacteriol., Vol. 180, No. 9. (1 May 1998), pp. 2373-2378.The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibilit y, parthenogenesi s, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterizati on of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibilit y phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.Henk Braig, Weiguo Zhou, Stephen Dobson, Scott O'Neill

Source: J. Bacteriol., Vol. 180, No. 9. (1 May 1998), pp. 2373-2378.
Identification and characterizati on of a biomineralizat ion related gene PFMG1 highly expressed in the mantle of Pinctada fucata.: Biochemistry, Vol. 46, No. 3. (23 January 2007), pp. 844-851.To elucidate the mechanism of nacre biomineralizat ion, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-bindin g protein CBP-1 and had two EF-hand calcium-bindin g domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallizatio n showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc- HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiatio n, and mineralization , and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralizat ion, and the SSH method can pave the way for the bulk cloning and characterizati on of new genes involved in biomineralizat ion in P. fucata and may accelerate research on the mechanism of pearl formation.HL Liu, SF Liu, YJ Ge, J Liu, XY Wang, LP Xie, RQ Zhang, Z Wang

Source: Biochemistry, Vol. 46, No. 3. (23 January 2007), pp. 844-851.
Environmental determinants of amphibian and reptile species richness in China: Ecography, Vol. 30, No. 4. (August 2007), pp. 471-482.Qian, Hong, Wang, Xihua, Wang, Silong, Li, Yuanliang

Source: Ecography, Vol. 30, No. 4. (August 2007), pp. 471-482.

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