Structural organization of the human vesicular monoamine transporter type-2 gene and promoter analysis using the jelly fish green fluorescent protein as a reporter.
The genomic structure of a human vesicle monoamine transporter, type-2 (hVMAT2) was determined from two overlapping cosmids, phVMAT2-cos1 and phVMT2-cos2, spanning more than 35 kb. The hVMAT2 open reading frame is encoded by 16 exons, with translation initiation and termination in exon 2 and exon 16, respectively. Several potential binding sites for transcriptional regulatory factors, including a cAMP response element (CRE) were identified in the 5'-upstream region of the gene. A promoter construct using the jellyfish green fluorescent protein (GFP) as reporter has been made and transfected into the human neuroblastoma cell line, SHSY-5Y. The cellular expression of the GFP was readily detected by fluorescence microscopy and cells expressing GFP could be sorted using a fluorescence-activated cell sorter (FACS), allowing the level of GFP expression in transfected SHSY-5Y cells to be quickly and reliably determined.
Xu
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W
Department of Neurobiology, Babraham Institute, Cambridge, UK. w.xu@ucl.ac.uk
Liu
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Mooslehner
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Emson
P C
PC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003360:Cosmids; D000242:Cyclic AMP / Q000378:metabolism; D005091:Exons; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D007438:Introns; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009479:Neuropeptides; D018377:Neurotransmitter Agents / Q000378:metabolism*; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D037401:Scyphozoa; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins
D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009479:Neuropeptides; D018377:Neurotransmitter Agents; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050492:Vesicular Biogenic Amine Transport Proteins; D050493:Vesicular Monoamine Transport Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP