Showing papers on "Complementary DNA published in 1989"

Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome

Abstract: A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.

Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.

Abstract: Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7 Transcripts, approximately 6500 nucleotides in size, were detectable in the tissues affected in patients with CF The predicted protein consists of two similar motifs, each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in ATP (adenosine triphosphate) binding A deletion of three base pairs that results in the omission of a phenylalanine residue at the center of the first predicted nucleotide-binding domain was detected in CF patients

Cloning immunoglobulin variable domains for expression by the polymerase chain reaction.

Abstract: We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

Cloning by functional expression of a member of the glutamate receptor family.

Abstract: We have isolated a complementary DNA clone by screening a rat brain cDNA library for expression of kainate-gated ion channels in Xenopus oocytes. The cDNA encodes a single protein of relative molecular mass (Mr) 99,800 which on expression in oocytes forms a functional ion channel possessing the electrophysiological and pharmacological properties of the kainate subtype of the glutamate receptor family in the mammalian central nervous system.

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

Abstract: We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family.

Abstract: A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers.

Abstract: An activating transcription factor (ATF)-binding site (consensus sequence 5'-GTGACGTACAG-3') is a promoter element present in a wide variety of viral and cellular genes. The two best-characterized classes of genes that contain ATF sites are E1A-inducible adenoviral genes and cAMP-inducible cellular genes. Here, we report the isolation of eight ATF cDNA clones, each of which is derived from a separate gene. All ATF cDNA clones examined contain a leucine zipper motif and are significantly similar to one another only within this region. The leucine zipper region of ATF proteins is also similar to that of the AP-1/c-jun family of transcription factors, whose DNA-binding site differs from the ATF-binding site at a single position. DNA binding studies reveal two mechanisms for generating further diversity from the ATF proteins. First, some, but not all, combinations of ATF proteins form heterodimers that efficiently bind to DNA. Second, although all ATF proteins bind to the ATF site, their precise interactions with DNA differ from one another, as evidenced by methylation interference analysis. Our results help to explain how a single promoter element, an ATF site, can be present in a wide variety of promoters.

Molecular cloning and characterization of an insulin-regulatable glucose transporter.

Abstract: A MAJOR mechanism by which insulin stimulates glucose transport in muscle and fat is the translocation of glucose transporters from an intracellular membrane pool to the cell surface1–5. The existence of a distinct insulin-regulatable glucose transporter was suggested by the poor cross-reactivity between antibodies specific for either the HepG2 or rat brain glucose transporters and the rat adipocyte glucose transporter6,7. More direct evidence was provided by the production of a monoclonal antibody (mAb 1F8) specific for the rat adipocyte glucose transporter that immunolabels a species of relative molecular mass 43,000 (43K) present only in tissues that exhibit insulin-dependent glucose transport8, suggesting that this protein may be encoded by a different gene from the previously described mammalian glucose transporters9–13. This antibody has been used to immunoprecipitate a 43K protein that was photoaffinity-labelled with cytochalasin B in a glucose displaceable way, and to immunolabel a protein in the plasma membrane of rat adipocytes, whose concentration was increased at least fivefold after cellular insulin exposure. Here we describe the cloning and sequencing of cDNAs isolated from both rat adipocyte and heart libraries that encode a protein recognized by mAb 1F8, and which has 65% sequence identity to the human HepG2 glucose transporter9. This cDNA hybridizes to an mRNA present only in skeletal muscle, heart and adipose tissue. Our data indicate that this cDNA encodes a membrane protein with the characteristics of the translocatable glucose transporter expressed in insulin-responsive tissues.

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase

Abstract: A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.

Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells

Abstract: Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.

Abstract: cDNA clones of a neuronal-specific mRNA encoding a novel 25-kD synaptosomal protein, SNAP-25, that is widely, but differentially expressed by diverse neuronal subpopulations of the mammalian nervous system have been isolated and characterized. The sequence of the SNAP-25 cDNA revealed a single open reading frame that encodes a primary translation product of 206 amino acids. Antisera elicited against a 12-amino acid peptide, corresponding to the carboxy-terminal residues of the predicted polypeptide sequence, recognized a single 25-kD protein that is associated with synaptosomal fractions of hippocampal preparations. The SNAP-25 polypeptide remains associated with synaptosomal membrane components after hypoosmotic lysis and is released by nonionic detergent but not high salt extraction. Although the SNAP-25 polypeptide lacks a hydrophobic stretch of residues compatible with a transmembrane region, the amino terminus may form an amphiphilic helix that may facilitate alignment with membranes. The predicted amino acid sequence also includes a cluster of four closely spaced cysteine residues, similar to the metal binding domains of some metalloproteins, suggesting that the SNAP-25 polypeptide may have the potential to coordinately bind metal ions. Consistent with the protein fractionation, light and electron microscopic immunocytochemistry indicated that SNAP-25 is located within the presynaptic terminals of hippocampal mossy fibers and the inner molecular layer of the dentate gyrus. The mRNA was found to be enriched within neurons of the neocortex, hippocampus, piriform cortex, anterior thalamic nuclei, pontine nuclei, and granule cells of the cerebellum. The distribution of the SNAP-25 mRNA and the association of the protein with presynaptic elements suggest that SNAP-25 may play an important role in the synaptic function of specific neuronal systems.

