Cloning and expression of the human erythropoietin gene.
Proceedings of the National Academy of SciencesPublished 1 November 1985Open access
F K Lin, Sidney V. Suggs, Chih-Wei Lin, Jeffrey K. Browne, Ralph Smalling, Joan C. Egrie
Citations1,148
Generate an AI Snapshot to get a quick, structured summary of this paper.
Study Snapshot
ObjectiveStudy objective
MethodsResearch methodology
PopulationPopulation studied
Sample sizeSample sizes
OutcomesStudy outcomes here
ResultsStudy results comes here
LimitationsResearch study limitations comes here
A concise AI-generated summary of the paper will appear here once you click Generate AI Snapshot.
TL;DR
The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes and encodes a 27-amino acid signal peptide and a 166-AMino acid mature protein with a calculated Mr of 18,399.
Abstract
The human erythropoietin gene has been isolated from a genomic phage library by using mixed 20-mer and 17-mer oligonucleotide probes. The entire coding region of the gene is contained in a 5.4-kilobase HindIII-BamHI fragment. The gene contains four intervening sequences (1562 base pairs) and five exons (582 base pairs). It encodes a 27-amino acid signal peptide and a 166-amino acid mature protein with a calculated Mr of 18,399. The erythropoietin gene, when introduced into Chinese hamster ovary cells, produces erythropoietin that is biologically active in vitro and in vivo.
Keywords
MedicineEngineering
Proceedings of the National Academy of SciencesDNA sequencing with chain-terminating inhibitors
69,306 Citations1977Frederick Sanger, S. Nicklen +1 more
A new method for determining nucleotide sequences in DNA is described, which makes use of the 2',3'-dideoxy and arabinon nucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase.
Journal of Molecular BiologyDetection of specific sequences among DNA fragments separated by gel electrophoresis
32,820 Citations1975E.M. Southern
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters that can be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography.
BiochemistryIsolation of biologically active ribonucleic acid from sources enriched in ribonuclease
22,203 Citations1979John M. Chirgwin, Alan Przybyla +2 more
Intact ribonucleic acid has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds.
Methods in enzymology on CD-ROM/Methods in enzymology[57] Sequencing end-labeled DNA with base-specific chemical cleavages
14,691 Citations1980Allan M. Maxam, Walter Gilbert
Proceedings of the National Academy of SciencesPurification of Biologically Active Globin Messenger RNA by Chromatography on Oligothymidylic acid-Cellulose
6,888 Citations1972Haim Aviv, Philip Leder
A convenient technique for the partial purification of large quantities of functional, poly(adenylic acid)-rich mRNA is described and should prove generally useful as an initial step in the isolation of specific mRNAs.
American Journal of NephrologyTargeting Hypoxia-Inducible Factors for the Treatment of Anemia in Chronic Kidney Disease Patients
4,816 Citations2017Francesco Locatelli, Steven Fishbane +2 more
Results from clinical studies of a number of HIF prolyl hydroxylase inhibitors are increasingly available and provide support for the continued evaluation of the risk-benefit ratio of this novel therapeutic approach to the treatment of anemia in CKD.
Nucleic Acids ResearchA catalogue of splice junction sequences
3,858 Citations1982Stephen M. Mount
The sequence CAAG/GTAGAGT was found to be a consensus of 139 exon-intron boundaries (or donor sequences) and (TC)nNCTAG/G was found of 130 intron-exon boundaries ( or acceptor sequences).
CellTransfer of purified herpes virus thymidine kinase gene to cultured mouse cells
1,404 Citations1977Michael Wigler, Saul J. Silverstein +4 more
The results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information and cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.
Molecular and Cellular BiologyHigh-Efficiency Cloning of Full-Length cDNA
1,278 Citations1982Hiroto Okayama, Paul Berg
The cloning procedure described here mitigates this shortcoming by attributing the high efficiency of cloning full- or nearly full-length cDNA to the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis.
American Journal of NephrologyHistory of Erythropoiesis-Stimulating Agents, the Development of Biosimilars, and the Future of Anemia Treatment in Nephrology
1,149 Citations2017Kamyar Kalantar‐Zadeh
NatureIsolation and characterization of genomic and cDNA clones of human erythropoietin
1,098 Citations1985Kenneth Jacobs, Charles B. Shoemaker +11 more
The cloning of the human erythropoietin gene and the expression of an erythrocytes cDNA clone in a transient mammalian expression system to yield a secreted product with biological activity are described.
CellThe isolation and characterization of linked δ- and β-globin genes from a cloned library of human DNA
1,052 Citations1978Richard M. Lawn, Edward F. Fritsch +3 more
The two independently isolated β-globin clones differ from each other by the presence of a Pst I restriction enzyme cleavage site within the large intervening sequence of the δ- globin gene of one of the clones, which suggests that the human DNA carried in the two clones was derived from two homologous chromosomes which were heterozygous for the PstI restriction enzyme recognition sequence.
