Characterization of the rat hippocalcin gene: the 5' flanking region directs expression to the hippocampus.
Hippocalcin is an EF-hand [Persechini A. et al. (1989) Trends Neurosci. 12, 462-467] Ca2+ binding protein encoded by a neuron-specific gene. A detailed atlas of hippocalcin messenger RNA expression in the adult rat brain was complied using in situ hybridization. Highest levels of messenger RNA are found in the hippocampus, where messenger RNA is localized in proximal dendrites of CA pyramidal cells. Expression is also seen in other brain regions, including the neocortex, caudate-putamen, taenia tecti, claustrum, olfactory tubercle, anterior olfactory nucleus, and granule cell and glomerular layers of the olfactory bulb. The rat hippocalcin gene spans approximately 9 kb and consists of three exons, separated by introns of 6.7 and 0.25 kb. Sequence analysis of the putative proximal promoter region identified two clusters of multiple E-box sites which may regulate the cell-specific expression. Two lacZ fusion constructs carrying 0.9 and 3.4 kb of rat hippocalcin gene upstream region were used to create transgenic mice. With the 3.4 kb construct, transgene expression varied between founder mice, but was always found in the dentate gyrus and CA1-CA4 regions of the hippocampus, thus partly mimicking the expression of the endogenous gene. For the 0.9 kb construct, the levels of lacZ expression were weaker and more variable. Neither construct showed expression in any peripheral tissues examined. To establish an in vitro model of transcriptional regulation, the 3.4 and 0.9 kb 5' upstream regions were fused to a promoterless reporter gene encoding chloramphenicol acetyltransferase and transiently transfected into the hippocalcin-positive NG-108 cells. The 3.4 kb construct was strongly expressed, whilst the 0.9 kb construct was not expressed. In this paper, we describe the detailed expression pattern of the rat hippocalcin gene, the gene structure and its neuron-specific promoter.
Grant
A L
AL
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Jones
A
A
Thomas
K L
KL
Wisden
W
W
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017931:DNA Primers; D005091:Exons; D051597:Hippocalcin; D006624:Hippocampus / Q000378:metabolism*; D017403:In Situ Hybridization; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D005810:Multigene Family; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic
D002135:Calcium-Binding Proteins; D017931:DNA Primers; C496692:Hpca protein, mouse; C496693:Hpca protein, rat; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D051597:Hippocalcin