A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program.

Abstract: MyoD1 is a nuclear phosphoprotein that is expressed in skeletal muscle in vivo and in certain muscle cell lines in vitro; it has been shown to convert fibroblasts to myoblasts through a mechanism requiring a domain with homology to the myc family of proteins. The BC3H1 muscle cell line expresses skeletal muscle-specific genes upon exposure to mitogen-deficient medium, but does not express MyoD1 at detectable levels. To determine whether BC3H1 cells may express regulatory genes functionally related to MyoD1, a cDNA library prepared from differentiated BC3H1 myocytes, was screened at reduced stringency with the region of the MyoD1 cDNA that shares homology with c-myc. From this screen, a cDNA was identified that encodes a major open reading frame with 72% homology to the myc domain and basic region of MyoD1. The mRNA encoded by this MyoD1-related gene is expressed in skeletal muscle in vivo and in differentiated skeletal myocytes in vitro and is undetectable in cardiac or smooth muscle, nonmuscle tissues, or nonmyogenic cell types. During myogenesis, the MyoD1-related mRNA accumulates several hours prior to other muscle-specific mRNAs and therefore represents an early molecular marker for entry of myoblasts into the differentiation pathway. Transient transfection of 10T1/2 or 3T3 cells with the MyoD1-related cDNA is sufficient to induce myosin heavy-chain expression and to activate a reporter gene under transcriptional control of the muscle creatine kinase 5' enhancer, which functions only in differentiated myocytes. Expression of this cDNA in stably transfected 10T1/2 cells also leads to fusion and muscle-specific gene expression upon exposure to mitogen-deficient medium. Thus, the product of this MyoD1-related gene is sufficient to activate the muscle differentiation program and may substitute for MyoD1 in certain developmental situations. Together, these results suggest the existence of a family of myogenic regulatory genes that share a conserved motif with c-myc.

Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format

Abstract: The in vitro amplification of biologically important nucleic acids has proceeded principally by a strategy of DNA replication. Polymerase chain reaction was the first such protocol to achieve this goal. In this report, a transcription-based amplification system (TAS) is described. Each cycle of the TAS is composed of two steps. The first is a cDNA synthesis step that produces one copy of a double-stranded DNA template for each copy of RNA or DNA target nucleic acid. During the course of this cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase is inserted into the cDNA copy of the target sequence to be amplified. The second step is the amplification of the target sequence by the transcription of the cDNA template into multiple copies of RNA. This procedure has been applied to the detection of human immunodeficiency virus type 1 (HIV-1)-infected cells. After four cycles of TAS, the amplification of the vif region of the HIV-1 RNA genome was measured to be, on the average, 38- to 47-fold per cycle, resulting in a 2-5 x 10(6)-fold increase in the copy number of the original target sequence. This amplification by the TAS protocol allows the detection of fewer than one HIV-1-infected CEM cell in a population of 10(6) uninfected CEM cells. Detection of the TAS-generated RNA from HIV-1-infected cells can easily be accomplished by means of a bead-based sandwich hybridization protocol, which provides additional specificity for the identification of the amplified HIV-1-specific sequence.

The leukocyte common antigen family.

Abstract: The leukocyte-common antigen (L-CA) family is a group of high molecular weight glycoproteins uniquely expressed on the surface of all leukocytes and their hemopoietic progenitors ( 114). Members of this family differ by both protein sequence and carbohydrate structures and are expressed by leukocyte populations in specific patterns ( 15-27). An example of the differential expression is shown for the rat L-CA family in Figure 1 ( 19). Thymocytes express the lowest apparent molecular weight form of 1 80 kd; B lymphocytes express the highest form, 220 kd; and T lymphocytes express multiple forms. Differences also exist between T-cell subsets (Figure 1 C); CD8 T cells (Te/s) express the higher molecular weight forms more abundantly than do CD4 T cells (T H) ' The cell-type-specific patterns of expression are conserved throughout mammalian evolution ( 1, 2, 91 1, 19, 28), and there appear to be similar patterns of expression in chicken lymphocytes (29). L-CA is referred to in the literature by different names, including T200 (30), B220 for the B cell form ( 1 2) , the mouse allotypic marker Ly-5 ( 3 1 ) and more recently CD45 (32). L-CA is the most accurate descriptive name and is used for the purpose of this review. The L-CA family is a major cell surface component of lymphocytes and carries much of the carbohydrate of these cells. It has been estimated that 10% of the lymphocyte surface is occupied by one or more L-CA members (33). Because of this abundance, L-CA was easily detected on SDS-poly­ acrylamide gels of lymphocyte membranes (36-39) (Figure lA). It was also initially characterized as the major specificity of antilymphocyte sera (34) and as an allotypic marker (31 , 35). The primary protein structure has been determined from the analysis of cDNA clones, and this information,

Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor.

