Isolation of hNap1BP which interacts with human Nap1 (NCKAP1) whose expression is down-regulated in Alzheimer's disease.

We previously reported the isolation of a novel apoptosis-related gene, human Nap1 (HGMW-approved symbol NCKAP1), the expression of which was strongly down-regulated in sporadic Alzheimer's disease (AD). Human Nap1 proved to be an orthologue of rat Nap1 which binds to the adaptor molecule Nck in signal transduction. In order to further elucidate the function of human Nap1, we performed yeast two-hybrid screening. As a result of screening, we discovered a protein designated hNap1BP (human Nap1 binding protein) which is a member of the tyrosine kinase-binding protein family. In addition, hNap1BP bound to the SH3 domain of c-Abl and Nck. hNap1BP is expressed ubiquitously in various tissues like human Nap1, and intriguingly these genes are co-expressed in hippocampus and cerebral cortex in mouse brain where AD pathological features are strongly evident. Further functional analysis of hNap1BP may clarify its contribution to AD pathology.     

Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer''s disease.

In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain. Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins. Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes. GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system. The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons. These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions. Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains. The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.     

Up-regulation of calcineurin Abeta mRNA in the Alzheimer's disease brain: assessment by cDNA microarray

Recent advances in cDNA microarray technology have made it possible to analyze expression of more than 8000 genes. Using this technology, gene expression in the hippocampus containing neurofibrillary tangle-associated lesions from an Alzheimer's disease (AD) patient was compared with expression in the parietal cortex from the same patient that lacked these lesions. We also compared gene expression using a control brain. The top 20 named genes significantly up-regulated or down-regulated only in the AD brain were determined. The most up-regulated gene proved to be calcineurin Abeta mRNA (CAbeta). In situ hybridization histochemistry revealed that CAbeta was significantly up-regulated in pyramidal neurons of the hippocampus in the AD brain. RT-PCR analysis revealed that CAbeta was up-regulated in the hippocampus from two out of three AD brains while there were no changes in three control brains. Our study suggests that CAbeta may play a crucial role in the pathophysiological mechanisms in AD.     

Cloning the full-length IL-2/15 receptor-beta cDNA sequence from mouse brain: evidence of enrichment in hippocampal formation neurons

Numerous studies have implicated interleukin-2 (IL-2) in various brain processes, and more recently, several studies have also attributed neurobiological actions to interleukin-15 (IL-15). On lymphocytes, receptors for IL-2 and IL-15 share a common subunit, the IL-2/15 receptor-beta (IL-2/15Rbeta) that is essential for intracellular signaling. Although a short segment of IL-2/15Rbeta has been cloned (0.35 kb) from normal brain cells, attempts to isolate the full-length cDNA have been unsuccessful, suggesting the possibility that the genes expressed by brain cells and lymphocytes may differ. Using conventional and anchored PCR cloning strategies, we isolated the full-length cDNA of IL-2/15Rbeta (2038 bp) from well-perfused, normal mouse forebrain. The coding sequence and the adjacent 5' and 3' UTR sequences from brain and lymphocyte were found to be fully homologous. Although evidence of expression of IL-2/15Rbeta can be found in many brain regions using PCR, clear evidence of gene expression by in situ hybridization was detectable only in the hippocampal formation, habenula and piriform cortex. This same pattern of mRNA expression in situ was also observed for the common gamma subunit shared by IL-2 and IL-15. In the hippocampus, IL-2/15Rbeta expression was localized to neurons by high resolution in situ hybridization and evidence of IL-2 receptor protein expression was also detected by radioligand receptor binding using hippocampal homogenates. Comparison of undifferentiated and differentiated, immortalized H19-7 hippocampal neurons showed that IL-2/15Rbeta was constitutively expressed across disparate stages of hippocampal neuronal differentiation. These data indicate that IL-2/15Rbeta may serve to modulate neuronal processes in the hippocampus and associated limbic brain regions.     

Characterization of a novel mouse brain gene (mbu-1) identified by digital differential display

Using in silico approaches, we cloned a novel mouse gene (mbu-1) that was strictly expressed in the central nervous system. mbu-1 was first identified as an EST after carrying out digital differential display for unigene libraries from various mouse tissues. The full-length cDNA sequence was obtained by extending the ends of EST by RACE. The cDNA sequence was 2611 bp long and contained an ORF of 597 AA. A positive cis-acting region was found in the neuroblastomaxglioma hybrid, NG108-15, and in human embryonic kidney HEK293 cell lines. RT-PCR and in situ hybridization analysis showed that the mbu-1 gene was only expressed in the brain and spinal cord during the embryonic stages, and throughout all regions of the adult brain, showing higher levels in the hippocampus and hypothalamus.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors

Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5'- and 3'-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein.     

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.     

Placental expression and chromosomal localization of the human Gcm 1 gene.

