Showing papers on "Complementary DNA published in 1991"
TL;DR: Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs), which will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing.
Abstract: Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.
2,375 citations
TL;DR: This article showed that VEGF is produced by cultured vascular smooth muscle cells by polymerase chain reaction and cDNA cloning, and showed that the three forms of the human vascular endothelial growth factor (VEGF) protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length.
1,992 citations
TL;DR: The expression of the TIS10 gene appears to be highly cell type-restricted in cultured cell lines; of 12 cell lines tested under superinducing conditions, only the rodent embryonic Swiss 3T3 and Rat1 cell lines expressed TIS12 gene.
1,781 citations
TL;DR: It is demonstrated that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium and, like VPF, proves to be a dimeric protein.
Abstract: A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.
1,055 citations
TL;DR: A complementary DNA clone encoding a human noradrenaline transporter is isolated and the predicted protein sequence demonstrates significant amino-acid identity with the Na+/γ-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins.
Abstract: AT most synapses, chemical signalling is terminated by a rapid reaccumulation of neurotransmitter into presynaptic terminals1–5. Uptake systems for the biogenic amines are the initial site of action for therapeutic antidepressants and drugs such as cocaine and the amphetamines. We have isolated a complementary DNA clone encoding a human noradrenaline transporter. The cDNA sequence predicts a protein of 617 amino acids, with 12–13 highly hydrophobic regions compatible with membrane-spanning domains. Expression of the cDNA clone in transfected HeLa cells indicates that noradrenaline transport activity is sodium-dependent and sensitive to selective noradrenaline transport inhibitors. Trans-porter RNA is localized to the brainstem and the adrenal gland. The predicted protein sequence demonstrates significant amino-acid identity with the Na+/γ-aminobutyric acid transporter, thus identifying a new gene family for neurotransmitter transporter proteins. Analysis of its structure and function may lead to structure-based drug design for the treatment of human depression and could help determine whether transporter abnormalities underlie affective disorders.
963 citations
TL;DR: Immunoblot analysis of the affinity- purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD.
Abstract: Human umbilical vein endothelial (HUVE) cells have been previously reported to express the genes for the A and B chains of PDGF and to secrete PDGF-related factors into culture media. Antihuman PDGF IgG affinity chromatography was used to purify PDGF-related activity from HUVE cell-conditioned media. Immunoblot analysis of the affinity-purified proteins with anti-PDGF IgG and antibodies specific for the A or B chain peptides of PDGF combined with chemotactic and mitogenic assays revealed that the major PDGF immunorelated molecule secreted by HUVE cells is a monomer of approximately 36-38 kD and that less than 10% of the purified biologically active molecules are PDGF A or B chain peptides. Screening of an HUVE cell cDNA library in the expression vector lambda gtl 1 with the anti-PDGF antibody resulted in the cloning and sequencing of a cDNA with an open reading frame encoding a 38-kD cysteine-rich secreted protein which we show to be the major PDGF-related mitogen secreted by human vascular endothelial cells. The protein has a 45% overall homology to the translation product of the v-src-induced CEF-10 mRNA from chick embryo fibroblasts. We have termed this new mitogen connective tissue growth factor.
890 citations
Journal Article•
TL;DR: The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
Abstract: Previously we have reported the purification and characterization of a novel cytokine from an EBV-transformed B cell line, RPMI 8866. This factor, termed natural killer cell stimulatory factor (NKSF), possessed pleiotropic activities including the induction of IFN-gamma from PBL, enhancement of cytotoxicity by NK cells, and stimulation of the proliferation of PBL. Purified NKSF was found to be a disulfide-linked heterodimeric protein composed of 35-kDa and 40-kDa subunits (p35 and p40). We now report the molecular cloning of cDNA for both subunits of NKSF from RPMI 8866 cellular RNA. The cDNA sequences indicate that both genes are novel, and Southern blot analysis confirmed that both cDNA are of human genomic origin. [35S]Methionine labeling indicated that cos-1 cells transfected with either p35 or p40 cDNA produced unique protein species of appropriate size. Methionine labeling of cos-1 cells cotransfected with p35 plus p40 cDNA yielded a broad band migrating between 70 and 90 kDa on a nonreducing gel. Reduction of this high molecular weight material yielded bands correlating with p35 and p40 gene products. Only culture supernatant from cotransfected cos-1 cells had a high level of NKSF biologic activity. That the high molecular weight material was responsible for this activity was indicated by the observation that biologic activity in the culture supernatant migrated at 70 to 90 kDa in a nonreducing gel. Furthermore, anti-p40 serum was able to block the biologic activities of both recombinant and natural NKSF, which indicates that it is a component of the active protein. In contrast, no activity could be detected in the supernatants of cos-1 cells transfected with p40 or p35 cDNA alone. The spectrum of biologic activity produced by cotransfected cos-1 cells was the same as NKSF purified to homogeneity from the RPMI 8866 cell line. A synergistic augmentation of some of these responses was found by the addition of IL-2 or the co-stimulators PHA or phorbol diester. The synergistic stimulation by NKSF plus IL-2 of T and NK function supports the possibility that these cytokines might prove useful in cancer therapy.
