Showing papers on "Complementary DNA published in 1990"
TL;DR: It is proposed that HLH proteins lacking a basic region may negatively regulate other HLHprotein through the formation of nonfunctional heterodimeric complexes.
2,203 citations
TL;DR: A contiguous stretch of DNA comprising 370 kilobase pairs has now been cloned from a region of chromosome 18q suspected to reside near the DCC gene, which may play a role in the pathogenesis of human colorectal neoplasia, perhaps through alteration of the normal cell-cell interactions controlling growth.
Abstract: Allelic deletions involving chromosome 18q occur in more than 70 percent of colorectal cancers. Such deletions are thought to signal the existence of a tumor suppressor gene in the affected region, but until now a candidate suppressor gene on this chromosomal arm had not been identified. A contiguous stretch of DNA comprising 370 kilobase pairs (kb) has now been cloned from a region of chromosome 18q suspected to reside near this gene. Potential exons in the 370-kb region were defined by human-rodent sequence identities, and the expression of potential exons was assessed by an "exon-connection" strategy based on the polymerase chain reaction. Expressed exons were used as probes for cDNA screening to obtain clones that encoded a portion of a gene termed DCC; this cDNA was encoded by at least eight exons within the 370-kb genomic region. The predicted amino acid sequence of the cDNA specified a protein with sequence similarity to neural cell adhesion molecules and other related cell surface glycoproteins. While the DCC gene was expressed in most normal tissues, including colonic mucosa, its expression was greatly reduced or absent in most colorectal carcinomas tested. Somatic mutations within the DCC gene observed in colorectal cancers included a homozygous deletion of the 5' end of the gene, a point mutation within one of the introns, and ten examples of DNA insertions within a 0.17-kb fragment immediately downstream of one of the exons. The DCC gene may play a role in the pathogenesis of human colorectal neoplasia, perhaps through alteration of the normal cell-cell interactions controlling growth.
1,716 citations
TL;DR: An adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells for expression of two cytokines and the copy number of the human GM-CSF gene in normal human cells is described.
Abstract: The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of the RNA to be assayed were added to serial dilutions of a competitor DNA fragment that differed from the cDNA of interest by having either a small intron or a mutated internal restriction enzyme site. Therefore, the same primers were used to coamplify the unknown and the competitor. The ratio of products remains constant through the amplification and can be readily quantitated. In unstimulated cells, no GM-CSF or IL-3 mRNA could be detected. However, with appropriate induction, mRNA for both cytokines was detected and quantitated in as few as 200 cells. Competitive PCR was also used to accurately quantitate the copy number of the human GM-CSF gene in normal human cells, in a clonal population of cells from a patient with 5q- syndrome, and in a human-hamster cell line known to have only one copy of the human GM-CSF gene.
1,564 citations
TL;DR: Using the hPR gene 5′‐flanking sequences as promoter region in chimeric genes, it is shown that a functional promoter directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15.
Abstract: The human progesterone receptor (hPR) cDNA, synthesized from T47D breast cancer cells, and the hPR gene 5'-flanking region were cloned and sequenced. Comparison of the cDNA-deduced amino acid sequence with other PR homologues demonstrated the modular structure characteristic of nuclear receptors. As in the case of the chicken homologue, there are two hPR forms, A and B, which originate from translational initiation at AUG2 (codon 165) and AUG1, respectively. Northern blot analysis of T47D mRNA using various cDNA derived probes identified two classes of hPR mRNAs, one of which could code for hPR form B, while the other one lacked the 5' region upstream of AUG1. S1 nuclease mapping and primer extension analyses confirmed that the second class of hPR transcripts are initiated between +737 and +842 and thus encode hPR form A, but not form B. By using the hPR gene 5'-flanking sequences as promoter region in chimeric genes, we show that a functional promoter (located between -711 and +31) directs initiation of hPR mRNAs from the authentic start sites located at +1 and +15. Most importantly, initiation of transcription from chimeric genes demonstrated the existence of a second promoter located between +464 and +1105. Transient co-transfection experiments with vectors expressing the human estrogen receptor showed that both promoters were estrogen inducible, although no classical estrogen responsive element was detected in the corresponding sequences. When transiently expressed, the two hPR forms similarly activated transcription from reporter genes containing a single palindromic progestin responsive element (PRE), while form B was more efficient at activating the PRE of the mouse mammary tumor virus long terminal repeat. Transcription from the ovalbumin promoter, however, was induced by hPR form A, but not by form B.
