Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.     

The octamer binding factor Oct6: cDNA cloning and expression in early embryonic cells.

We have cloned a cDNA encoding a novel octamer binding factor Oct6 that is expressed in undifferentiated ES cells. Expression of the Oct6 gene is downregulated upon differentiation of these cells by aggregate formation. Furthermore the gene is transiently up regulated during retinoic acid induced differentiation of P19 EC cells, reaching maximum levels of expression one day after RA addition. Sequence analysis of the cDNA encoding the Oct6 protein indicated that the Oct6 gene is a member of the POU-HOMEO domain gene family. The gene expresses a 3 kb mRNA encoding a 449 amino acid protein with an apparent molecular weight of 45 kD. The sequence of the Oct6 POU domain is identical to that of the rat SCIP (Tst-1) gene. The Oct6 expression pattern suggests a role for this DNA binding protein in neurogenesis as well as early embryogenesis.     

cDNA sequence of the rat U snRNP-associated protein N: description of a potential Sm epitope.

Anti-Sm antibodies from a patient with systemic lupus erythematosus (SLE) were used to isolate cDNA clones encoding the snRNP-associated protein N from a rat brain derived cDNA library. The predicted primary structure of the 240 amino acid protein has a proline rich carboxyl terminus and shares a region of sequence similarity with other snRNP polypeptides, A and B/B'. Anti-Sm sera recognize a beta-galactosidase fusion protein containing only the carboxyl-terminal 80 amino acids of N; antibodies eluted from this fusion protein also react with A, B/B' and N on immunoblots, suggesting that these proteins share an Sm epitope located within this segment. Polyclonal antibodies raised against a 23 amino acid synthetic peptide derived from this conserved region of N recognize A, N and B/B' on immunoblots and can immunoprecipitate the Sm class of U snRNAs. These results confirm that this sequence defines a potential Sm epitope. RNA blotting analyses demonstrate that a 1.6 kb mRNA expressed predominantly in brain encodes the N polypeptide in both rats and humans. At low stringency rat N cDNA also hybridizes to a 1.3 kb mRNA species which encodes B/B', suggesting that N is structurally related to, but distinct from B/B'. Although B/B' proteins are thought to be expressed in all human cells, only N and B, but not B', are observed on immunoblots of human brain proteins probed with anti-Sm sera. The apparent difference in the complement of proteins associated with snRNP particles in human brain versus elsewhere suggests a possible mechanism for the regulation of brain-specific mRNA splicing.     

Isolation, expression and characterization of a human apolipoprotein B 100-specific cDNA clone

The isolation and characterization of a human apolipoprotein B 100-specific cDNA clone (lambda gt-B1) containing a 1321 base pairs (bp) spanning insert is described. It encodes the 3'-nontranslated 281 bp long region up to the polyadenylation site and 1040 bp of the C-terminal coding region of 345 amino-acid residues of human apo B 100 and the stop codon. The lambda gt-B1 cDNA clone has been isolated from a human hepatoma cDNA expression library by immunoscreening using affinity-purified polyclonal anti apo B 100 antibodies. The nucleotide sequence of the apo B 100 insert has been determined. A part of the polypeptide sequence derived from this nucleotide sequence was identical with the amino-acid sequence obtained by protein sequencing of a purified cyanogen bromide fragment of apo B 100. The fusion protein consisting of beta-galactosidase and the 345 amino-acid residue long C-terminus of apo B 100 had an apparent molecular mass of 148 kDa in NaDodSO4 polyacrylamide gel electrophoresis. In Northern blot hybridization analysis the insert of the apo B 100-cDNA clone hybridized to a 20 to 22 kb mRNA from adult human liver.     