874 citations
TL;DR: The primary structure of a voltage-dependent cal-cium channel from rabbit brain has been deduced by cloning and sequencing the complementary DNA and it is suggested that it is expressed predominantly in cerebellar Purkinje cells and granule cells.
Abstract: The primary structure of a voltage-dependent cal-cium channel from rabbit brain has been deduced by cloning and sequencing the complementary DNA. Calcium channel activity expressed from the cDNA is dramatically increased by coexpression of the α2 and β subunits, known to be associated with the dihydropyridine receptor. This channel is a high voltage-activated calcium channel that is insensitive both to nifedipine and to ω-conotoxin. We suggest that it is expressed predominantly in cerebellar Purkinje cells and granule cells.
860 citations
TL;DR: A candidate AN cDNA (D11S812E) that is completely or partially deleted in two patients with AN is cloned and the concordance between expression and pathology, map location, structure, and predicted function argues that the cDNA corresponds to the AN gene.
827 citations
TL;DR: Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but B CRFI lacks comparable activity in this assay, suggesting that BCRFI may have conserved only a subset of hIL -10 activities.
Abstract: We have demonstrated the existence of human cytokine synthesis inhibitory factor (CSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BCRFI. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this assay, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.
796 citations
TL;DR: An adult rat cerebellar cDNA library in search of novel protein tyrosine-kinase (PTK) cDNAs showed that the trkB gene is expressed predominantly in the brain and that trkB expresses multiple mRNAs, ranging from 0.7 to 9 kb, and hybridization of cerebral mRNAAs with a variety of probes indicates that there are m RNAs encoding truncated trkB receptors.
Abstract: We have screened an adult rat cerebellar cDNA library in search of novel protein tyrosine-kinase (PTK) cDNAs. A cDNA for a putative PTK, trkB, was cloned, and its sequence indicates that it is likely to be derived from a gene for a ligand-regulated receptor closely related to the human trk oncogene. Northern (RNA) analysis showed that the trkB gene is expressed predominantly in the brain and that trkB expresses multiple mRNAs, ranging from 0.7 to 9 kb. Hybridization of cerebral mRNAs with a variety of probes indicates that there are mRNAs encoding truncated trkB receptors. Two additional types of cDNA were isolated, and their sequences are predicted to encode two distinct C-terminally truncated receptors which have the complete extracellular region and transmembrane domain, but which differ in their short cytoplasmic tails.
TL;DR: The expression cloning of a complementary DNA encoding a bovine angiotensin II receptor is reported to overcome the difficulties faced in purifying the receptor owing to its instability and low concentration.
Abstract: Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.
TL;DR: Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini.
Abstract: CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5' untranslated nucleotide sequence, an 807-bp open reading frame, and approximately 2 kilobases of 3' untranslated sequence containing a polyadenylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DNA sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.
TL;DR: The cloned MAD-3 cDNA encodes an I kappa B-like protein that is likely to be involved in regulation of transcriptional responses to NF-kappa B, including adhesion-dependent pathways of monocyte activation.
TL;DR: This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.
Abstract: THROMBOXANE A2 is a very unstable arachidonate metabolite, yet a potent stimulator of platelet aggregation and a constrictor of vascular and respiratory smooth muscles1. It has been implicated as a mediator in diseases such as myocardial infarction, stroke and bronchial asthma2. Using a stable analogue of this compound we recently purified the human platelet thromboxane A2 receptor to apparent homogeneity3. Using an oligonucleotide probe corresponding to its partial amino-acid sequence, we have obtained a complementary DNA clone encoding this receptor from human placenta and a partial clone from cultured human megakaryocytic leukaemia cells. The placenta cDNA encodes a protein of 343 amino acids with seven putative transmembrane domains. The protein expressed in COS-7 cells binds drugs with affinities identical to those of the platelet receptor, and that in Xenopus oocytes opens Ca2+-activated Cl- channel on agonist stimulation. Northern blot analysis and nucleotide sequences of the two clones suggest that an identical species of the thromboxane A2 receptor is present in platelets and vascular tissues. This first report on the molecular structure of an eicosanoid receptor will promote the molecular pharmacology and pathophysiology of these bioactive compounds.