Proceedings of the National Academy of SciencesIsolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.
910 Citations1980G Urlaub, Lawrence A. Chasin
Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase were isolated after mutagenesis and exposure to high-specific-activity [3H]deoxyuridine as a selective agent.
Journal of Biological ChemistryPurification of human erythropoietin.
885 Citations1977Takahito Miyake, Charles Kung +1 more
Human erythropoietin, derived from urine of patients with aplastic anemia, has been purified to apparent homogeneity by seven-step procedure, which yielded a preparation with a potency of 70,400 units/mg of protein in 21% yield.
Nucleic Acids ResearchCompilation of published signal sequences
756 Citations1984Marion Watson
A number of secreted and membrane-bound proteins have now been shown to have N-terminal sequences which resemble the transient peptides, but which are not cleaved, which require that the signal peptide must be transient.
Journal of Molecular BiologyBase sequence studies of 300 nucleotide renatured repeated human DNA clones
603 Citations1981Prescott L. Deininger, Douglas J. Jolly +3 more
The nucleotide sequences of 15 clones constructed from these 300 nucleotide S 1 -resistant repeats are determined and ten of these cloned sequences are members of the Alu family of interspersed repeats, a dimeric structure that was evidently formed from a head to tail duplication of an ancestral monomeric sequence.
CellThree regions upstream from the cap site are required for efficient and accurate transcription of the rabbit β-globin gene in mouse 3T6 cells
591 Citations1983Peter M. Dierks, Albert van Ooyen +4 more
Cloned rabbit beta-globin genes, modified in vitro by restructuring or site-directed mutagenesis, were introduced into mouse 3T6 cells, and the resulting transcripts were analyzed by nuclease S1 mapping to find the first 109 bp preceding the cap site sufficed for maximal beta- globin transcription.
Annual Review of BiochemistryTHE PATHWAY OF EUKARYOTIC mRNA FORMATION
431 Citations1983Joseph R. Nevins
This paper presents a monograph on the construction of a Transcriptional Unit and some of the properties of this unit, as well as some examples of use cases and applications.
Proceedings of the National Academy of SciencesUse of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin.
422 Citations1981Sidney V. Suggs, R. Bruce Wallace +3 more
Two sets of 15-base-long oligodeoxyribonucleotides corresponding to all possible coding sequences for a small portion of human beta 2-microglobulin are synthesized and detected in the 535 bacterial plasmid clones screened.
Journal of VirologyRegulatory and coding potential of the mouse mammary tumor virus long terminal redundancy
240 Citations1981L A Donehower, An-Li Huang +1 more
Molecular clones containing the 3' half of newly integrated mouse mammary tumor virus (MMTV) DNA with adjacent mouse cellular sequences were characterized and specificity of integration with respect to viral sequences was precise.
BloodThe Liver as a Source of Extrarenal Erythropoietin Production
239 Citations1972W Fried
It is suggested that the liver plays an important role in the production of extrarenal erythropoietin and is barely detectable but significantly lower than in nephrectomized rats when exposed to 0.435 atmos.
Journal of Clinical InvestigationStudies on the Liver to Kidney Switch of Erythropoietin Production
175 Citations1981Esmail D. Zanjani, João L. Ascensão +3 more
In sheep, the liver to kidney switch of Ep production is initiated in utero during the last third of the gestation period, but is completed after birth, and this transition occurs gradually; the assumption of Ep producing capacity by the kidney is not preceded by an abrupt loss of hepatic Ep formation; and the switch is profoundly influenced by alterations in thyroid hormone and oxygen supply-demand levels.
Molecular and Cellular BiologyAnalysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids.
165 Citations1983Christian C. Simonsen, Arthur D. Levinson
Annual Review of MedicineErythropoietin and the Control of Red Cell Production
154 Citations1978Stanley E. Gräber, Sanford B. Krantz
The aim of the present review is to provide the reader with a general background of Ep biology, and to update knowledge of the hormone to the present time with particular attention directed to its mechanism of action and role in clinical medicine.
Methods in enzymology on CD-ROM/Methods in enzymology[26] A sensitive and rapid method for recombinant phage screening
149 Citations1979Savio L.C. Woo
By using this procedure, recombinant phages containing various Eco RI fragments of a unique-sequence chick ovalbumin gene are identified and analyses of these cloned deoxyribonucleic acid (DNA) fragments have revealed that structural ovalbum in gene sequences are separated into eight portions by seven intervening DNA sequences.
PubMedPlasma erythropoietin in health and disease.
136 Citations1980A. J. Erslev, J. Jaime +2 more
The results with the use of this in vivo bioassay to measure plasma erythropoietin in patients with uncomplicated anemia, aplasticAnemia, anemia of renal disease, anemias of chronic inflammatory or neoplastic disorder polycythemia vera, and secondary polycyhimia are charted and their diagnostic significance discussed.
