Human Fetal Brain cDNA Library Research Articles

Identification and Characterization of a Retinoic Acid-regulated Human Homologue of the unc-33-like Phosphoprotein Gene (hUlip) from Neuroblastoma Cells

A cDNA, 7G1, was isolated from retinoic acid (RA) differentiated neuroblastoma cells whose expression was high in human fetal brain and spinal cord mRNA but undetectable in adult brain or non-neuronal tissues. Sequence analysis indicates that 7G1 is homologous to the Caenorhabditis elegans gene unc-33. A 5.5-kilobase pair full-length cDNA from a human fetal brain cDNA library contains an 1710-base pair open reading frame. Because the predicted 570 amino acid sequence of 7G1 shares 98% identity with the murine Ulip gene product, an unc-33-like-phosphoprotein, we refer to 7G1 as the human Ulip (hUlip). hUlip is also similar to the bacterial enzyme D-hydantoinase and the recently described vertebrate gene products CRMP62, TOAD-64, CRMP1, CRMP2, and mUNC. RA stimulates an increase in hUlip mRNA that is transcriptionally regulated. RA stimulates an increase in polypeptides of 58, 60, 65, and 70 kDa with the 58- and 65-kDa species being dephosphorylated forms of the 60- and 70-kDa species. This study presents a model in which to study the regulation and expression of the hUlip gene, a member of an emerging family of molecules that potentially mediates signals involved in axonal outgrowth.

CDNA Cloning, Chromosomal Localization, and Expression Pattern ofEPLG8,a New Member of theEPLGGene Family Encoding Ligands of EPH-Related Protein-Tyrosine Kinase Receptors

By screening a human fetal brain cDNA library under low stringency using cDNA encoding the mouse ligand of Cek5 as a probe, we have isolated a novel cDNA belonging to theEPLGgene family. This family encodes ligands of EPH-related tyrosine kinase receptors. Since the novel gene is the eighth member of theEPLGgene family, it is designatedEPLG8.The deduced amino acid sequence ofEPLG8suggests that it encodes a transmembrane protein that is most related to those encoded byEPLG2andEPLG5.We mapped theEPLG8gene to human chromosome 17p11.2–p13.1 by PCR screening of human–rodent somatic cell hybrid panels. In the midterm fetus,EPLG8mRNA is expressed at the highest level in brain, followed by heart, kidney, and lung. In the adult,EPLG8mRNA expression is restricted to brain. These data suggest that LERK-8, the protein encoded byEPLG8,is important in brain development as well as in its maintenance. Moreover, since levels ofEPLG8expression were particularly high in several forebrain subregions compared to other brain subregions, LERK-8 may play a pivotal role in forebrain function.

Cloning of a Novel Homeobox-Containing Gene,PKNOX1,and Mapping to Human Chromosome 21q22.3

To contribute to the development of the transcript map of human chromosome 21 and to the understanding of the pathogenesis of Down syndrome, we have used exon trapping to identify portions of genes from pools of HC21-specific cosmids. More than 550 potential exons have been isolated to date. One such trapped exon, hmc37a09 (GenBank Accession No. X88106), was identical to a region of a human EST, L12425 (GenBank Accession No. D31072). Its predicted amino acid sequence was homologous to the homeodomain region of homeobox-containing genes. Using the trapped sequence and the EST as probes to screen human fetal brain and kidney cDNA libraries, we have cloned the corresponding full-length cDNA. This novel gene encodes a homeodomain-containing polypeptide of 436 amino acids. The most closely related sequence is that of the mouseMeis1,aPBX-like homeobox gene. The homeodomain of the novel gene is closely related to those of the mammalianPBXfamily and the plantKnotted1family (involved in plant development). This gene is namedPKNOX1by the Human Nomenclature Committee. By PCR amplification, hybridization, and genetic linkage analysis using a (GT)npolymorphism in the 3′UTR, we have precisely localizedPKNOX1to chromosome 21q22.3 between markers D21S212 and D21S25 on YAC350F7.PKNOX1is expressed in many human tissues tested by Northern blot analysis. The involvement of thePKNOX1gene in Down syndrome and/or monogenic disorders associated with dysfunction of this gene is presently unknown. Targeted disruption of thePKNOX1homolog in mice will enhance our understanding of its biological function in normal mammalian development.

Cloning of the human puromycin-sensitive aminopeptidase and evidence for expression in neurons.

The puromycin-sensitive aminopeptidase (PSA) is thought to contribute to the degradation of enkephalins. Besides being the most abundant aminopeptidase in the brain, PSA is expressed in other organs as well. From a human fetal brain cDNA library, we have isolated a cDNA encoding the human PSA (huPSA) protein. The isolated cDNA gave rise to a protein with a molecular mass of 99 kDa. Compared with mouse PSA, homology at the amino acid and cDNA level was 98 and 93%, respectively. Translation of the huPSA was found to be initiated at the second of two possible start codons, as shown by studies with antibodies directed against peptide sequences of both potential N-terminal regions. Northern blot analysis with RNA isolated from different human organs demonstrated that the huPSA transcript is strongest but not exclusively expressed in the brain. Vesicular stomatitis virus epitope-tagged huPSA protein was expressed in HeLa cells and found to be localized in the cytoplasm, especially in the perinuclear region. By in situ hybridization, huPSA transcript could be identified in cortical and cerebellar neurons, whereas glial cells and blood vessels remained negative.

Identification and Characterization of a Novel Human Cortistatin-like Peptide

Expressed sequence tags (ESTs) that showed significant homology to rat cortistatin (CST) were found in a human fetal brain cDNA library. A protein coded by the cDNA showed 55% identity to rat preprocortistatin in amino acid. Similarly in the generation of mature peptides from rat preprocortistatin, it was expected that cleavage at dibasic amino acids in the C-terminal portion of the coded protein might produce at least two different sizes of mature peptides with 29 and 17 amino acid residues, respectively. We chemically synthesized the predicted mature peptide with 17 amino acid residues (hCS-17) and examined its biological activities. It bound to all human somatostatin receptor (SSTR) subtypes in almost the same manner as rat CST-14. It also inhibited cAMP production induced by forskolin through SSTRs. Administration of hCS-17 to the cerebral ventricle showed flattening of cortical and hippocampal electroencephalograms in rats. These results indicate that a bioactive peptide encoded by the cDNA is a human counterpart corresponding to rat CST.

The novel human HNF-3/fork head-like 5 gene: chromosomal localization and expression pattern.

Analysis of cDNA clones, isolated from a human fetal brain cDNA library, that hybridized with the rat HNF-3 alpha fork head homolog domain revealed the 3.6-kb HFKL5 cDNA. The transcript of HFKL5 is 4.4 kb long and represents a novel member of the HNF-3/fork head transcription factor family. Comparison of the amino acid sequence of the fork head domain reveals a relatively low level of homology to other members of this family of genes, the closest related sequence being rat HFH7 with 68% homology. The HFKL5 cDNA codes for a putative 500-amino-acid protein. Southern analysis revealed that the HFKL5 gene homolog is present as a single copy in the human genome. Zoo Southern analysis showed strong evolutionary conservation of HFKL5 among mammalian and possibly avian species. Expression of HFKL5 in neurons is restricted to the fully differentiated neurons in fetal and adult brain as well as in the parasympathic ganglia of the small intestine. We also observed expression in lymphocytes, kidney tubule cells, and a subset of hepatocytes. The HFKL5 gene homolog was mapped to chromosome 22q13-qter by cell panel hybridization.

Isolation and mapping of karyopherin α3 (KPNA3), a human gene that is highly homologous to genes encoding <i>Xenopus</i> importin, yeast SRP1 and human RCH1

From a human fetal-brain cDNA library, we isolated and characterized a novel gene (KPNA3) encoding a protein highly homologous to certain nuclear transport proteins of Xenopus and human. The complete cDNA clone, designated karyopherin alpha 3, contained an open reading frame of 1,563 nucleotides encoding 521 amino acids. The predicted amino acid sequence showed 48%, 45% and 48% identity with Xenopus importin, yeast SRP1 and human RCH1, respectively. The similarities among these proteins suggest that karyopherin alpha 3 may be involved in the nuclear transport system. Eight repeats of the arm motif were well conserved among these proteins. The N-terminal region of the predicted karyopherin alpha 3 product was highly basic and the C-terminal region was strongly acidic. A 4.3-kb transcript was expressed in all adult human tissues examined by Northern blotting. The cDNA clone was assigned to chromosome band 13q14.3 by fluorescence in situ hybridization.

