Everybody's got something to hide except me and my patented monkey: patentability of cloned organisms.
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Animals↗
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Results for “Cloning, Organism”
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[Basic studies on mitochondria--cloned organisms and gene therapy].
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Adenosine Triphosphate↗
Uptake of the mycotoxin ochratoxin A in liver cells occurs via the cloned organic anion transporting polypeptide.
Ochratoxin A (OTA) is a mycotoxin produced by mold. It mainly causes nephropathies in humans and domestic animals as a major pathogenic contaminant of cereals and animal feed. Upon p.o. uptake and intestinal absorption, a large part of OTA is taken up by hepatocytes and eliminated into bile. In the present study, hepatocellular uptake of radiolabeled [3H]OTA in isolated rat hepatocytes was characterized; a saturable (K(m) = 18.9 microM, Vmax = 473 pmol/mg/min), temperature (Aapp = 30.4 and 76.6 kilo Joule/mol) and energy-dependent mycotoxin transport was found. This OTA uptake was inhibited by various bile acids, sulfobromophthalein and the thrombin inhibitor CRC 220. Because all inhibitors are substrates of the organic anion-transporting polypeptide (oatp), a recently cloned hepatic carrier, uptake experiments were performed in oatp-cRNA-injected Xenopus laevis oocytes. These studies revealed an oatp-specific OTA uptake (K(m) = 16.6 microM). In contrast, OTA was not transported by the hepatic Na+/taurocholate-cotransporting polypeptide. Known oatp substrates cis-inhibited OTA uptake in oatp-cRNA-injected oocytes in close correlation with the results derived from isolated hepatocytes. These results identify OTA as a new substrate for oatp. They further support the multispecific nature of oatp-mediated transport and stress the importance of this carrier for hepatic clearance of xenobiotics.
Animals↗
Molecular cloning of alpha-amylase genes from Drosophila melanogaster. II. Clone organization and verification.
Restriction maps of an alpha-amylase structural gene clone, lambda Dm65, and of four putative alpha-amylase pseudogene clones are presented. Two alpha-amylase structural genes, inverted with respect to each other, are contained in lambda Dm65. Subregions of internal DNA sequence homology within lambda Dm65 and of cross-homology between the presumptive pseudogene clones and lambda Dm65 were determined. Subregions of cross-homology between the Drosophila clones and the mouse alpha-amylase cDNA clone, pMSa104, were also determined. The presence of functional alpha-amylase structural genes in lambda Dm65 was verified by injection of appropriate subclones into the germinal vesicle of Xenopus oocytes, followed by incubation of the oocytes under conditions that allowed coupled transcription and translation of injected genes to occur. Subclones of the 3.8- and 5.6-kb EcoRI fragments of lambda Dm65 were shown to code for alpha-amylase isozymes 1 and 3, respectively, of Drosophila melanogaster Canton-S. Both subclones are homologous to RNA of a size sufficient to accommodate the alpha-amylase-coding information. No RNA species homologous to other subcloned EcoRI fragments of lambda Dm65 was detected.
Animals↗
Rat multidrug resistance protein 4 (Mrp4, Abcc4): molecular cloning, organ distribution, postnatal renal expression, and chemical inducibility.
In the present study, we report cloning of the rat Mrp4 cDNA. The cDNA is 4526 bp, containing a 3975 bp open reading frame. The deduced polypeptide has 1325 amino acids and is 83% and 91% identical to human MRP4 and mouse Mrp4, respectively. Phylogenetic analysis revealed that the cloned rat cDNA is closely related to human MRP4 and mouse Mrp4. Additionally, an alternatively spliced variant, 111 bp shorter than the full-length form, was cloned. Rat Mrp4 mRNA was detectable in 11 tissues examined, with levels being highest in kidney, and lowest in liver. Mrp4 mRNA levels in kidney were higher in males than females, and at birth were about half of adult levels. Mrp4 expression in liver and kidney of rats treated with six classes of microsomal enzyme inducers was examined. Mrp4 mRNA in liver was induced by two electrophile response element activators, namely ethoxyquin and oltipraz.
