Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1989-04-24
1993-07-06
Yarbrough, Amelia Burgess
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 29, 435 691, 435 701, 435 703, 4352401, 4352402, 43524023, 4351723, 435284, 514 44, 536 235, 935 9, 935 11, 935 13, 935 34, 935 70, 935 71, 935 78, 935 88, C12Q 168
Patent
active
052253236
DESCRIPTION:
DESCRIPTION
cells and L-D1 and L-D2, but not against those proteins encoded by transfected human genes. Immunoglobulins will be separated from total serum by ammonium sulfate precicpitation at 50% saturation. Resuspended immunoglobulins will be used to precipitate common antigens from solubilized cell lysates and plasma membrane preparations. Proteins remaining in the supernatant after several rounds of precipitation will be assayed for their capacity to bind [H.sup.3 ]-dopamine and analyzed by two dimensional gel electrophoresis using the method of O'Farrell (O'Farrell (1975) J. Biol. hem. 250:4007).
For two dimensional electrophoresis, cells will be transferred to a minimum essential methionine-culture medium and incubated with 10-50 .mu.Ci of S.sup.35 -methionine per ml of media for 20-24 hours. Cells will be harvested and suspended in SDS solubilization buffer (Anderson, Anderson & Tollaksen, Argonne Nat'l Lab., Publication 79-2) (0.05M CHES pH 9.5, 2% SDS, 10% glycerol, 2% 2-mercaptoethanol). Samples will be centrifuged at 100,00.times.g to remove insoluble material and supernatant will be treated with antiserum described above. Proteins remaining in the supernatant after treatment with antiserum will be concentrated so that approximately 40 .mu.g containing 100,000 cpm in 25 .mu.l will be loaded in the first dimension. Upon completion of electrophoresis in the second dimension, gels will be fixed, patterns will be evaluated visually from staining and from autoradiographs. Peptides present in lysates or membranes from putative transgenic clones but not in preparations from control cells will be used as antigens for further specific antibody production.
It is evident from the above results that novel methods and compositions are provided for studying neurotransmitter transport in cell culture. Thus, an important new tool is provided for dissecting the mechanism of neurotransmitter transport, while also allowing for rapid screening of a large number of compounds for their ability to bind to the receptor and be transported intracellularly or compete with compounds bound by the receptor.
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
1. An in-vitro cell culture consisting essentially of transgenic mammalian cells comprising: transporter, said transporter being characterized as capable of transporting said neurotransmitter into said cells.
2. The cell culture according to claim 1, wherein said cells are of the genus rodentiae.
3. The cell culture according to claim 1, wherein said cells are other than neuronal cells.
4. The cell culture according to claim 1, wherein said neurotransmitter is 5-hydroxytryptamine, dopamine or glycine.
5. An in-vitro composition consisting essentially of: neurotransmitter transporter, wherein uptake of a neurotransmitter into said cells via said transporter is characterized as sodium ion dependent, saturable and temperature sensitive with the proviso that when said primate cells are human cells, said DNA is heterologous to said cells.
6. The composition according to claim 5, wherein said cells are further characterized as capable of being grown in vitro.
7. The composition according to claim 5, wherein said uptake is further characterized as being at least substantially inhibited by known agonists or antagonists of said neurotransmitter.
8. The composition according to claim 5, wherein said uptake is further characterized as being of high affinity.
9. The composition according to claim 6, wherein said cells are mouse fibrobla
REFERENCES:
patent: 4675285 (1987-01-01), Clark et al.
patent: 4985352 (1991-01-01), Julius et al.
Lowe et al. (1988) Journal of Cell Biology, vol. 106, pp. 51-59.
Yorek et al. (1987) Journal of Biological Chemistry, vol. 262, No. 23, pp. 10986-10993.
Blin et al.; "A General Method for Isolation of High Molecular Weight DNA from Eukaryotes"; Nucleic Acids Research; vol. 3, No. 9, Sep. 1976, pp. 2303-2308.
Claudio et al; "Genetic Reconstitution of Functional Acteylcholine Receptor Channels in Mouse Fibroblasts"; Science, vol. 238, Dec. 1987, pp. 1688-1694.
Fargin et al; "The Genomic Clone G-21 Which Resembles a Betz-Adrenergic Receptor Sequence Enclodes the 5-Ht Receptor"; Nature; vol. 335, Sep. 1988, pp. 358-360.
Frnka et al; "Pharmacological Characteristics of High-Affinity Serotonin Uptake Systems Established Through Gene Transfer"; The Hournal of Pharmacology and Experimental Therapeutics; vol. 256, No. 2, pp. 734-740.
Julius et al; "Molecular Characterization of a Functional cDNA Encoding the Serotonin Ic Receptor"; Science, vol. 241, pp. 558-564.
Old, R. W., "Principles of Gene Manipulation" (1985) 2, pp. 231-254, Blackwell Scientific Publications, London.
Yorek et al., "Synthesis and High Affinity Uptake of Serotonin and Dopamine by Human Y79 Retinoblastoma Cells" (Dec. 1, 1987) Biological Abstracts, 84(11) Abstract No. 124514.
Allen et al., "Isoproterenol response following transfection of the mouse .beta..sub.2 -adrenergic receptor gene into Y1 cells", EMBO Journal (1988) 7:133-138.
Asano et al., "Rabbit Brain Glucose Transporter Responds to Insulin When Expressed in Insulin-sensitive Chinese Hamster Ovary Cells", The Journal of Biological Chemistry (1989) 264:3416-3420.
Blakely, et al., "Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes", Proc. Natl. Acad. Sci. USA (1988) 85:9846-9850.
Burch and McBride, "Human Gene Expression in Rodent Cells after Uptake of Isolated Metaphase Chromosomes", Proc. Nat. Acad. Sci. USA (1975) 72:1797-1801.
Burk et al., "cDNA Cloning, Functional Expression, and mRNA Tissue Distribution of a Third Organellar Ca.sup.2+ Pump", The Journal of Biological Chemistry (1989) 264:18561-18568.
Change et al., "Characterization of a genetically reconstituted high-affinity system for serotonin transport", Proc. Natl. Acad. Sci. USA (1989) 86:9611-9615.
Fraser et al., "Continuous High Density Expression of Human .beta..sub.2 -Adrenergic Receptors in a Mouse Cell Line Previously Lacking .beta.-Receptors", The Journal of Biological Chemistry (1987) 262:14843-14846.
Hara et al., "Expression of sodium pump activities in BALB/c3T3 cells transfected with cDNA encoding .alpha..sub.3 -subunits of rat brain Na.sup.+, K.sup.+ -ATPase", FEBS (1988) 238:27-30.
Jones et al., "Electrophysiological characterization of cloned ml muscarinic receptors expressed in A9 L cells", Proc. Natl. Acad. Sci. USA (1988) 85:4056-4060.
Sardet et al., "Molecular Cloning, Primary Structure, and Expression of the Human Growth Factor-Activatable Na.sup.+ /H.sup.+ Antiporter", Cell (1989) 56:271-280.
Sarthy, ".gamma.-
This invention was made with government support under NIH Grant No. EY02423 awarded by the National Institutes of Health through the National Eye Institute. The government has certain rights in the invention.
This application is a continuation-in-part application of U.S. application Ser. No. 274,328 which was filed Nov. 21, 1988, and now U.S. Pat. No. 5,188,954.
Baylor College of Medicine
Burgess Yarbrough Amelia
Marschel Ardin H.
LandOfFree
Human high-affinity neurotransmitter uptake system does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Human high-affinity neurotransmitter uptake system, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Human high-affinity neurotransmitter uptake system will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1688956