Proceedings of the National Academy of SciencesSpecific binding of erythropoietin to spleen cells infected with the anemia strain of Friend virus.
123 Citations1984S B Krantz, Eugene Goldwasser
These studies indicate that erythropoietin attaches to specific binding sites, which are most likely receptors since they manifest high affinity and specificity, and that the biologic effect of the hormone may be produced by attachment of a very small number of erythroblast molecules.
Proceedings of the National Academy of SciencesExpression of abbreviated mouse dihydrofolate reductase genes in cultured hamster cells.
119 Citations1982Charles S. Gasser, Christian C. Simonsen +2 more
A number of abbreviated dihydrofolate reductase (DHFR) genes are constructed by using cloned mouse genomic and cDNA sequences and contain the first two introns of the DHFR gene; the other three lack introns.
British Journal of HaematologyI. EVIDENCE FOR THE VALIDITY OF THE ASSAY METHOD AND THE PHYSIOLOGICAL RELEVANCE OF ESTIMATES
118 Citations1982P. Mary Cotes
A radioimmunoassay for erythropoietin (Ep) in human serum with parallelism of dilution curves of the 2nd International Reference Preparation (IRP) is described, showing a fall in serum immunoreactive Ep following blood transfusion, and an increase with increasing severity of anaemia except in anaemias associated with severe renal failure.
Annals of Internal MedicineErythropoietin Radioimmunoassay in Evaluating Patients with Polycythemia
105 Citations1981H. Phillip Koeffler, EUGENE GOLDWASSER
Serum erythropoietin levels as ascertained by radioimmunoassay can distinguish between most polycythemia vera and secondary polycyhimia patients and should prove useful in the differential diagnosis of polyCythemia.
NatureInactivation of Erythropoietin by Neuraminidase and by Mild Substitution Reactions
102 Citations1960Peter H. Lowy, Geoffrey Keighley +1 more
It is shown that exposure to neuraminidase8 completely inactivates the mucoprotein, and it is possible that other bonds (sugar, amino-acid) were broken.
Journal of Clinical InvestigationThe anemia of chronic renal failure in sheep. Response to erythropoietin-rich plasma in vivo.
98 Citations1984J. W. Eschbach, J Mladenovic +3 more
Dose-response effects, coupled with the identical erythropoietic response in normal and uremic sheep given the same amount of Ep-rich plasma, imply that there are no physiologically significant erythrocyte mass changes in uremia.
EndocrinologyAn Assay for Erythropoietin in Vitro at the Milliunit Level
94 Citations1975Eugene Goldwasser, James F. Eliason +1 more
A method is described for the assay of erythropoietin using primary cultures of adult rat bone marrow cells, which has the disadvantage of detecting asialoerythropOietin which has no activity in vivo.
PubMedErythropoietin and the differentiation of red blood cells.
88 Citations1975Eugene Goldwasser
Erythropoietin is the primary factor regulating red blood cell formation in mammals and some other animals and its mode of action as the inducer of red cell differentiation has been studied in marrow cell and fetal liver cell cultures.
Proceedings of the National Academy of SciencesIsolation of a cDNA clone for human X-linked 3-phosphoglycerate kinase by use of a mixture of synthetic oligodeoxyribonucleotides as a detection probe.
84 Citations1983Judith Singer–Sam, Robert L. Simmer +6 more
A cDNA clone encoding most of human X-linked 3-phosphoglycerate kinase (PGK) was obtained and DNA sequence analysis has established that the 1.2-kilobase-pair fragment encodes PGK from amino acid 121 through the COOH terminus.
Proceedings of the National Academy of SciencesCloning and expression of human erythropoietin cDNA in Escherichia coli.
77 Citations1984Sylvia Lee‐Huang
Human erythropoietin cDNA has been cloned in Escherichia coli by using pBR322 as a vector and three positive clones were identified that express the Ep gene sequences as a beta-lactamase fusion protein.
British Journal of HaematologyEffect of Erythropoietin Therapy on the Red Cell Volume of Uraemic and Non‐uraemic Rats
34 Citations1977A Anagnostou, J Barone +2 more
After 13 doses of Ep, started either 5 or 21 d after removal of five‐sixths of the renal mass, the increase in the red cell volume of uraemic rats was as great as that of non‐uraemic ones.
NatureIs renin substrate an erythropoietin precursor?
32 Citations1984Frej Fyhrquist, Katarina Rosenlöf +3 more
It is shown here that purified renin substrate from human plasma is immunologically related to human erythropoietin and causes the dose-dependent increase of haemoglobin F in culturedhuman erythroid leukaemia K562 cells.
BMJPlasma erythropoietin in chronic uraemia.
26 Citations1965Robert N. Brown
The clinic will be held for two weeks, mainly to deal with unsatisfied demand but also to enable women who may be menstruating to be examined for cervical carcinoma and Instruction will be given in self-examination of the female breast.