Human kinesin light (beta) chain gene: DNA sequence and functional characterization of its promoter and first exon.
Kinesins are tubulin molecular motors whose function is to transport organelles within cells. Very little is known about the regulation of expression of these proteins. We have characterized the gene product of one differentially spliced mRNA of the human light chain kinesin and cloned its promoter region. A full-length kinesin cDNA was translated in vitro in a cell-free system, producing a 70-kDa protein. Using this cDNA as a probe, we isolated and sequenced the promoter, first exon, and part of the first intron of this gene from a genomic lambda EMBL3 human placental DNA library. The whole gene spans more than 90 kb. The beta kinesin promoter region confers only constitutive transcription to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In permanently transfected human HeLa and NB100 neuroblastoma cells, a reporter gene containing the promoter and part of the first exon of beta kinesin was 75-fold more active than the HSV-tk promoter. The first exon contains the 5'-untranslated sequence capable of forming a stable double-hairpin loop, which functions as a translational enhancer. Its deletion decreases the efficiency of in vitro translation of beta kinesin mRNA and confers increased translation to a CAT reporter gene.
Chernajovsky
Y
Y
Kennedy Institute of Rheumatology, Molecular Biology Laboratory, Hammersmith, London, UK.
Brown
A
A
Clark
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D004285:Dogs; D005091:Exons; D005260:Female; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000096:biosynthesis* / Q000235:genetics*; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010179:Pancreas / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D046911:Macromolecular Substances; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D016547:Kinesins