Alternative Splicing↗
Membrane localization and pH-dependent transport of a newly cloned organic cation transporter (PMAT) in kidney cells.
Plasma membrane monoamine transporter (PMAT) is a novel membrane transporter recently cloned and characterized in our laboratory. We previously demonstrated that PMAT functions as a polyspecific organic cation transporter and efficiently transports many organic cations such as monoamine neurotransmitters and 1-methyl-4-phenylpyridinium (MPP(+)). In this study, we explored the role of PMAT in the renal handling of organic cations. Using a polyclonal antibody generated toward the NH(2)-terminal 66 amino acid residues of human PMAT, we showed that the PMAT protein (approximately 55 kDa) is expressed in the human kidney and is primarily targeted to the apical membranes when expressed in polarized Madin-Darby canine kidney (MDCK) cells. Using MDCK cells stably expressing human PMAT, we showed that PMAT-mediated MPP(+) uptake is strongly dependent on extracellular pH. Lowering extracellular pH from 7.4 to 6.6 greatly stimulated PMAT-mediated MPP(+) uptake, whereas elevating extracellular pH to 8.2 abolished transporter activity. Kinetic analysis revealed that the apparent V(max) at pH 6.6 is about fourfold higher than that at pH 7.4, whereas the apparent K(m) values were not statistically different at these two conditions. Under acidic conditions (pH 6.6), the proton ionophore, carbonyl cyanide p-trifluormethoxyphenylhydrazone, drastically reduced PMAT-mediated MPP(+) uptake, suggesting that the stimulatory effect of proton may be due to transporter coupling with a proton gradient. Taken together, our data suggest that PMAT is expressed on the apical membranes of renal epithelial cells and may use luminal proton gradient to drive organic cation reabsorption in the kidney.
1-Methyl-4-phenylpyridinium↗
Molecular cloning, organization and localization of the gene for the mouse neuropeptide Y-Y5 receptor.
A cDNA clone homologous with the human neuropeptide Y (NPY)-Y5 receptor has been isolated from a mouse brain cDNA library. Analysis of the predicted amino acid sequence indicates that the polypeptide encoded by this cDNA is 89% and 97% identical to the human and rat NPY-Y5 receptors, respectively, but has a repeat sequence of 21-amino acid residues, unlike the human and rat NPY-Y5 receptors, in its N-terminal region. Northern blot analysis indicated that the mouse NPY-Y5 receptor mRNA was highly expressed in the brain, but not in the heart, kidney, spleen, lung, liver, skeletal muscle, and testis. A genomic DNA clone encoding the mouse NPY-Y5 receptor has also been cloned and used as a probe for fluorescence in situ hybridization analysis. The mouse NPY-Y5 receptor gene consists of at least two exons with a 7.0 kbp-long intronic sequence and is localized in the chromosome 8B3-C2 region. Restriction mapping, partial sequencing, and hybridization analysis of the mouse NPY-Y5 and NPY-Y1 receptor genes were used to determine the relationship between the two genes in the chromosome 8B3-C2 region. The mouse NPY-Y5 receptor gene can map about 10 kbp upstream of the transcriptional initiation sites of the NPY-Y1 receptor gene with an opposite orientation.
Amino Acid Sequence↗
Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.
The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.
Amino Acid Sequence↗
Cloning, organization, and expression of the bioluminescence genes of Xenorhabdus luminescens.
The lux genes of Xenorhabdus luminescens, a symbiont of the nematode Heterorhabditis bacteriophora, were cloned and expressed in Escherichia coli. The expression of these genes in E. coli was qualitatively similar to their expression in X. luminescens. The organization of the genes is similar to that found in the marine luminous bacteria. Hybridization studies with the DNA that codes for the two subunits of luciferase revealed considerable homology among all of the strains of X. luminescens and with the DNA of other species of luminous bacteria, but none with the nonluminous Xenorhabdus species. Gross DNA alterations such as insertions, deletions, or inversions do not appear to be involved in the generation of dim variants known as secondary forms.
Cloning, Molecular↗
[Cloning, organization and expression of the genes for eukaryotic ribosomal proteins].
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Animals↗
Three concepts of cloning in human beings.
Human cloning, organ cloning and tissue cloning are various types of cloning that occur at different levels with different methodologies. According to three standards of terminology for an embryo (fertilization through germ cells, development in the uterus and having the potential to produce a human life), tissue cloning and type I organ cloning will not produce an embryo. In contrast, human cloning and type II organ cloning will produce an embryo. Thus, only non-germinal tissue cloning and type I organ cloning are beyond the ethical question and will not change human beings as a species. Using cloned tissues to make new tissues or organs is promising for the future of medicine.
Cloning, Organism↗
Mechanism of electrogenic cation transport by the cloned organic cation transporter 2 from rat.
The organic cation transporter 2 (OCT2) is expressed in plasma membranes of kidney and brain. Its transport mechanism and substrates are debated. We studied substrate-induced changes of electrical current with the patch clamp technique after expression of rat OCT2 in oocytes. Activation of current, corresponding to efflux, was observed for small organic cations, e.g. choline. In contrast, the bigger cations quinine and tetrabutylammonium elicited no change in current. However, transport of choline could be inhibited by applying quinine or tetrabutylammonium to the cytoplasmic side. Inhibition of organic cation efflux by quinine was competitive with substrates. Quinine at the inside also inhibited substrate influx from the outside. Current-voltage analysis showed that both maximal turnover and apparent affinity to substrates are voltage-dependent. Substrate-induced currents with organic cations on both membrane sides reversed as predicted from the Nernst potential. Our results clearly identify the electrochemical potential as driving force for transport at neutral pH and exclude an electroneutral H(+)/organic cation(+) exchange. We suggest the existence of an electroneutral organic cation(+) exchange and propose a model for a carrier-type transport mechanism.
Animals↗
Cloning, organization and functional analysis of ilvA, ilvB and ilvC genes from Corynebacterium glutamicum.
Corynebacterium glutamicum is an industrially important bacterium for the manufacture of amino acids. We constructed genomic libraries of this Gram+ bacterium and screened for clones carrying isoleucine biosynthesis genes (ilv) by complementation of Escherichia coli mutants. Clones complementing ilvA, ilvB, and ilvC were isolated. As based on the functional analysis of the corresponding plasmids in C. glutamicum, the DNA fragments isolated encode threonine dehydratase, acetohydroxy acid synthase, and isomeroreductase, catalyzing three subsequent reactions in Ile synthesis. Subcloning and transposon mutagenesis revealed that ilvB and ilvC reside on a 7-kb chromosomal fragment and that these genes are transcribed in the same direction. A shuttle vector was constructed to allow exonuclease treatment and assay subsets of plasmids for gene expression in the original C. glutamicum background. These constructs and their enzyme activity determinations revealed that despite close linkage ilvC is expressed independently from ilvB. Using Southern blots, a 15-kb fragment of chromosomal DNA carrying the ilvBC cluster was characterized. This fragment does not contain ilvA, demonstrating the entirely different organization of the isoleucine biosynthesis genes in C. glutamicum from that in enterobacteria.
Acetolactate Synthase↗
Microarray Assistant clone organizer and array simulator.
