Elsevier

Molecular Brain Research

Volume 42, Issue 2, December 1996, Pages 202-212
Molecular Brain Research

Variable subcellular localization of a neuron-specific protein during NTera 2 differentiation into post-mitotic human neurons

https://doi.org/10.1016/S0169-328X(96)00115-5Get rights and content

Abstract

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 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 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.

Introduction

Over the last decade, positional cloning technology has been used to identify more than 40 inherited disease genes 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 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 the HD gene (the IT15 gene) [26]. These included the α-adducin gene [10], a fibroblast growth factor receptor gene [27], the α-L-iduronidase gene [15], the β-subunit of the cGMP phosphodiesterase gene [19], and more than 20 anonymous transcribed probes [3,12]

In a previous study, we identified a series of putative genes that mapped near the HD gene (within the 4p16.3 chromosomal segment) [3]. One of these putative genes, derived from cosmid clone cKO2-32, contained a probe (the D4S234 marker) that detected evolutionarily conserved 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 neuron-specific rat (i.e. p1A75) [24,25] and mouse (i.e. p19) genes [22]. Whereas sequence analysis shows that the mouse m234 and rat p1A75 genes are interspecies homologs, the divergence in the m234 and mouse p19 sequences suggests that these genes are nonidentical members of a multigene family. Furthermore, transcriptional analyses revealed that the rodent D4S234 gene was preferentially expressed in adult neurons, whereas the p19 gene exhibited an embryonic neuronal expression pattern [22]. Since previous immunostaining studies of the rodent brain localized the p1A75 and p19 proteins to the neuronal cytoplasm [22,24], the subcellular localization of the D4S234 protein was examined in the mitotic NTera 2 (NT2) cell line and in post-mitotic NT2-N neurons. In mitotic NT2 cells, an antipeptide antibody mapped the D4S234 protein to the nucleus. In contrast, post-mitotic NT2-N neurons often retained the D4S234 protein in the cytoplasm. Therefore, the D4S234 gene appears to be a member of a multigene family of neuron-specific genes, that can be distinguished from its other family member by its preferential expression in adult neurons and its transport to the nuclei of mitotic cells.

Section snippets

Cell culture

Astrocyte-enriched cultures were prepared from Fisher 344 rats essentially as described [23]. In summary, total brains from newborn (1 day) animals were dissected and mechanically disrupted in Trypsin-EDTA. These samples were seeded in MEM containing 10% fetal calf serum (FCS) and incubated for 24 h. The cells were then maintained in medium supplemented with 1 μM cytosine arabinoside, 10 μM flurodeoxyuridine and 10 μM uridine to minimize fibroblast contamination. Cultures were kept for 2 to 4 weeks

Characterization of the human and mouse D4S234 genes

Our previous study showed that the 32B10 subclone from human cosmid cKO2-32 mapped close to the Huntington disease gene within the 4p16.3 chromosome segment and contained transcribed and evolutionarily conserved sequences [3]. A 1964-bp segment from cKO2-32 (encompassing the 32B10 probe) was sequenced (GenBank Accession number M98528) and used to search GenBank for related sequences. One entry, a rat cDNA clone (named p1A75; GenBank Accession number J00755) exhibited significant homology to the

Discussion

The gene described in this study (the D4S234 gene) was isolated during positional cloning analysis of the Huntington disease gene region [3]. The current report describes the molecular characterization of the human homolog (the D4S234 gene) and the corresponding mouse gene (the m234 gene) and shows that these genes are homologous to a previously identified neuron-specific rat gene (the p1A75 partial cDNA sequence) [24]. Cell culture and in vivo animal studies demonstrated that the rodent m234

Acknowledgements

We thank A. Pandrangi and R. Vijesurier for their technical support and Dr. Bor Luen Tang for observing the sequence similarity between the D4S234 and p19 genes. This work was supported by NIH Grant NS24236 (L.C.).

References (33)

  • T. Boulikas

    Nuclear localization signals

    Crit. Rev. Eukaryote Gene Exp.

    (1993)
  • L. Carlock et al.

    Transcription of the Huntington disease gene during the quinolinic acid excitotoxic cascade

    NeuroReport

    (1995)
  • P. Cohen

    The structure and regulation of protein phosphatases

    Annu. Rev. Biochem.

    (1989)
  • F.S. Collins

    Positional cloning moves from perditional to traditional

    Nature Genet

    (1995)
  • Y. Goldberg et al.

    Cloning and mapping of the α-adducin gene close to D4S95 and assessment of its relationship to Huntington disease

    Hum. Mol. Genet.

    (1992)
  • J. Humphrey et al.

    Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence

    J. Cell Biol.

    (1993)
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