HDIP — a potential transcriptional regulator related to murine TSC-22 and Drosophila shortsighted ( shs) — is expressed in a large number of human tissues

We have cloned a 420 bp cDNA from a human fetal brain cDNA library in lambda encoding the human homologue of a DSIP-immunoreactive leucine zipper protein (DIP) isolated from porcine brain. The derived human protein (hDIP) shares a significant sequence identity with parts of the murine TSC-22 and Drosophila shs, both proteins which are discussed as functioning as transcriptional regulators. A similar role of hDIP is partially confirmed by the results of an RT-PCR analysis, demonstrating the widespread distribution of the protein among different human tissues.

ICAAR, a Novel Member of a New Family of Transmembrane, Tyrosine Phosphatase-like Proteins

We have isolated a cDNA from human foetal brain cDNA library which encodes a putative transmembrane protein bearing an intracellular protein tyrosine phosphatase (PTPase) like domain. The PTPase like domain contains an alanine to aspartate amino acid change relative to other PTPases in the catalytic core domain. This amino acid change is found in only three other known proteins, islet cell autoantigens; human, murine and rat IA-2, murine IA-2b and its rat orthologue phogrin, which have a similar overall structure to ICAAR, and the recently identified X-linked myotubular myopathy (MTM1) gene. ICAAR, IA-2 and IA-2b clearly represent a new family of PTP-like proteins for which catalytic activity has yet to be demonstrated. An abundant ICAAR mRNA is detectable in the brain and pancreas but not in the other normal human tissues surveyed. We have localised ICAAR to human chromosome 7q36.

Cloning and Characterization of Two Novel Human cDNAs (NELL1 and NELL2) Encoding Proteins with Six EGF-like Repeats

From a human fetal-brain cDNA library we isolated two novel genes encoding peptides containing six EGF-like repeats. Both showed significant homologies with nel, a gene strongly expressed in neural tissues of chicken. The cDNAs, designated NELL1 (nel-like, type 1) and NELL2 (nel-like, type 2), contained open reading frames encoding 810 and 816 amino acids, respectively. NELL2 is strongly expressed in brain of adult and fetus but only weakly in fetal kidney. NELL1 and NELL2 were mapped by FISH to chromosomal bands 11p15.1–p15.2 and 12q13.11–q13.12, respectively.

Evidence for the direct interaction of the insulin-like growth factor I receptor with IRS-1, Shc, and Grb10.

We have used the yeast two-hybrid system to study the interaction between the IGF-I receptor and two putative substrates, IRS-1 and Shc. In addition, we have identified Grb10 as a protein that binds to the insulin-like growth factor I (IGF-I) receptor. This two-hybrid system (the interaction trap) utilizes a hybrid protein containing the LexA DNA-binding domain fused to the intracellular portion of the IGF-I receptor (LexA-IGFIR beta) and hybrids containing an activation domain fused to either IRS-1 (Ad-IRS-1), Shc (Ad-Shc), or a cDNA library. A positive interaction of LexA-IGFIR beta with the activation domain hybrid results in activation of reporter genes, LacZ and LEU2, in the yeast. Western blotting of extracts from transformed yeast demonstrated that the LexA-IGFIR beta fusion protein was expressed and phosphorylated on tyrosine residues. Coexpression of LexA-IGFIR beta with Ad-IRS-1 resulted in strong activation of both reporter genes; activation did not occur with a kinase-negative receptor mutant. IRS-1 residues 160-516 were sufficient for this strong interaction. Coexpression of LexA-IGFIR beta with Ad-Shc also resulted in strong activation of both LacZ and LEU2 reporter genes. This interaction was also dependent upon a tyrosine kinase-active receptor and required tyrosine 950 in the juxtamembrane region of the receptor. An N-terminal fragment of Shc (amino acids 1-232) interacted almost as strongly as full-length Shc whereas the Shc SH2 domain only activated the more sensitive LEU2 reporter. Full-length Shc was phosphorylated on tyrosine when coexpressed with IGFIR beta but not when coexpressed with the kinase-negative receptor mutant. To identify additional proteins that interact with the IGFIRs, a human fetal brain cDNA library was screened using the interaction trap system. This analysis identified partial cDNAs for Grb10. Coexpression of LexA-IGFIR beta with Ad-Grb10 resulted in strong activation of both LacZ and LEU2 reporter genes; this interaction was dependent upon a tyrosine kinase-active receptor but did not require tyrosine 950.

