Cloning and expression of the cDNA coding for a human lymphocyte IgE receptor.

Low-affinity receptors (Fc epsilon R) and secreted factors (IgE-BF) which bind to immunoglobulins of the IgE isotype play a key role in the regulation of human IgE synthesis. We report here the cloning of a cDNA coding for the Fc epsilon R of the human B-lymphoblast cell line RPMI 8866. The nucleotide sequence of this cDNA predicts a polypeptide with 321 amino acids and a mol. wt of 36,281 daltons. A functional Fc epsilon R capable of binding IgE was expressed in Chinese hamster ovary cells after stable transformation with the cDNA which had been cloned into a mammalian expression vector. Amino acid sequence analysis of IgE-BF purified from RPMI 8866 cells revealed an amino-terminal sequence of 19 residues which coincides with the predicted amino acid sequence of the Fc epsilon R, starting at residues 148 and 150. A computer search with the translated amino acid sequence of the Fc epsilon R revealed a domain of 120 amino acids having striking homology to the human asialoglycoprotein receptors.     

Cloning of several cDNA segments coding for human liver proteins

A human cDNA library was constructed using M13 derivative vectors. The simple and rapid procedures for sequencing single-stranded DNA by the dideoxy chain termination method allowed a screening of individual clones directly by DNA sequence analysis. Some of these clones were identified as coding for: serum albumin, alpha1-antitrypsin, retinol-binding protein, prothrombin, haptoglobin, and metallothionein. Furthermore, a clone coding for aldolase B was tentatively identified on the basis of high sequence homology with rabbit muscle aldolase.     

Cloning and expression of cDNA coding for bouganin.

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.     

Cytokine and chemokine expression in a rat endometriosis is similar to that in human endometriosis

The pathogenesis of endometriosis, a gynecologic disorder associated with infertility, appears to involve immune responses. However, the details involved have not been clarified. In this study, we analyzed expression levels of interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1, eosinophil chemotactic protein, macrophage inflammatory protein-1α, and regulated on activation normal T cell expressed and secreted (RANTES) and CC chemokine receptor 1 in endometriotic lesions in a rat model in which endometrium is autotransplanted onto peritoneal tissue and found that they were remarkably increased, while those of IL-2, IL-4, and interferon-γ were not. These results were obtained in a rat model induced by autologous, not allogeneic, transplantation of endometrial epithelium to the peritoneum. Expression of these factors is consistent with that of endometriosis in humans. Therefore, this model may be useful in the investigation of the pathogenesis and treatment of endometriosis.     

A murine cDNA encodes a pan-epithelial glycoprotein that is also expressed on plasma cells.

Using a subtractive cDNA approach, we have identified a number of genes expressed in murine plasmacytomas, but not B or pre-B lymphomas. One of these genes, 289A, expresses a 1.8-kb microsomally localized mRNA that encodes a 314-amino-acid protein containing a signal sequence and a hydrophobic transmembrane domain. Sequence comparison suggests that the predicted protein is the murine homologue of a human cell surface pan-epithelial glycoprotein known variously as EGP, GA733-2, KSA, and KS1/4, recognized by mAb HEA125, GA733, KS1/4, CO17-1A, M74, and 323/A3. The 289A mRNA is highly expressed in normal murine tissues containing epithelial cells, and at a low level in plasma cells induced by LPS stimulation of spleen B lymphocytes. It is expressed in 15 of 16 plasmacytomas, but at a much lower level, if at all, in pre-B or B lymphomas. In human B cell lines, 289A detects a 1.5-kb mRNA in the myeloma cell line 8226, but not in Burkitt's lymphoma or lymphoblastoid cell lines. Subsequent FACS analysis of human cell lines with the mAb GA733 and KS1/4 demonstrated concordant expression of the mRNA and the protein. We conclude that 289A is the murine homologue of EGP, GA733-2, KSA, and KS1/4 Ag. Although its expression was previously thought to be restricted to epithelial cells, it is also expressed in plasma cells and is a B lymphocyte differentiation Ag. Because of the multiplicity of names, we propose calling the human gene hEGP314, and the murine gene mEGP314.     

Localization of the ecto-ATPase (ecto-nucleotidase) in the rat hepatocyte plasma membrane. Implications for the functions of the ecto-ATPase

The surface distribution of the plasma membrane Ca2+ (Mg2+)-ATPase (ecto-ATPase) in rat hepatocytes was determined by several methods. 1) Two polyclonal antibodies specific for the ecto-ATPase were used to examine the distribution of the enzyme in frozen sections of rat liver by immunofluorescence. Fluorescent staining was observed at the bile canalicular region of hepatocytes. 2) Plasma membranes were isolated from the canalicular and sinusoidal regions of rat liver. The specific activity of ecto-ATPase in the canalicular membranes was 22 times higher than that of sinusoidal membranes. The enrichment of the ecto-ATPase activity in the canalicular membrane is closely parallel to that of two other canalicular membrane markers, gamma-glutamyltranspeptidase and leucine aminopeptidase. 3) By immunoblots with polyclonal antibodies against the ecto-ATPase and the Na+,K+-ATPase, it was found that the ecto-ATPase protein was only detected in canalicular membranes and not in sinusoidal membranes, while the Na+,K+-ATPase protein was only detected in sinusoidal membranes and not in canalicular membranes. These results indicate that the ecto-ATPase is enriched in the canalicular membranes of rat hepatocytes.     

Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).     

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum.

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.     