1,506 citations
TL;DR: Interestingly, NF‐IL6 was shown to bind to the regulatory regions for various acute‐phase protein genes and several other cytokine genes such as TNF, IL‐8 and G‐CSF, implying that NF‐ IL6 has a role in regulation not only for the IL‐6 gene but also for several other genes involved in acute‐ phase reaction, inflammation and hemopoiesis.
Abstract: NF-IL6 is a nuclear factor that specifically binds to an IL1-responsive element in the IL-6 gene. In this study the gene encoding NF-IL6 has been cloned by direct screening of a lambda gt11 library using NF-IL6 binding sequence as a ligand. The full-length cDNA encoded a 345 amino acid protein with a potential leucine zipper structure and revealed a high degree of homology to a liver-specific transcriptional factor, C/EBP, at the C-terminal portion. The bacterial fusion protein bound to the CCAAT homology as well as the viral enhancer core sequences as in the case of C/EBP. Recombinant NF-IL6 activated the human IL-6 promoter in a sequence-specific manner. Southern blot analysis demonstrated the high-degree conservation of the NF-IL6 gene through evolution and the existence of several other related genes sharing the DNA-binding domain. NF-IL6 mRNA was normally not expressed, but induced by the stimulation with either LPS, IL-1 or IL-6. Interestingly, NF-IL6 was shown to bind to the regulatory regions for various acute-phase protein genes and several other cytokine genes such as TNF, IL-8 and G-CSF, implying that NF-IL6 has a role in regulation not only for the IL-6 gene but also for several other genes involved in acute-phase reaction, inflammation and hemopoiesis.
1,453 citations
TL;DR: A method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA and sequences for cyclophilin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in aRNA derived from single cerebellar tissue sections.
Abstract: The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.
1,388 citations
TL;DR: The physiological importance of these two proteins in cellular signal transduction is discussed, and partial purification of GSK‐3 activity from bovine brain results in the isolation of active alpha and beta proteins.
Abstract: Glycogen synthase kinase-3 (GSK-3) is a protein-serine kinase implicated in the hormonal control of several regulatory proteins including glycogen synthase and the transcription factor c-jun. Two classes of rat brain cDNA for this enzyme have been isolated termed GSK-3 alpha and GSK-3 beta. The alpha-type encodes a 51 kd polypeptide, the sequence of which includes all of the tryptic peptides determined by protein sequence analysis of purified skeletal muscle GSK-3. The novel beta-type cDNA has the potential to encode a 47 kd protein with 85% amino acid identity to GSK-3 alpha. The two types of cDNA are the products of distinct genes as determined by genomic organization and nucleic acid sequence analysis. Both alpha and beta clones exhibit kinase activity when expressed in COS-1 cells and type-specific antibodies to GSK-3 alpha and beta detect proteins of 51 and 47 kd, respectively, in a variety of rat tissue extracts, with highest levels of both in brain. Partial purification of GSK-3 activity from bovine brain results in the isolation of active alpha and beta proteins. The physiological importance of these two proteins in cellular signal transduction is discussed.
1,293 citations
TL;DR: A cloned gp130 could associate with a complex of IL-6 and solubleIL-6-R and transduce the growth signal when expressed in a murine IL-3-dependent cell line and confirmed that a gp130 is involved in the formation of high affinity IL- 6 binding sites.
1,292 citations
TL;DR: YTOGENETIC analysis has identified chromosome Ilpl3 as the smallest overlap region for deletions found in individuals with WAGR syndrome, which includes Wilms tumour, anirida, genito-urinary abnormalities and mental retardation.