TL;DR: The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).
Abstract: A cDNA clone encoding a receptor protein which presents all the characteristics of a guanine-nucleotide-binding protein (G-protein)-coupled receptor was isolated from a human brain stem cDNA library. The probe used (HGMP08) was a 600 bp DNA fragment amplified by a low-stringency PCR, using human genomic DNA as template and degenerate oligonucleotide primers corresponding to conserved sequences amongst the known G-protein-coupled receptors. The deduced amino acid sequence encodes a protein of 472 residues which shares 97.3% identity with the rat cannabinoid receptor cloned recently [Matsuda, Lolait, Brownstein, Young & Bronner (1990) Nature (London) 346, 561-564]. Abundant transcripts were detected in the brain, as expected, but lower amounts were also found in the testis. The same probe was used to screen a human testis cDNA library. The cDNA clones obtained were partially sequenced, demonstrating the identity of the cannabinoid receptors expressed in both tissues. Specific binding of the synthetic cannabinoid ligand [3H]CP55940 was observed on membranes from Cos-7 cells transfected with the recombinant receptor clone. In stably transfected CHO-K1 cell lines, cannabinoid agonists mediated a dose-dependent and stereoselective inhibition of forskolin-induced cyclic AMP accumulation. The ability to express the human cannabinoid receptor in mammalian cells should help in developing more selective drugs, and should facilitate the search for the endogenous cannabinoid ligand(s).
TL;DR: It is shown that RCCl specifically catalyses the exchange of guanine nucleo-tides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras), and that the protein binds GDP and GTP.
Abstract: THE product of the gene RCC1 (regulator of chromosome condensation) in a BHK cell line is involved in the control of mitotic events1. Homologous genes have been found in Xenopus2, Drosophila3 and yeast4,5. A human genomic DNA fragment and complementary DNA that complement a temperature-sensitive mutation ofRCC1 in BHK21 cells6,7 encode a protein of relative molecular mass 45,000 (Mr45K) which is located in the nucleus and binds to chromatin8. We have recently isolated a protein from HeLa cells that strongly binds an anti-RCCl antibody and has the same molecular mass, DNA-binding properties, and amino-acid sequence as the 205 residues already identified9. HeLa cell RCCl is complexed to a protein of Mr 25K (ref. 9). We have shown10 that this 25K protein has a sequence homologous to the translated reading frame ofTC4, a cDNA found by screening a human teratocarcinoma cDNA library with oligonucleotides coding for a ras consensus sequence11, and that the protein binds GDP and GTP. We have referred to this protein as the Ran protein (ras-related nuclear protein). In addition to the fraction of Ran protein complexed to RCCl, a 25-fold molar excess of the protein over RCCl was found in the nucleoplasm of HeLa cells. Here we show that RCCl specifically catalyses the exchange of guanine nucleo-tides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras).
TL;DR: The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood, and could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.
TL;DR: Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino- terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19.
Abstract: Processing of the putative structural proteins of hepatitis C virus was examined by using an in vitro expression system. An RNA transcript for cell-free translation was prepared from a cDNA construct that encompasses the region encoding the 980 amino-terminal residues of the viral polyprotein precursor. Processing of the in vitro translation product proceeded cotranslationally in the presence of microsomal membranes and generated four major membrane-associated products. Two of these four major products, named gp35 and gp70, were shown to be transported into microsomes and heavily glycosylated, suggesting that the processing events are partly mediated by the signal peptidase of the endoplasmic reticulum. The other two products, p19 and p21, were probably associated with the outer surface of the microsomal membrane. Analysis of processed proteins translated from a series of truncated forms of the cDNA construct as well as determination of amino-terminal amino acid sequences of gp35 and gp70 indicated that these four products are arranged from the amino-terminal end of the polyprotein precursor in the order: NH2-p22-gp35-gp70-p19. Both gp35 and gp70 could be candidates of initially processed forms of envelope proteins of the hepatitis C virus.
TL;DR: MTNF-R2 showed strong specificity for recombinant murine TNF-alpha, suggesting that the various activities of human tumor necrosis factor alpha reported in mice or in murine cell lines are probably mediated by mT NF-R1.