Cloning and Functional Expression of a Human Ca 2+-Permeable Cation Channel Activated by Calcium Store Depletion

Depletion of intracellular calcium stores generates a signal that activates Ca 2+-permeable channels in the plasma membrane. We have identified a human cDNA, TRPC1A, from a human fetal brain cDNA library. TRPC1A is homologous to the cation channels trp and trpl in Drosophila and is a splice variant of the recently identified cDNA Htrp-1. Expression of TRPC1A in CHO cells induced nonselective cation currents with similar permeabilities for Na +, Ca 2+, and Cs +. The currents were activated by intracellular infusion of myo inositol-1,4,5-trisphosphate or thapsigargin. Expression of TRPC1A significantly enhanced increases in the intracellular free calcium concentration induced by Ca 2+ restitution after prolonged depletion. Similar results were obtained in Sf9 cells. We conclude that TRPC1A encodes a Ca 2+-permeable cation channel activated by depletion of intracellular calcium stores.

Cloning of human neuronatin gene and its localization to chromosome-20q11.2–12: the deduced protein is a novel ‘proteolipid’

Human brain development is a continuum governed by differential gene expression. Therefore, we proceeded to identify genes selectively expressed in the developing brain. Using differential display and library screening, a novel rat cDNA, neuronatin, was identified and used to screen a human fetal brain cDNA library. Human neuronatin cDNA was isolated and sequenced. The cDNA was 1159 by long and corresponded in size to the 1.25 kb message detected on Northern analysis. Neuronatin mRNA was selectively expressed in human brain during fetal development, but became repressed in adulthood. When studied in the rat, neuronatin mRNA first appeared at mid-gestation in association with the onset of neurogenesis, becoming most pronounced later in development when neuroepithelial proliferation and neuroblast commitment are manifest, and declined postnatally coinciding with the completion of neurogenesis. The deduced protein has two distinct domains, a hydrophobic N-terminal and basic C-terminal rich in arginine residues. Both the amino acid sequence and secondary structure of this amphipathic polypeptide exhibited homology to PMPI and phospholamban, members of the ‘proteolipid’ class of proteins which function as regulatory subunits of membrane channels. The neuronatin gene, 3973 bases long, contains in its 5′-flanking region a neural restrictive silencer element which may govern neuron-specific expression. Based on screening a somatic cell hybrid panel, neuronatin gene was assigned to chromosome-20. And, using deletion constructs of chromosome-20 and fluorescence in situ hybridization, neuronatin was localized to chromosome-20811.2-12. In conclusion, neuronatin is a novel human gene that is developmentally regulated and expressed in the brain. The deduced protein is a proteolipid that may function as a unique regulator of ion channels during brain development. The definitive localization of neuronatin to human chromosome 20811.2-12 provides the basis to investigate this gene as a candidate in neuro-developmental diseases that may also map to this region.

Molecular Cloning of Human Hyaluronan Synthase

We report here the cDNA sequence of human hyaluronan synthase. The cDNA clone was isolated from a human fetal brain cDNA library by screening with a DNA probe generated from the coding sequence of murine hyaluronan synthase. It contains a 2107 bp cDNA insert which is composed of a 1629 bp open reading frame and a short segment of the 3′-untranslated region. Comparative analysis of the predicted coding region with the murine hyaluronan synthase sequence indicates 84.4% and 96.0% identity in nucleotide sequence and amino acid sequences, respectively. Northern analysis indicated that the gene was ubiquitously expressed in various human tissues.

Isolation of Inositol 1,3,4-Trisphosphate 5/6-Kinase, cDNA Cloning, and Expression of the Recombinant Enzyme

Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate. The final preparation contained proteins of 48 and 36-38 kDa. All of these proteins had the same amino-terminal sequence and were enzymatically active. The smaller species represent proteolysis products with carboxyl-terminal truncation. The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein. The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues. The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa. The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue. The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity. Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1. This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions. Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain. The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.