Abstract: Cytogenetic analysis has identified chromosome 11p13 as the smallest overlap region for deletions found in individuals with WAGR syndrome, which includes Wilms tumour (a recessive childhood nephroblastoma), aniridia, genito-urinary abnormalities and mental retardation. The underlying loci have since been resolved into an aniridia (AN2) locus at a telomeric position, and a locus of closely spaced genes or a single pleiotropic gene involved in genito-urinary tract abnormalities and Wilms tumour at a more centromeric position. Pulsed-field gel analysis of the 11p13 region has revealed the presence of several putative CpG islands, structures which are frequently associated with the 5' ends of expressed sequences, mainly housekeeping genes and some tissue-specific genes. Starting from a CpG island, we have now isolated four neighbouring CpG islands, all within 650 kilobases (kb), by means of two consecutive bidirectional jumps in rare-cutting restriction-enzyme jumping libraries. In two instances, flanking sequences were conserved in other species and RNA transcripts were identified. A complementary DNA clone isolated for one of them derives from an RNA highly expressed in fetal kidney, and is predicted to encode a Kruppel-like zinc-finger protein that is probably a transcription factor. The entire cDNA region is included in two partially overlapping homozygous deletions found in Wilms tumour DNA samples. Cloning of the breakpoints in one tumour revealed a deletion size of 170 kb, one-third of which is covered by the cDNA. The expression pattern and sequence of this cDNA could point to an important role for its corresponding gene in the normal development of the renal system as well as in Wilms tumour.
1,260 citations
TL;DR: The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.
Abstract: Complementary DNA clones encoding mouse cytokine synthesis inhibitory factor (CSIF; interleukin-10), which inhibits cytokine synthesis by TH1 helper T cells, were isolated and expressed. The predicted protein sequence shows extensive homology with an uncharacterized open reading frame, BCRFI, in the Epstein-Barr virus genome, suggesting the possibility that this herpes virus exploits the biological activity of a captured cytokine gene to enhance its survival in the host.
1,167 citations
TL;DR: Northern analysis indicates a single species of mRNA for the TNF-R in a variety of cell types, therefore, the soluble TNF binding protein found in human serum is probably proteolytically derived from the T NF-R.
Journal Article•
TL;DR: A new human gene encoding a receptor-type tyrosine kinase was isolated by a weak cross-hybridization with v-ros oncogene and designated as flt (fms-like tyrosin kinase) gene, which was strongly suppressed in most of the tumor cell lines examined so far.
Abstract: A new human gene encoding a receptor-type tyrosine kinase was isolated by a weak cross-hybridization with v-ros oncogene. A cDNA of about 7.7 kb carried a 4.2 kb open reading frame, and the predicted amino acid sequence of 1338 residues contained extracellular, transmembrane and tyrosine kinase domains. Although its extracellular domain is approximately 220 amino acids longer than those of the products of the fms family, i.e. c-fms, c-kit and platelet-derived growth factor receptor genes, the overall structure including cysteine motifs in its extracellular domain and a long peptide insertion in its tyrosine kinase domain indicates that this new gene is closely related to the fms family. Consequently, the gene was designated as flt (fms-like tyrosine kinase) gene. The expression of the flt gene was strongly suppressed in most of the tumor cell lines examined so far, whereas this mRNA was expressed in a variety of normal tissues of adult rat.
TL;DR: The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.
Abstract: Tumor necrosis factor alpha and beta (TNF-alpha and TNF-beta) bind surface receptors on a variety of cell types to mediate a wide range of immunological responses, inflammatory reactions, and anti-tumor effects. A cDNA clone encoding an integral membrane protein of 461 amino acids was isolated from a human lung fibroblast library by direct expression screening with radiolabeled TNF-alpha. The encoded receptor was also able to bind TNF-beta. The predicted cysteine-rich extracellular domain has extensive sequence similarity with five proteins, including nerve growth factor receptor and a transcriptionally active open reading frame from Shope fibroma virus, and thus defines a family of receptors.
TL;DR: Human monocytes induced with adherent IgG secrete an interleukin-1 receptor antagonist which could be important for the in vivo regulation of IL-1 activity and analysis of monocyte RNA indicates that the gene is transcriptionally regulated.
Abstract: Human monocytes induced with adherent IgG secrete an interleukin-1 receptor antagonist which could be important for the in vivo regulation of IL-1 activity. A complementary DNA for this molecule has been isolated from a human monocyte library. Analysis of monocyte RNA indicates that the gene is transcriptionally regulated. The sequence of the receptor antagonist indicates that it is structurally similar to IL-1 beta. Expression of the cDNA in Escherichia coli yields IL-1 receptor antagonist activity.
TL;DR: Evidence suggests that NF1 encodes a cytoplasmic GAP-like protein that may be involved in the control of cell growth by interacting with proteins such as the RAS gene product.
TL;DR: Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd were expressed at low levels by various cells as discussed by the authors, and partial amino acid sequences were determined Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction.
TL;DR: It is concluded that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis.