The transmembrane domain of glycoprotein Ibbeta is critical to efficient expression of glycoprotein Ib-IX complex in the plasma membrane

Lack of expression of glycoprotein (GP) Ib-IX-V complex in platelets often results from mutations in its three subunits: GP Ibalpha, GP Ibbeta, or GP IX. The requirement of all three subunits in the efficient surface expression of the receptor complex has been reproduced in Chinese hamster ovary cells. Here, we probed the role of the transmembrane domains in expression of the GP Ib-IX complex and potential interactions between these domains. Replacing the transmembrane domains of either GP Ibalpha or GP Ibbeta, but not that of GP IX, with unrelated sequences markedly diminished surface expression of the GP Ib-IX complex in transiently transfected Chinese hamster ovary cells. Replacement of the Ibbeta transmembrane domain produced the largest effect. Furthermore, several single-site mutations in the Ibbeta transmembrane domain were found to significantly decrease overall expression as well as surface expression of GP Ibalpha, probably by perturbing the interaction between the Ibalpha and Ibbeta transmembrane domains and in turn reducing the stability of GP Ibalpha in the cell. Mutations S503V and S503L in the Ibalpha transmembrane domain partly reversed the expression-decreasing effect of mutation H139L, but not the others, in the Ibbeta transmembrane domain, suggesting a specific interaction between these two polar residues. Together, our results have demonstrated the importance of the Ibbeta transmembrane domain, through its interaction with the Ibalpha counterpart, to the proper assembly and efficient surface expression of the GP Ib-IX complex.     

Cloning and sequencing of a full length cDNA coding for human retinol-binding protein.

We have isolated and sequenced a cDNA clone coding for human Retinol Binding Protein. The sequence indicates that Retinol Binding Protein is synthesized as a single polypeptide chain precursor which is then matured to the secreted protein by removal of a leader peptide. Southern and Northern blot analysis suggest that the gene is present in one or few copies per haploid genome and is transcribed in a single mRNA species.     

Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor

A Drosophila cDNA clone was obtained using the human insulin receptor cDNA sequence as a probe. The 3586 bp nucleotide sequence predicted a single polypeptide of 1095 amino acid residues which showed considerable homology (35.2%) with the human insulin receptor precursor. Although the cDNA was incomplete at its 5'-terminal region, it encodes a transmembrane glycoprotein as a single precursor of a two subunit molecule having a structural architecture similar to that of the human insulin receptor precursor. The presumptive beta subunit carries a well conserved Tyr kinase domain which showed 63.5% homology with that of human insulin receptor; however the protein of the alpha subunit is only weakly conserved (25%).     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

Identification and characterization of a human cytomegalovirus gene coding for a membrane protein that is conserved among human herpesviruses.

A rabbit antiserum was raised against envelope material from purified human cytomegalovirus strain AD169. The serum recognized polypeptides 200, 170, 160, 75, 58, and 45 kilodaltons in size. It was used to screen a cDNA library constructed from poly(A)+ RNA from human cytomegalovirus-infected cells in the expression vector lambda gt11. A recombinant bacteriophage expressing cytomegalovirus-specific sequences was identified, and the corresponding gene was mapped to the HindIII R fragment. The gene is transcribed into a late 1.5-kilobase RNA. The nucleotide sequence of the coding region was determined. Computer analysis of the gene product revealed a polypeptide containing multiple potential membrane-spanning domains, representing a type of protein not identified in the envelope of herpesviruses before. The protein shows homology on the amino acid level to hypothetical proteins from reading frames BBRF3 of Epstein-Barr virus, UL10 of herpes simplex virus type 1, and ORF50 of varicella-zoster virus. By using an antiserum raised against procaryote-expressed parts of the cytomegalovirus membrane protein, a 45-kilodalton structural component of the virus was identified as the gene product.     

Cloning a rat meprin cDNA reveals the enzyme is a heterodimer.

The structure of the kidney microvillar membrane metallopeptidase meprin (EC 3.4.24.18) from rats has been examined. Previously reported to be a homotetramer, we demonstrate that the enzyme is composed of two similar but distinct subunits through tryptic peptide mapping and the sequencing of peptides of the papain solubilized form of the enzyme. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the native rat meprin tetramer is dissociated by detergent into disulfide-linked heterodimers. A full-length cDNA clone encoding one of the meprin subunits has been isolated and sequenced. The cDNA contains an open reading frame of 668 amino acids, coding for a polypeptide of molecular weight 75,054. The enzyme contains the zinc binding sequence HEFLH and a potential membrane-spanning region near its amino terminus. Comparison of this clone with peptide sequences from mouse meprins A and B shows that the clone is a B type or beta subunit. Northern blot analysis is consistent with the existence of two distinct subunits and further indicates that rat meprin subunits may be differentially expressed in various rat tissues.     

Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein

An extended synthetic oligonucleotide (58-mer) has been used to identify and characterize a human liver gap junction cDNA. The cDNA is 1,574 bases long and contains the entire coding region for a gap junction protein. In vitro translation of the RNA products of this cDNA is consistent with it coding for a 32,022-D protein. Southern blot analysis indicates that the gap junction gene is present as a single copy, and that it can be detected in a variety of organisms using the human liver cDNA as a probe. The human cDNA has been used to screen a rat liver cDNA library, and a rat liver junction cDNA clone has been isolated. The rat liver clone is 1,127 bases in length, and it has strong sequence homology to the human cDNA in the protein-coding region, but less extensive homology in the 3'-untranslated region.