Abstract: Complementary DNA clones encoding two distinct tumor necrosis factor receptors were isolated from a mouse macrophage cDNA library. The cDNA for murine tumor necrosis factor receptor type 1 (mTNF-R1) predicts a mature polypeptide of 425 amino acids that is 64% identical to its human counterpart, whereas the cDNA of murine tumor necrosis factor receptor type 2 (mTNF-R2) predicts a mature protein of 452 amino acids that is 62% identical to human tumor necrosis factor receptor type 2. The two murine tumor necrosis factor receptors have limited sequence homology (approximately 20% identity) in their extracellular regions but no apparent similarity in their cytoplasmic portions. Northern (RNA) analysis indicates a single 2.6-kilobase (kb) transcript for mTNF-R1; a 3.6-kb and a more predominant 4.5-kb transcript are observed for mTNF-R2. A human cell line transfected with either mTNF-R1 or mTNF-R2 expression vectors specifically bound 125I-labeled recombinant murine tumor necrosis factor alpha (TNF-alpha). Although mTNF-R1 had a similar affinity for both recombinant murine TNF-alpha and human TNF-alpha, mTNF-R2 showed strong specificity for recombinant murine TNF-alpha. This result suggests that the various activities of human tumor necrosis factor alpha reported in mice or in murine cell lines are probably mediated by mTNF-R1.
TL;DR: The results verify the existence of at least two 5α-reductases in man and provide insight into a fundamental hormone-mediated event in male sexual differentiation.
Abstract: THE conversion of testosterone into dihydrotestosterone by steroid 5α-reductase is a key reaction in androgen action, and is essential both for the formation of the male phenotype during embryogenesis and for androgen-mediated growth of tissues such as the prostate1,2. Single gene defects that impair this conversion lead to pseudohermaphroditism in which 46 X, Y males have male internal urogenital tracts, but female external genitalia3. We have described the isolation of a human 5α-reductase complementary DNA from prostate4. Subsequent cloning and genetic studies showed that this gene (designated 5α-reductase 1) was normal in patients with Sor-reductase deficiency26. We report here the isolation of a second 5α-reductase cDNA by expression cloning and the polymerase chain reaction. The biochemical and pharmacological properties of this cDNA-encoded enzyme (designated 5α-reductase 2) are consistent with it being the major isozyme in genital tissue. A deletion in this gene is present in two related individuals with male pseudohermaphroditism caused by 5α-reductase deficiency. These results verify the existence of at least two 5α-reductases in man and provide insight into a fundamental hormone-mediated event in male sexual differentiation.
TL;DR: In this paper, a fetal liver kinase 1 (Flk-1) was cloned from mouse cell populations enriched for hematopoietic stem and progenitor cells.
Abstract: We have cloned a receptor tyrosine kinase cDNA, designated fetal liver kinase 1 (Flk-1), from mouse cell populations enriched for hematopoietic stem and progenitor cells. Sequence analysis of this clone reveals strong homology to the c-Kit subfamily of receptor kinases, and in particular to the Flt gene product. Chromosomal mapping shows that the Flk-1, Kit, and Pdgfra genes are closely linked. Flk-1 mRNA is expressed in primitive and more mature hematopoietic cells as well as in a wide variety of nonhematopoietic tissues.
TL;DR: A complementary DNA clone for a serotonin (5HT) transporter has been isolated from rat basophilic leukemia cells and predicts a 653-amino acid protein with 12 to 13 putative transmembrane domains.
Abstract: A complementary DNA clone for a serotonin (5HT) transporter has been isolated from rat basophilic leukemia cells. The complementary DNA sequence predicts a 653-amino acid protein with 12 to 13 putative transmembrane domains. The 5HT transporter has significant homology to the gamma-aminobutyric acid, dopamine, and norepinephrine transporters. Uptake by CV-1 cells expressing the transporter complementary DNA resembles 5HT uptake by platelets and brain synaptosomes; it is sensitive to antidepressants, amphetamine derivatives, and cocaine.
TL;DR: Data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation.
Abstract: Lymphoid-specific cDNA clones were isolated that encode a nuclear protein with homology to the chromosomal nonhistone protein HMG-1 and to putative regulators of cell specialization, including the mammalian testis-determining factor SRY and fungal mating-type proteins The gene represented by the isolated cDNA clones, termed LEF-1 (lymphoid enhancer-binding factor 1), is developmentally regulated and expressed in pre-B and T lymphocytes but not in later-stage B cells or nonlymphoid tissues Both endogenous and recombinant LEF-1 were shown to bind to a functionally important site in the T-cell antigen receptor (TCR) alpha enhancer Maximal TCR alpha enhancer activity was found to parallel the cell type-specific expression pattern of LEF-1 Moreover, forced expression of recombinant LEF-1 in late stage B cells increases TCR alpha enhancer function Taken together, these data suggest that LEF-1 is a regulatory participant in lymphocyte gene expression and differentiation
TL;DR: DUK-1 was designated as the alpha subunit of the hIL-3R, indicating that different alpha subunits compete for a common beta subunit in human interleukin-3 cells.