Abstract: Cloning and sequencing of the complementary DNA for platelet-derived endothelial cell growth factor indicates that it is a novel factor distinct from previously characterized proteins. The factor, a protein with a relative molecular mass of about 45,000, stimulates endothelial cell growth and chemotaxis in vitro and angiogenesis in vivo.

Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY).

Abstract: The polymerase chain reaction (PCR) is used as part of a new approach to the absolute quantification of mRNA. We describe a PCR aided transcript titration assay (PATTY) which is based on the co-amplification of an in vitro generated transcript differing by a single base exchange from the target mRNA. Identical portions of a total RNA sample are "spiked" with different amounts of this mutated standard RNA, converted to cDNA and amplified by PCR. Because the base exchange creates a novel restriction endonuclease site, the ratio of co-amplified DNA derived from target mRNA to amplified DNA derived from standard RNA can be determined after restriction endonuclease digestion and separation by gel electrophoresis. This method gives accurate results within 24 hours and is useful especially for the quantification of either low-abundance mRNA or more abundant mRNA present in very small amounts of total RNA. The low-abundance mRNA encoding 4-coumarate:CoA ligase (4CL) in cultured potato cells (Solanum tuberosum L.) was measured in a case study. About 100 molecules per assay could be accurately detected by the new method.

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Abstract: Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor (hGM-CSF-R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach. The cloned GM-CSF-R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin-6, erythropoietin and interleukin-2 (beta-chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity (KD = 2(-8) nM) and specificity for human GM-CSF but not interleukin-3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding, and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.

Selective amplification and cloning of four new members of the G protein-coupled receptor family.

Abstract: An approach based on the polymerase chain reaction has been devised to clone new members of the family of genes encoding guanosine triphosphate-binding protein (G protein)-coupled receptors. Degenerate primers corresponding to consensus sequences of the third and sixth transmembrane segments of available receptors were used to selectively amplify and clone members of this gene family from thyroid complementary DNA. Clones encoding three known receptors and four new putative receptors were obtained. Sequence comparisons established that the new genes belong to the G protein-coupled receptor family. Close structural similarity was observed between one of the putative receptors and the 5HT1a receptor. Two other molecules displayed common sequence characteristics, suggesting that they are members of a new subfamily of receptors with a very short nonglycosylated (extracellular) amino-terminal extension.

Mammalian genes coordinately regulated by growth arrest signals and DNA-damaging agents.

Abstract: More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Primary structure and functional expression from complementary DNA of the rod photoreceptor cyclic GMP-gated channel

Abstract: The complete amino-acid sequence of the cyclic GMP-gated channel from bovine retinal rod photo-receptors, deduced by cloning and sequencing its complementary DNA, shows that the protein contains several putative transmembrane segments, followed by a region that is similar to the cyclic GMP-binding domains of cyclic GMP-dependent protein kinase. Expression of the complementary DNA produces cyclic GMP-gated channel activity in Xenopus oocytes.

A third human retinoic acid receptor, hRAR-gamma

Abstract: Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-alpha and hRAR-beta) cDNAs and have recently cloned their murine cognates (mRAR-alpha and mRAR-beta) together with a third RAR (mRAR-gamma) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-gamma cDNA was used here to clone its human counterpart (hRAR-gamma) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-gamma cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either alpha, beta, or gamma) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-alpha, -beta, and -gamma may perform specific functions. We show also that hRAR-gamma RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-gamma mediates some of the retinoid effects in this tissue.

Illegitimate transcription: transcription of any gene in any cell type.

Abstract: Using in vitro amplification of cDNA by the polymerase chain reaction, we have detected spliced transcripts of various tissue-specific genes (genes for anti-Mullerian hormone, beta-globin, aldolase A, and factor VIIIc) in human nonspecific cells, such as fibroblasts, hepatoma cells, and lymphoblasts. In rats, erythroid- and liver-type pyruvate kinase transcripts were also detected in brain, lung, and muscle. The abundance of these "illegitimate" transcripts is very low; yet, their existence and the possibility of amplifying them by the cDNA polymerase chain reaction provide a powerful tool to analyze pathological transcripts of any tissue-specific gene by using any accessible cell.