Microarrays are extensively used in molecular biology experiments. While several vendors offer microarrays on a variety of platforms, many researchers prefer to use custom microarrays with a selected list of clones for their experiments. Many research centers have established core facilities for the production of custom microarrays. Microarray production involves a number of steps, including maintaining a master list of stock clones, selecting required clones for custom microarrays, subculturing selected clones, amplifying inserts, recording results, and identifying the orientation of clones in the microarray. We have created a simple, user-friendly, and versatile Microsoft Excel spreadsheet-based software, Microarray Assistant, which can assist the user in all the steps of microarray design and synthesis. In addition, the program gives options to insert, delete, or interchange clones during various steps. The program also gives a visual picture of the locations of the clones in the plates, as well as in the microarray. The program can also be used to assist in the transfer of clones between plates of different configuration.
Algorithms↗
BFIT, a unique acyl-CoA thioesterase induced in thermogenic brown adipose tissue: cloning, organization of the human gene and assessment of a potential link to obesity.
We hypothesized that certain proteins encoded by temperature-responsive genes in brown adipose tissue (BAT) contribute to the remarkable metabolic shifts observed in this tissue, thus prompting a differential mRNA expression analysis to identify candidates involved in this process in mouse BAT. An mRNA species corresponding to a novel partial-length gene was found to be induced 2-3-fold above the control following cold exposure (4 degrees C), and repressed approximately 70% by warm acclimation (33 degrees C, 3 weeks) compared with controls (22 degrees C). The gene displayed robust BAT expression (i.e. approximately 7-100-fold higher than other tissues in controls). The full-length murine gene encodes a 594 amino acid ( approximately 67 kDa) open reading frame with significant homology to the human hypothetical acyl-CoA thioesterase KIAA0707. Based on cold-inducibility of the gene and the presence of two acyl-CoA thioesterase domains, we termed the protein brown-fat-inducible thioesterase (BFIT). Subsequent analyses and cloning efforts revealed the presence of a novel splice variant in humans (termed hBFIT2), encoding the orthologue to the murine BAT gene. BFIT was mapped to syntenic regions of chromosomes 1 (human) and 4 (mouse) associated with body fatness and diet-induced obesity, potentially linking a deficit of BFIT activity with exacerbation of these traits. Consistent with this notion, BFIT mRNA was significantly higher ( approximately 1.6-2-fold) in the BAT of obesity-resistant compared with obesity-prone mice fed a high-fat diet, and was 2.5-fold higher in controls compared with ob/ob mice. Its strong, cold-inducible BAT expression in mice suggests that BFIT supports the transition of this tissue towards increased metabolic activity, probably through alteration of intracellular fatty acyl-CoA concentration.
Adipose Tissue↗
Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.
We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .
4-Butyrolactone↗
A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization.
Using the bacteriophage P1 cloning system, we have constructed a two to three times coverage, high-molecular-weight (HMW) genomic library from mouse C127 fibroblast cells. The library consists of about 127,500 clones with an average insert size of about 70 kb that are organized into 300 primary pools containing approximately 425 clones per pool. For screening purposes the primary pools are combined into secondary pools (approximately 4250 clones each) and tertiary pools (approximately 21,250 clones each). Screening is performed by the polymerase chain reaction (PCR) with DNA isolated from the secondary and tertiary pools. We have screened the library for 13 different mouse sequences and have detected 11. Clones generated from two of the eleven positive screens were isolated from the library (those containing the c-fos and G alpha i2 genes) and were further characterized. Direct double-stranded sequencing of DNA from P1 clones with primers bordering the insert provided sequence information from each end of the cloned DNA.
Animals↗
The serotonin 5-HT2B receptor from the puffer fish Tetraodon fluviatilis: cDNA cloning, genomic organization and alternatively spliced variants.
We cloned the 5-HT2B serotonin receptor from the puffer fish Tetraodon fluviatilis. Two cDNAs differing in length because of the use of alternative polyadenylation sites were isolated. We partly characterized the genomic organization of the 5-HT2B gene and we found two introns conserved in position between the puffer fish and mammals. In addition, four splice variants which would generate truncated forms of the receptor were detected.
Alternative Splicing↗