Abstract: Immunohistological staining of primary colorectal carcinomas with antibodies specific to p53 demonstrated gross overexpression of the protein in approximately 50% of the malignant tumors examined. Benign adenomas were all negative for p53 overexpression. To determine the molecular basis for this overexpression we examined p53 protein expression in 10 colorectal cancer cell lines. Six of the cell lines expressed high levels of p53 in ELISA, cell-staining, and immunoprecipitation studies. Direct sequencing and chemical-mismatch-cleavage analysis of p53 cDNA by using the polymerase chain reaction in these cell lines showed that all cell lines that expressed high levels of p53 were synthesizing mRNAs that encoded mutant p53 proteins. In two of those four cell lines where p53 expression was lower, point mutations were still detected. Thus, we conclude that overexpression of p53 is synonymous with mutation, but some mutations would not be detected by a simple immunohistochemical analysis. Mutation of the p53 gene is one of the commonest genetic changes in the development of human colorectal cancer.
TL;DR: The full sequence for PEM, as deduced from cDNA sequences, is reported, with length variations in the tandem repeat result in PEM being an expressed variable number tandem repeat locus.
TL;DR: An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions.
Abstract: An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions. This antibody identified a complementary DNA clone from an endothelial cell library. The 130-kilodalton translated sequence contained six extracellular immunoglobulin (Ig)-like domains and was most similar to the cell adhesion molecule (CAM) subgroup of the Ig superfamily. This is the only known member of the CAM family on platelets. Its cell surface distribution suggests participation in cellular recognition events.
TL;DR: A cDNA clone that codes for a new tissue-specific DNA binding protein, PU.1, was isolated and was shown to be a transcriptional activator that is expressed in macrophages and B cells.
TL;DR: Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins.
Abstract: A complementary DNA clone (designated GAT-1) encoding a transporter for the neurotransmitter gamma-aminobutyric acid (GABA) has been isolated from rat brain, and its functional properties have been examined in Xenopus oocytes. Oocytes injected with GAT-1 synthetic messenger RNA accumulated [3H]GABA to levels above control values. The transporter encoded by GAT-1 has a high affinity for GABA, is sodium-and chloride-dependent, and is pharmacologically similar to neuronal GABA transporters. The GAT-1 protein shares antigenic determinants with a native rat brain GABA transporter. The nucleotide sequence of GAT-1 predicts a protein of 599 amino acids with a molecular weight of 67 kilodaltons. Hydropathy analysis of the deduced protein suggests multiple transmembrane regions, a feature shared by several cloned transporters; however, database searches indicate that GAT-1 is not homologous to any previously identified proteins. Therefore, GAT-1 appears to be a member of a previously uncharacterized family of transport molecules.
TL;DR: The cloning of cDNAs encoding the MGF protein is reported, a novel mast cell growth factor that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10.
TL;DR: Evidence is presented that the candidate cDNA is the murine homologue of bovine phosphodiesterase β cDNA and that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterases β subunit.
Abstract: MICE homozygous for the rd mutation display hereditary retinal degeneration and the classic rd lines serve as a model for human retinitis pigmentosa1,2. In affected animals the retinal rod photo-receptor cells begin degenerating at about postnatal day 8, and by four weeks no photoreceptors are left3. Degeneration is preceded by accumulation of cyclic GMP in the retina4 and is correlated with deficient activity of the rod photoreceptor cGMP-phospho-diesterase5. We have recently isolated a candidate complementary DNA for the rd gene6 from a mouse retinal library and completed the characterization of cDNAs encoding all subunits of bovine photoreceptor phosphodiesterase7. The candidate cDNA shows strong homo logy with a cDNA encoding the bovine phospho-diesterase β subunit. Here we present evidence that the candidate cDNA is the murine homologue of bovine phosphodiesterase β cDNA. We conclude that the mouse rd locus encodes the rod photoreceptor cGMP-phosphodiesterase β subunit.
TL;DR: A complementary cDNA coding for KBF1 is isolated and the DNA binding and dimerization domain of the protein is identified, which suggests functional homologies between KBF2 and v-rel and the Drosophila maternal morphogen dorsal.
TL;DR: It is shown that antisense RNA, which has previously been used only to reduce the expression of genes of known function when applied to pTOM13, reduces ethylene synthesis in a gene dosage-dependent manner.