USE OF THE YEAST TWO-HYBRID SYSTEM TO INVESTIGATE IGF-I SIGNAL TRANSDUCTION.▴ 516

The anabolic affects of the IGFs are mediated through the type I IGF receptor (IGFIR), a member of the tyrosine-kinase family of growth factor receptors. Activation of the IGFIR results in its autophosphorylation. This creates motifs containing phosphotyrosine (PY) residues which are recognized by cytosolic proteins involved in IGF signal transduction. A number of such proteins have been characterized, including IRS-1, IRS-2, and Shc, but it is clear that additional proteins remain unidentified. We have previously shown that a yeast two-hybrid system (the interaction trap) can be used to characterize the interaction between the IGFIR and IRS-1 and Shc. We have now used the interaction trap to screen a human fetal brain cDNA library for novel proteins which interact with the IGFIR. This analysis identified three clones which contained src homology 2 (SH2) domains. SH2 domains bind to PY containing motifs and are found in a number of signaling molecules. Each of these clones interacted with the wild type (i.e., tyrosine-kinase active) IGFIR cytoplasmic domain and the insulin receptor cytoplasmic domain but not with a kinase-defective IGFIR mutant. One clone encoded the C-terminal portion of Grb10, a protein recently identified as interacting weakly with the EGF receptor. The other two clones encoded previously unidentified SH2 domains, both of which are most similar to the N-terminal SH2 domain of p85, the regulatory subunit of PI-3 kinase. Additional studies will be necessary to characterize the interactions between these molecules and the IGFIR and to determine if these molecules are involved in IGFIR signal transduction in mammalian cells.

Characterization and Sequence Analysis of the Human Homeobox-Containing GeneGBX2

Polymerase chain reaction (PCR) was used to amplify portions of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be theGastrulation and brain specific-2gene (GBX2). Screening this human fetal brain cDNA library with probes specific forGBX2led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of theGBX2homeodomain is identical (100%) to the that of homologous gene,Gbx2,expressed in the developing mouse embryo and virtually identical (97%) to a gene expressed in the developing chicken embryo,CHox7.The 5′ end of theGBX2gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. A single 2.2-kb transcript was detected by Northern analysis in the developing human CNS as well as in other tissues. The human genomic clone forGBX2was also isolated, characterized, and mapped to 2q36(d)–q37 by somatic cell hybrid analysis and fluorescencein situhybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development.

Transformation of NIH 3T3 Cells by HER3 or HER4 Receptors Requires the Presence of HER1 or HER2

Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF.

Identification of a novel human gene containing the tetratricopeptide repeat domain from the Down syndrome region of chromosome 21.

The Down syndrome (DS) region on chromosome 21, which is responsible for the DS main features, has been defined by analysis of DS patients with partial trisomy 21. Within the DS region, we constructed a 1.6-Mb P1 contig map previously. To isolate gene fragments from the 1.6-Mb region, we performed direct cDNA library screening and exon trapping using the P1 clones and a human fetal brain cDNA library, and obtained 67 cDNA fragments and 52 possible exons. Among them, 23 cDNA fragments and 4 exons were interpreted to be derived from a single gene by localization on P1 clones and by Northern analysis. To obtain the full-length cDNA sequence, longer cDNA clones were further screened from another human cDNA library which was enriched with longer cDNA species. These clones were sequenced and assembled to a sequence of 9045 bp. This transcribed sequence encodes a novel 2025 amino-acid protein containing tetratricopeptide repeat (TPR) motifs and therefore the gene was designated as TPRD (a gene containing the TPR motifs on the Down syndrome region). The TPR domain has been found in a certain protein phosphatase and in other proteins involved in the regulation of RNA synthesis or mitosis. The TPRD gene, the novel gene which was proved to be in the 1.6-Mb region and to have the interesting features described above, is a candidate for genes responsible for the DS phenotypes.

Cloning, expression pattern and mapping to Xq of NAP1L3, a gene encoding a peptide homologous to human and yeast nucleosome assembly proteins.

From a human fetal brain cDNA library, we isolated a novel gene sharing significant homology with the genes of nucleosome assembly proteins (NAPs). This cDNA clone, designated NAP1L3 (nucleosome assembly protein 1-like 3), contained an open reading frame of 1518 nucleotides encoding 506 amino acids. Its predicted amino acid sequence showed 46% identity and 65% similarity with NAP1L. In its C-terminal half NAP1L3 contained several characteristic motifs strictly conserved with NAP1L and yeast NAP1, but the N-terminal half showed little conservation. Northern-blot analysis revealed strong expression of a 3.0-kb transcript in human adult brain and weak expression in heart. NAP1L3 was closely linked to a marker (DXS990) mapped to chromosome bands Xq21.3-->q22, where genes responsible for several X-linked mental retardation syndromes have been localized.