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Cloning Techniques (cloning + techniques)
Selected AbstractsDiversity of bacteriorhodopsins in different hypersaline waters from a single Spanish salternENVIRONMENTAL MICROBIOLOGY, Issue 11 2003R. Thane Papke Summary Haloarchaeal rhodopsins are a diverse group of transmembrane proteins that use light energy to drive several different cellular processes. Two rhodopsins, bacteriorhodopsin and halorhodopsins, are H+ and Cl, ion pumps, respectively, and two rhodopsins, sensory rhodopsin I and II, regulate phototaxis. Bacteriorhodopsin is of special interest as it is a non-chlorophyll-based type of phototrophy (i.e. generation of chemical energy from light energy). However, very little is known about the diversity and distribution of rhodopsin genes in hypersaline environments. Here, we have used environmental PCR and cloning techniques to directly retrieve rhodopsin genes from three different salinity ponds located in a sea salt manufacturing facility near Alicante, Spain. Our survey resulted in the discovery of previously concealed variation including what is hypothesized to be bacteriorhodopsin genes from the uncultivated square morphotype that dominates these environments. In some instances, identical genes were discovered in seemingly different habitats suggesting that some haloarchaea are present over widely varying concentrations of salt. [source] Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyiFEBS JOURNAL, Issue 10 2000Rosario Maida Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K., Krieger, J. & Breer, H. (1989) FEBS Lett.256, 2215,2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta1088, 277,284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6,11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively. [source] Companion Animal Medicine in the Age of Medical GeneticsJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2000Donald F. Patterson We'd like to explain what pathology means, In terms of what's wrong with the structure of genes; Know if a control or a structural locus Constitutes the exact pathological focus. With the help of the enzymes that slice DNA, And cloning techniques, we now have a way To study the actual sequence of bases; To know when those purines are not in their places.1 [source] Comparison of chorionic gonadotropin expression in human and macaque (Macaca fascicularis) trophoblastsAMERICAN JOURNAL OF PRIMATOLOGY, Issue 2 2002Jason A. Wilken Abstract We have designed novel DNA primers that allow us to detect the expression of the subunits of chorionic gonadotropin (CG) from a variety of species of the order Primates. Using these primers, reverse transcriptase-polymerase chain reaction (RT-PCR), and standard cloning techniques, we detected the expression of a single gene for the common glycoprotein hormone (GPH) ,-subunit and at least two genes for the CG ,-subunit in trophoblasts of Macaca fascicularis (cynomolgous macaque (cm)) at gestational day (GD) = 26 (± 2d). No cmCG expression was detected at GD = 35,40. When sequences of cmGPH-, and cmCG-, genes were compared to the corresponding genes of other primates, we found that the ,-subunit of M. fascicularis was highly conserved compared to other primate species. However, cmCG ,-subunits appeared to be less conserved, residing between those of human CG-, and baboon CG-, when analyzed phylogenetically. Of particular interest was a three amino acid stretch in one of the expressed cmCG-, genes that is distinct from all other primates studied. Our findings imply that not only does the expression of multiple CG ,-subunit genes appear to be common to Old World monkeys, but that the presented methodology will greatly facilitate our ability to understand primate evolution. Am. J. Primatol. 56:89,97, 2002. © 2002 Wiley-Liss, Inc. [source] Patent Policy for Human Embryonic Stem Cell Research in TaiwanTHE JOURNAL OF WORLD INTELLECTUAL PROPERTY, Issue 4 2010Jerry I.-H. The potential of human embryonic stem cell (ESC) research could prove to provide immense therapeutic value for illnesses not curable under currently existing therapies. However, human ESC research is controversial as it touches the fundamental value of human life. Taiwan has been aiming to become the biotech hub of Asia-Pacific and is becoming a major player in human ESC research. Whether or not the research results from human ESC are patentable could have a profound impact on the progress in this field. In this article, the science of human ESC research is clarified and tested against the existing murky Taiwan patent standards. In particular, this article distinguishes between therapeutic cloning and reproductive cloning techniques, asks questions about the patentability of totipotent human ESCs and explores the meaning of the word embryo. This article draws comparison with the European practice on ethical standards and concludes that patenting human ESC research might not be so controversial, but Taiwan has to make its patent law clearer in this field to fulfill the country's intended goal. [source] cDNA cloning and expression analysis of the myosin heavy chain (MYH) gene of the mandarin fish Siniperca kneriAQUACULTURE RESEARCH, Issue 4 2009Jianshe Zhang Abstract In this study, we applied RT-PCR and cDNA cloning techniques to clone myosin heavy chain (MYH) cDNA from muscle tissues of the mandarin fish Siniperca kneri. The cDNA was determined to be of 6987 base pairs in length, encoding a peptide of 1937 amino acids (Genbank accession no. EF446616). A search of encoded protein sequences in the NCBI conserved domain database indicated the presence of all known protein domains for MYH proteins, i.e. the myosin motor domain in the N-terminal region, the DIL domain at the C-terminus, and the ATPase domain. The MYH gene and its protein were expressed predominantly in muscle tissues and weakly in cardiac tissues. Developmentally, the MYH gene was first expressed in the muscle formation stage and continued later on. Our work provided a novel mypsin heavy chain gene sequence in fish biology and the results indicate that the MYH gene and the protein it encodes are important for the growth and development of the mandarin fish, as well as its muscle characterization. [source] | ||||||||||