The newly identified human nuclear protein NXP-2 possesses three distinct domains, the nuclear matrix-binding, RNA-binding, and coiled-coil domains

Using a monoclonal antibody that recognizes a nuclear matrix protein, we selected a cDNA clone from a lambdagt11 human placenta cDNA library. This cDNA encoded a 939-amino acid protein designated nuclear matrix protein NXP-2. Northern blot analysis indicated that NXP-2 was expressed in various tissues at different levels. Forcibly expressed green fluorescent protein-tagged NXP-2 as well as endogenous NXP-2 was localized in the nucleus and distributed to the nuclear matrix. NXP-2 was released from the nuclear matrix when RNase A was included in the buffer for nuclear matrix preparation. Mapping of functional domains was carried out using green fluorescent protein-tagged truncated mutants of NXP-2. The region of amino acids 326-353 was responsible for nuclear matrix binding and contained a cluster of hydrophobic amino acids that was similar to the nuclear matrix targeting signal of acute myeloleukemia protein. The central region (amino acids 500-591) was demonstrated to be required for RNA binding by Northwestern analysis, although NXP-2 lacked a known RNA binding motif. The region of amino acid residues 682-876 was predicted to have a coiled-coil structure. The RNA-binding, nuclear matrix-binding, and coiled-coil domains are structurally separated, suggesting that NXP-2 plays important roles in diverse nuclear functions, including RNA metabolism and maintenance of nuclear architecture.     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

Heterologous expression of recombinant human glutathione transferase A1-1 from a hepatoma cell line.

A cDNA clone, lambda GTHA1, encoding human glutathione transferase A1-1 has been isolated from a hepatoma HepG2 cDNA library. At the nucleotide level, the new clone showed minor differences from cDNA deriving from normal liver, but the deduced amino acid sequence was identical to the structure previously described. The protein was expressed from a plasmid, pKHA1, and isolated by a single-step affinity purification on an S-hexylglutathione Sepharose matrix. The yield of the recombinant protein was 165 mg from a 3-liter culture of bacteria.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Cloning of STK13, a Third Human Protein Kinase Related toDrosophilaAurora and Budding Yeast Ipl1 That Maps on Chromosome 19q13.3–ter

This report describes the identification of a cDNA encoding STK13, a third human protein kinase related to theDrosophilaAurora and the budding yeast Ipl1 kinases. After screening of a human placental cDNA library with aXenopus laeviscDNA encoding the pEg2 protein kinase and 5′ RACE on testis mRNA, a full-length cDNA was isolated. The chromosomal localization of STK13 on 19q13.3–ter between the markers D19S210 and D19S218 was established by a combination of somatic cell and radiation hybrid panel PCR screening. The localization of STK13 on human chromosome 19 was confirmed by fluorescencein situhybridization (FISH) using a genomic clone containing STK13 as a probe.     

BH-protocadherin-c, a member of the cadherin superfamily, interacts with protein phosphatase 1 alpha through its intracellular domain

Using a yeast two-hybrid system, we isolated eight cDNA clones which interacted with BH-protocadherin-c (BH-Pcdh-c) from the human brain cDNA library. One clone encoded protein phosphatase type I isoform alpha (PP1alpha) and another two PP1alpha2. PP1alpha was co-immunoprecipitated from the extract of a gastric adenocarcinoma cell line MKN-28 with anti-BH-Pcdh-c antibody. PP1alpha activity towards glycogen phosphorylase was inhibited by the intracellular domain of BH-Pcdh-c. Inhibition of the phosphatase required more than the minimal domain of BH-Pcdh-c which could associate with PP1alpha. In situ hybridization revealed that BH-Pcdh-c mRNA was predominantly expressed in cerebral cortex neurons in the adult mouse brain.     