Abstract: ETHYLENE controls many physiological and developmental processes in higher plants, including ripening of fruit, abscission, senescence and responses to wounding1. Although the accumulation of messenger RNAs in ripening fruit and senescing leaves has been correlated with ethylene production and perception2–4, the regulatory mechanisms governing ethylene synthesis and the stimulation of gene expression by ethylene are not understood. We have previously shown that the complementary DNA, pTOM13, corresponds to an mRNA whose synthesis is correlated with that of ethylene in ripening fruit and wounded leaves5,6,8. The pTOM13 mRNA encodes a protein of relative molecular mass 35,0006. The cDNA and three related genomic clones have been sequenced, but the function of the protein is unknown7–9. We show here that antisense RNA, which has previously been used only to reduce the expression of genes of known function10–12, when applied to pTOM13, reduces ethylene synthesis in a gene dosage-dependent manner. Analysis of these novel mutants suggests that pTOM13 encodes a polypeptide involved in the conversion of 1-amino-cyclopropane-1-carboxylic acid to ethylene by the ethylene-forming enzyme (ACC-oxidase).
TL;DR: It is indicated that the NF1 gene product can interact with RAS proteins and structural and functional similarities and differences among the GAP, IRA1, IRA2, and NF1 proteins are demonstrated.
TL;DR: It is demonstrated that the expression of both the MK-886-binding protein and 5-lipoxygenase is necessary for leukotriene synthesis in intact cells.
Abstract: LEUKOTRIENES, the biologically active metabolites of arachidonic acid, have been implicated in a variety of inflammatory responses, including asthma, arthritis and psoriasis1,2. Recently a compound, MK-886, has been described that blocks the synthesis of leukotrienes in intact activated leukocytes, but has little or no effect on enzymes involved in leukotriene synthesis, including 5-lipoxygenase, in cell-free systems3. A membrane protein with a high affinity for MK-886 and possibly representing the cellular target for MK-886 has been isolated from rat and human leukocytes4. Here, we report the isolation of a complementary DNA clone encoding the MK-886-binding protein. We also demonstrate that the expression of both the MK-886-binding protein and 5-lipoxygenase is necessary for leukotriene synthesis in intact cells. Because the MK-886-binding protein seems to play a part in activating this enzyme in cells, it is termed the five-lipoxygenase activating protein (FLAP).
TL;DR: Hydropathy analysis suggests that the Na(+)-Ca2+ exchanger of the cardiac sarcolemma can rapidly transport Ca2+ during excitation-contraction coupling and has multiple transmembrane helices, and a small region of the sequence is similar to that of the Na- and K-dependent adenosine triphosphatase.
Abstract: The Na(+)-Ca2+ exchanger of the cardiac sarcolemma can rapidly transport Ca2+ during excitation-contraction coupling. To begin molecular studies of this transporter, polyclonal antibodies were used to identify a complementary DNA (cDNA) clone encoding the Na(+)-Ca2+ exchanger protein. The cDNA hybridizes with a 7-kilobase RNA on a Northern blot and has an open reading frame of 970 amino acids. Hydropathy analysis suggests that the protein has multiple transmembrane helices, and a small region of the sequence is similar to that of the Na(+)- and K(+)-dependent adenosine triphosphatase. Polyclonal antibodies to a synthetic peptide from the deduced amino acid sequence react with sarcolemmal proteins of 70, 120, and 160 kilodaltons on immunoblots. RNA, synthesized from the cDNA clone, induces expression of Na(+)-Ca2+ exchange activity when injected into Xenopus oocytes.
TL;DR: Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated and exhibit potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.
TL;DR: Results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure, including leading and lagging strand synthesis at the replication fork.
Abstract: The proliferating cell nuclear antigen, PCNA, has recently been identified as the polymerase delta accessory protein. PCNA is essential for cellular DNA synthesis and is also required for the in vitro replication of simian virus 40 (SV40) DNA where it acts to coordinate leading and lagging strand synthesis at the replication fork. The cDNA for rat PCNA was cloned into a series of bacterial expression vectors and the resulting protein used to immunize mice. Eleven new monoclonal antibodies to PCNA have been isolated and characterized. Some of the antibodies recognize epitopes conserved from man to fission yeast. Immunocytochemical analysis of primate epithelial cell lines showed that the antibodies recognized antigenically distinct forms of PCNA and that these forms were localized to different compartments of the nucleus. One antibody reacted exclusively with PCNA in the nucleolus. These results suggest that the PCNA protein may fulfil several separate roles in the cell nucleus associated with changes in its antigenic structure.