Reversible interconversion between primitive endoderm- and parietal endoderm-like F9 cells demonstrated by mRNAs expression

The differentiation of retinoic acid-treated F9 cells (primitive endoderm-like F9 cells) into parietal endoderm-like F9 cells induced by dibutyryl cAMP was studied as a culture model of the morphogenesis of early mouse embryo. For this purpose, 6 cDNA clones coding for mRNAs specifically expressed in parietal endoderm-like F9 cells were selected. Northern hybridization of RNA extracted from variously treated F9 cells to nick-translated plasmid DNA of these clones demonstrated the reversible expression of many mRNAs depending on the presence of dibutyryl cAMP in the culture medium. This result suggested that the differentiated state of parietal endoderm, which is formed from primitive endoderm at a position adjacent to the trophectoderm in mouse embryo, can be reversed if the local signal is removed. One of the selected clones, pLAM, hybridized to an mRNA of 6.3 kb and selected mRNA producing a laminin B subunit in an in vitro translation system. This clone has an inserted sequence of 3.1 kb. Among the restriction sites in this sequence, six were consistent with those in a 1.7 kb inserted sequence of pPE 49 and pPE 386, which were isolated by Barlow et al. as laminin B1 clones. An XbaI site found in both pPE 49 and pPE 386 was, however, not found at the corresponding position of pLAM. Dot hybridization of RNA with pLAM showed that expression of laminin B in F9 cells is stimulated more than 100-fold during differentiation of F9 stem cells into parietal endoderm-like F9 cells.     

Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.

CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).     

Isolation of cDNAs encoding the CD19 antigen of human and mouse B lymphocytes. A new member of the immunoglobulin superfamily

The CD19 (B4) molecule is a m.w. 95,000 cell-surface protein of human B lymphocytes that is expressed before Ig and persists throughout differentiation. In this report, cDNA clones that encode the CD19 molecule were isolated and the amino acid sequence of CD19 was determined. A cDNA clone that selectively hybridized to RNA from CD19+ cell lines was selected from a human tonsilar cDNA library using differential hybridization. This cDNA was used to isolate additional cDNA clones. Four of the five longest cDNA clones isolated were sequenced and found to contain unique sequences presumed to be introns. One clone, pB4-19, was near full length (2.1 kb) and did not contain these putative introns. pB4-19 contained an 1685 bp open reading frame that could encode a protein of about 60 kDa. COS cells that were transfected with pB4-19 expressed a nascent cell surface structure reactive with the anti-B4 antibody. Immunoprecipitation of this structure from surface-iodinated COS cells with the anti-B4 antibody revealed a m.w. 85,000 protein. Northern blot analysis indicated that pB4-19 hybridized with a predominant mRNA species of 2.4 kb and a minor species of 1.5 kb, found in only CD19+ cells. The pre-B cell line, PB-697, also expressed four larger RNA species that hybridized with pB4-19. cDNA clones that encode the putative cytoplasmic portion (247 amino acids) of the mouse CD19 molecule were also isolated and found to be highly homologous (79 and 75%) with the human CD19 nucleotide and amino acid sequences. The deduced amino acid sequence of the CD19 cytoplasmic tail shared no significant homology with other known proteins but the putative extracellular region contained two Ig-like domains indicating that CD19 is a new member of the Ig superfamily.     

Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin.

A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.     

Expression of cloned cDNA for a major surface antigen of Plasmodium falciparum merozoites

A cDNA library of P. falciparum was constructed. Using size-selected mRNA as a probe several clones were isolated which hybridized to mRNAs larger than 5 kilobases (kb). The cDNA insert of pFC 17, which hybridizes to 5.6-kb mRNA was expressed by fusion to anthranilate synthetase I in a plasmid expression vector. The expressed fusion protein was shown to contain epitopes of a 195-kDa protein which is the precursor to 3 major surface antigens of P. falciparum merozoites.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

Cloning and expression of a novel human galectin cDNA, predominantly expressed in placenta(1)

A novel human galectin cDNA (PPL13) was isolated by screening a human 18-week fetal brain library. The mRNA was predominantly expressed in placenta, while the expression of it was not or barely detectable in heart, brain, lung, liver, skeletal muscle, kidney, and pancreas by Northern blot. COS-7 cells transfected with cDNA encoding human PPL13 sequestered the protein in nuclei although it lacked any known nuclear localization signal. STS of Unigene Hs. 24236 placed the cDNA to human chromosome 19q13.2.     

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.     

Molecular cloning of a tumor-associated antigen recognized by monoclonal antibody 3H11

Monoclonal antibody (MAb) 3H11 can bind specifically to different cancer cells from different tissues. MAb 3H11 labeled with radioactive isotopes has been used clinically to detect primary cancer and metastatic cancer. Molecular cloning of the antigen recognized by MAb 3H11 is important in studying tumor occurrence and in developing new biotherapy for cancer. Using MAb 3H11, we screened cDNA library made from the human gastric cancer cell line MGC 803, which reacts with MAb 3H11, and isolated one positive clone specifically recognized by the antibody. The insert cDNA fragment was 0.5 kb. After recombining with glutathione-S-transferase expression vector pGEX-4T, the cDNA fragment could be expressed into a fusion protein that specifically reacted with MAb 3H11. Moreover, the fusion protein could competitively inhibit MAb 3H11 binding to MGC 803 cells. Based on the nucleotide sequence of the cDNA fragment, the full length of the cDNA (2156 bp) was obtained by Rapid-Amplification-cDNA-End (RACE) and nested PCR. Its reading frame was 1767 bp encoding a protein of 589 amino acids. Sequence analysis indicated that there is no highly homologous gene in the GenBank. Northern blot and RT-PCR showed that the mRNA of MAb 3H11 antigen was extensively distributed in embryonic tissue and in different cancerous tissues, but not in corresponding normal tissues. Moreover, in producing antibodies to the antigen expressed prokaryotically, we found that the immunogenicity of the antigen was low in mammalian. Thus we believe that this novel antigen acts as an expression regulator in embryo cells and regains expression in tumor cells. In addition, this antigen is characterized by low differentiation and high proliferation. Molecular function of the antigen needs to be investigated.     

Isolation and characterization of two types of cDNA for mitochondrial adenylate kinase and their expression in Escherichia coli

Two cDNA clones for mitochondrial adenylate kinase were isolated from a cDNA library of bovine liver poly(A)+ RNA by using synthetic oligodeoxynucleotides as probes. The clone containing a 0.9-kilobase insert had the reading frame for a 241-residue protein (AK2A), while the other clone containing a 1.6-kilobase insert had the frame for a 234-residue protein (AK2B). Nucleotide sequences of these two clones were the same in the 5' portion up to the coding sequence for the 233rd residue, but different in the remaining 3' portions. The reported amino acid sequence of mitochondrial adenylate kinase from bovine heart corresponded to AK2A. Neither AK2A nor AK2B had a cleavable NH2-terminal presequence as that found in other imported mitochondrial proteins. RNA blot analysis of poly(A)+ RNAs from bovine liver and heart revealed three species of mRNA with approximate sizes of 0.9, 1.4, and 1.7 kilobases. The 1.7- and 1.4-kilobase species were specific for AK2B, whereas the 0.9-kilobase species was specific for AK2A. In the liver, the 1.7-kilobase mRNA was more abundant, whereas in the heart the 0.9-kilobase mRNA was predominant. The 1.4-kilobase mRNA was present only in the heart. The AK2A- and AK2B-coding sequences were expressed in Escherichia coli cells under the control of trc promoter. Both the products reverted the temperature-sensitive phenotype of the adenylate kinase mutant of E. coli.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell–cell and cell–neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes. Dev. Genet. 21:187–200, 1997. © 1997 Wiley-Liss, Inc.     

Cloning and expression of the human myeloid cell nuclear differentiation antigen: regulation by interferon alpha.

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product was sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow lambda gt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5' and 3' untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon-inducible genes: 204 and interferon regulatory factor 2. In addition, a 12-base sequence matching the interferon-stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5' untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon alpha. The MNDA mRNA was not induced by interferon alpha in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell-specific response to interferon.