Cloning, structure and expression of a cDNA encoding the human androgen receptor

A cDNA clone has been isolated from a library prepared of mRNA of human breast cancer T47D cells with an oligonucleotide probe homologous to part of the region encoding the DNA-binding domain of steroid receptors. The clone has a size of 1505 bp and sequence analysis revealed an open reading frame of 1356 bp. The deduced amino acid sequence displays two highly conserved regions identified as the putative DNA-binding and hormone binding domains respectively of steroid receptors. Expression of this cDNA clone in COS cells produces a nuclear protein with all the binding characteristics of the human androgen receptor (hAR). The gene encoding the cDNA is assigned to the human X-chromosome. High levels of three hybridizing mRNA species of 11, 8.5 and 4.7 kb respectively are found in the human prostate cancer cell line (LNCaP), which contains elevated levels of hAR. The present data provide evidence that we have isolated a cDNA that encodes a major part of the human androgen receptor.     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

Identification of AMSH-LP containing a Jab1/MPN domain metalloenzyme motif

We have isolated a cDNA clone encoding a new AMSH (associated molecule with the SH3 domain of STAM) family protein, termed AMSH-like protein (AMSH-LP). AMSH-LP has similar characteristics to AMSH; both AMSH-LP and AMSH are expressed ubiquitously in various human tissues, contain a putative nuclear localization signal (NLS), an Mpr/Pad1/N-terminal (MPN) domain, and a Jab1/MPN domain metalloenzyme (JAMM) motif in their structures, and are excluded from the nucleus when lacking either the NLS or MPN domain. Moreover, we observed an enhancement of interleukin 2 (IL-2)-mediated c-myc induction in AMSH-LP-transfected cells similar to that seen in AMSH-transfected cells, suggesting a functional similarity between AMSH-LP and AMSH. However, the present study demonstrated that AMSH-LP, unlike AMSH, fails to bind to the SH3 domains of STAM1 (signal transducing adaptor molecule 1) and Grb2. These results suggest that AMSH-LP and AMSH may have different functions.     

TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins.

A murine monoclonal antibody was identified by its ability to induce a reversible antiproliferative effect on a human lymphoma cell line. Immunoprecipitation studies revealed that the antibody reacted with a 26-kilodalton cell surface protein (TAPA-1). A diverse group of human cell lines, including hematolymphoid, neuroectodermal, and mesenchymal cells, expressed the TAPA-1 protein. Many of the lymphoid cell lines, in particular those derived from large cell lymphomas, were susceptible to the antiproliferative effects of the antibody. TAPA-1 may therefore play an important role in the regulation of lymphoma cell growth. A cDNA clone coding for TAPA-1 was isolated by using the monoclonal antibody to screen an expression library in COS cells. Analysis of the deduced amino acid sequence indicated that the protein is highly hydrophobic and that it contains four putative transmembrane domains and a potential N-myristoylation site. TAPA-1 showed strong homology with the CD37 leukocyte antigen and with the ME491 melanoma-associated antigen, both of which have been implicated in the regulation of cell growth.     

Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X

A 1.5-kb cDNA (FX) encoding full-length human coagulation factor X was isolated from a human fetal liver cDNA library. The identity of the insert in a selected phage lambda clone was confirmed to be FX by nucleotide (nt) sequence analysis and restriction mapping. This FX cDNA clone contained 1467 bp of coding sequence, no 5'-untranslated sequence, a short 3'-untranslated sequence of 10 nt and a poly(A) tail at the 3'-end. The FX cDNA was inserted into a mammalian expression vector and transfected into COS-1 monkey kidney cells. Media from transfected cells showed evidence of factor X antigen and, following addition of Russel's viper venom factor X activator, enhanced amidolytic activity toward a synthetic peptide rho-nitroanilide substrate. Immunoprecipitation with an anti-factor X monoclonal antibody of [35S]methionine-labeled cell-conditioned media showed evidence of polypeptides of 74, 55, and 17 kDa, as determined by SDS-PAGE followed by autoradiography. Together, these results indicate that an active factor X can be successfully expressed in a recombinant DNA expression system. This approach will allow the systematic structure/function investigation of this important blood-clotting enzyme.     

Evaluation of MUC6 mucin tandem repeats

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.