Although gcm was first recognized for its role in specifying glial cell fate in Drosophila melanogaster, its mammalian counterparts are expressed predominantly in non-neural tissues. Here we demonstrate expression of the mouse and human GCM 1 proteins in placenta. We have prepared a highly specific antibody that recognizes the GCM 1 protein and have used it to assess the temporal and spatial expression profile of the protein. In both mouse and human placenta, the protein is associated with cells that are involved with exchange between maternal and fetal blood supplies: the labyrinthine cells of the mouse placenta and the syncytio- and cytotrophoblasts of the human placenta. Using the full-length hGcm 1 cDNA as a probe, we have mapped the gene on human chromosome 6p12 by fluorescent in situ hybridization.     

A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene

To search for genes involved in determining the morphology of individual neuronal types, a cDNA library was constructed from postnatal day 13 mouse cerebellum. From this library, 2 clones, L7 and L19, were isolated by a differential hybridization procedure and shown by in situ hybridization to be Purkinje cell-specific within the cerebellum. Both RNAs appear between postnatal days 4 and 8 and continue into adulthood, coinciding with terminal differentiation of the Purkinje cells. L7 seems to be expressed exclusively in the cerebellum, whereas L19 is expressed throughout the brain. Consistent with the RNA localization, L7 protein is found only in the cerebellum and is confined to the Purkinje cells. The L7 amino acid sequence has been deduced from the cDNA sequence, and a pseudo-repeat within the L7 protein sequence is homologous to the amino acids sequence in the primary translation product of the gene for human sis/PDGF.     

The sequence, expression, and chromosomal localization of a novel polycystic kidney disease 1-like gene, PKD1L1, in human

Polycystin-1 and polycystin-2 are the products of PKD1 and PKD2, genes that are mutated in most cases of autosomal dominant polycystic kidney disease. Since the first two polycystins were cloned, three new members, polycystin-L, -2L2, and -REJ, have been identified. In this study, we describe a sixth member of the family, polycystin-1L1, encoded by PKD1L1 in human. The full-length cDNA sequence of PKD1L1, determined from human testis cDNA, encodes a 2849-amino-acid protein and 58 exons in a 187-kb genomic region. The deduced amino acid sequence of polycystin-1L1 has significant homology with all known polycystins, but the longest stretches of homology were found with polycystin-1 and -REJ over the 1453- and 932-amino-acid residues, respectively. Polycystin-1L1 is predicted to have two Ig-like PKD, a REJ, a GPS, a LH2/PLAT, a coiled-coil, and 11 putative transmembrane domains. Several rhodopsin-like G-protein-coupled receptor (GPCR) signatures are also found in polycystin-1L1. Dot-blot analysis and RT-PCR revealed that human PKD1L1 is expressed in testis and in fetal and adult heart. In situ hybridization analysis showed that the most abundant and specific expression of Pkd1l1 was found in Leydig cells, a known source of testosterone production, in mouse testis. We have assigned PKD1L1 to the short arm of human chromosome 7 in bands p12--p13 and Pkd1l1 to mouse chromosome 11 in band A2 by fluorescence in situ hybridization. We hypothesize a role for polycystin-1L1 in the heart and in the male reproductive system.     

Analysis of the human cysteine-rich protein gene (CSRP), assignment to chromosome 1q24-1q32, and identification of an associated MspI polymorphism.

The human cysteine-rich protein (hCRP) is encoded by a highly conserved and widely expressed serum-inducible immediate early response gene. hCRP contains two copies of the "LIM/double zinc-finger" motif. Using a characterized hCRP cDNA probe, we demonstrate that the human CRP gene (CSRP) is present in a single copy and that both mouse and human genomes contain one or more CRP-related genes detected by hybridization at low stringency. Using a panel of human x rodent somatic cell hybrids, the hCRP locus is assigned to chromosome 1. In situ hybridization of 3H-labeled CRP cDNA to human metaphase chromosomes confirms this assignment and permits regional localization to bands 1q24-1q32. A common MspI polymorphism is identified and mapped to intron 4 of the hCRP gene. The chromosomal localization and restriction site polymorphism should prove useful in future studies of the function of this gene.     

Cloning and expression pattern of a novel microspore-specific gene encoding hypersensitive-induced response protein (LjHIR1) from the model legume, Lotus japonicus

In order to understand the microspore and pollen development, recently, we have isolated a number of anther-specific genes in the model legume, Lotus japonicus. From these anther-specific genes, we identified one novel microspore-specific gene, LjImfb-c82. In order to determine the molecular characterization of LjImfb-c82, full-length cDNA clone was first isolated and sequenced. It encoded a protein of 286 amino acids (LjHIR1), which had sequence similarity to Hypersensitive-Induced Response like protein. LjHIR1 was specifically expressed in microspore on the in situ hybridization experiment. From the sequence similarity to prohibitin-domain protein, the LjHIR1 might be related to ion channel regulation in microspore development.