Ž . Molecular Brain Research 42 1996 202–212 Research report Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons Leon Carlock a, ) , Trieu Vo a , Matthew Lorincz a , Paul D. Walker b , Denise Bessert a , Douglas Wisniewski a , Joan C. Dunbar a a Center for Molecular Medicine and Genetics, 9115 Scott Hall, Wayne State UniÕersity School of Medicine, Detroit, MI 48201, USA b Department of Anatomy and Cell Biology, Wayne State UniÕersity School of Medicine, Detroit, MI 48201, USA Accepted 23 April 1996 Abstract Ž . Ž . The current report describes the molecular characterization of the human the D4S234 locus and mouse the m234 homologs of a gene that was isolated during our genomic analysis of the Huntington disease gene region. Sequence comparisons of full-length cDNA clones revealed that the mouse and human homologs encoded evolutionarily conserved 21-kDa proteins with greater than 90% amino acid Ž sequence identity. Extensive sequence identity between the D4S234 gene and the rat p1A75 gene a previously identified rat . neuron-specific gene showed that these genes are interspecies homologs. Furthermore, the D4S234 protein exhibited significant amino acid similarity to a 19-kDa mouse protein that localizes to the Golgi apparatus of embryonic neurons. However, nonconservative sequence differences suggested that these genes are independent members of a multigene family. Northern analyses revealed that rodent D4S234 expression occurred predominantly in the brain and included all brain regions. Neuron-specific expression was demonstrated using Northern analysis of cultured glial cells and quinolinic acid-treated rat brain samples. Minimal amounts of the rodent D4S234 mRNA were detected prenatally; however, elevated adult levels were detected within 1 month of birth. Sequence analyses of the human and mouse D4S234 proteins identified an evolutionarily conserved hydrophobic sequence and a consensus nuclear localization signal in both genes. Immunofluorescence microscopy, using an antipeptide antibody, established that the human D4S234 protein preferentially localized to the nucleus of mitotic cultured cells. Since the rat p1A75 protein was previously mapped to the neuronal cytoplasm by in situ Ž . hybridization, the subcellular localization of the D4S234 protein was subsequently examined during differentiation of the NTera 2 NT2 cell line. Following differentiation into postmitotic NT2-N neurons, the D4S234 protein demonstrated cytoplasmic staining and reduced or undetectable nuclear staining in many cells. The variation in the intracellular localization of the D4S234 protein in mitotic and nonmitotic cells suggests that the subcellular localization of this protein is developmentally regulated and provides clues about the biochemical function of this protein. Keywords: Neuron-specific expression; D4S234; m234; p1A75; p19; Multigene family; Evolutionary conservation; Golgi apparatus; Nuclear localization 1. Introduction Over the last decade, positional cloning technology has been used to identify more than 40 inherited disease genes wx based upon their location in the human genome 6. In many of these studies, a large number of candidate disease genes were identified prior to the isolation of the disease- specific mutation. This was also true for the chromosomal Ž . region containing the Huntington disease HD gene, in that a number of transcribed sequences and candidate disease genes were isolated prior to the identification of Ž . w x the HD gene the IT15 gene 26 . These included the ) Ž . Corresponding author. Fax: q1 313 577-5218. w x a-adducin gene 10 , a fibroblast growth factor receptor w x w x gene 27 , the a-L-iduronidase gene 15 , the b-subunit of w x the cGMP phosphodiesterase gene 19 , and more than 20 w x anonymous transcribed probes 3,12 In a previous study, we identified a series of putative Ž genes that mapped near the HD gene within the 4p16.3 . wx chromosomal segment 3 . One of these putative genes, derived from cosmid clone cKO2-32, contained a probe Ž . the D4S234 marker that detected evolutionarily con- served genomic DNA and mRNA species. In the current report, we describe the complete sequence of the human D4S234 and mouse m234 transcripts and show that these sequences are homologous to the previously characterized Ž . w x Ž neuron-specific rat i.e. p1A75 24,25 and mouse i.e. . w x p19 genes 22 . Whereas sequence analysis shows that the 0169-328Xr96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. Ž . PII S0169-328X 96 00115-5