TL;DR: The DNA binding activities of some basic region and putative helix-loop-helix-containing transcriptional factors can be inhibited by the Id protein, which selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3.
Abstract: The DNA binding activities of some basic region and putative helix-loop-helix (bHLH)-containing transcriptional factors can be inhibited by the Id protein. Because Id contains the HLH motif for dimerization but not the basic amino acid region for DNA binding, heterodimers of Id with bHLH transcriptional factors may not bind to DNA. We have isolated and characterized the gene and cDNA clones for a new Id protein, designated Id2. The Id2 protein contains a helix-loop-helix motif similar to that of the previously described Id protein (referred to here as Id1), but the two proteins are different elsewhere. Id1 and Id2 are encoded by two unlinked genes, as shown by chromosome mapping. The two Id proteins have similar inhibitory activities. They selectively bind to and inhibit the function of one set of bHLH proteins, typified by E2A.E47 and E2B.m3, but not that of the other set, including TFE3, USF, and AP4. The Id proteins also homodimerize poorly. Expression of both Id genes is down-regulated during differentiation in a variety of cell types.
TL;DR: In analyzing the glucocorticoid effects on PGHS synthesis in C127 mouse fibroblasts, a novel 4-kilobase mRNA is detected that is related to a PGHS cDNA cloned from an ovine seminal vesicle library and encodes a 70-kDa protein that is specifically immunoprecipitated by anti-PGHS serum.
TL;DR: The availability of platelet PGG/H synthase cDNA should enhance the understanding of the important structure/function domains of this protein and its gene regulation.
Abstract: Platelets metabolize arachidonic acid to thromboxane A2, a potent platelet aggregator and vasoconstrictor compound. The first step of this transformation is catalyzed by prostaglandin (PG) G/H synthase, a target site for nonsteroidal antiinflammatory drugs. We have isolated the cDNA for both human platelet and human erythroleukemia cell PGG/H synthase using the polymerase chain reaction and conventional screening procedures. The cDNA encoding the full-length protein was expressed in COS-M6 cells. Microsomal fractions from transfected cells produced prostaglandin endoperoxide-derived products which were inhibited by indomethacin and aspirin. Mutagenesis of the serine residue at position 529, the putative aspirin acetylation site, to an asparagine reduced cyclooxygenase activity to barely detectable levels, an effect observed previously with the expressed sheep vesicular gland enzyme. Platelet-derived growth factor and phorbol ester differentially regulated the expression of PGG/H synthase mRNA levels in th...
TL;DR: A rat dopamine transporter complementary DNA has been isolated with combined complementary DNA homology and expression approaches and Binding of the cocaine analog [3H]CFT to transfected COS cell membranes yields a pharmacological profile similar to that in striatal membranes.
Abstract: A rat dopamine (DA) transporter complementary DNA has been isolated with combined complementary DNA homology and expression approaches. The DA transporter is a 619-amino acid protein with 12 hydrophobic putative membrane-spanning domains and homology to the norepinephrine and gamma-aminobutyric acid transporters. The expressed complementary DNA confers transport of [3H]DA in Xenopus oocytes and in COS cells. Binding of the cocaine analog [3H]CFT ([3H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane) to transfected COS cell membranes yields a pharmacological profile similar to that in striatal membranes.
TL;DR: DNA sequence analysis identified an open reading frame of 1440 base pairs encoding a protein of 480 amino acids that was supported by the synthesis of a Mr 58,000 protein in an in vitro translation system that used RNA transcribed from cloned cDNAs with SP6 RNA polymerase.
Abstract: A partial cDNA was isolated that encoded a protein kinase, termed rac (related to the A and C kinases). This cDNA was subsequently used to screen libraries derived from the human cell lines MCF-7 and WI38 and led to the isolation of full-length cDNA clones. DNA sequence analysis identified an open reading frame of 1440 base pairs encoding a protein of 480 amino acids (Mr, 55,716). This result was supported by the synthesis of a Mr 58,000 protein in an in vitro translation system that used RNA transcribed from cloned cDNAs with SP6 RNA polymerase. The predicted protein contains consensus sequences characteristic of a protein kinase catalytic domain and shows 73% and 68% similarity to protein kinase C and the cAMP-dependent protein kinase, respectively. Northern (RNA) analysis revealed a single mRNA transcript of 3.2 kilobases that varied up to 300-fold between different cell lines. Specific antisera directed towards the carboxyl terminal of the rac protein kinase were prepared and used to identify that phosphorylated several substrates in immunoprecipitates prepared with the rac-specific antisera.