Cloning of the gene encoding the human erythropoietin receptor

The genomic and complementary DNAs of the human erythropoietin receptor (hEpo-R) have been isolated and characterized from a genomic placental library and from two cDNA libraries prepared from bone marrow and fetal liver. The five different partial cDNAs isolated were aberrant in the predicted reading frames as compared with the Epo-R protein sequence, because all retained insert sequences that may represent splicing intermediates (three clones), cloning artifact (one clone), or a new sequence at a splice junction (one clone) of the gene. The cDNAs were used to isolate several genomic clones encompassing the complete hEpo-R gene. This gene, which encodes a 508-amino acid polypeptide chain of predicted M(r) 55,000, is organized into eight exons spread over 6 kb of DNA and exhibited a high degree of sequence homology (81.6% in the coding region) and structural organization with its murine counterpart. Primer extension analysis indicated that the transcription initiation site is located 141 bp upstream of the initiation codon. Sequence homology 320 bp upstream of the cap site was significantly lower (60%) and diverged completely further upstream as compared with the murine gene. Similarly, the human and murine sequences were largely divergent downstream of the stop codon, indicating that a strong conservation during evolution was restricted to the coding sequence of the Epo-R protein. The 320-bp region upstream of the cap site does not contain the typical TATA or CAAT boxes present in many tissue-specific genes, but does include potential binding sites for the ubiquitous Sp1 and the erythroid-specific GATA-1 trans-activating factors. These boxes are well conserved in sequence and position relative to the cap site within the promoter region of the human and murine genes, but the CACCC boxes present in the murine gene are absent in the human gene.

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Cloning of the gene encoding the human erythropoietin receptor

Blood ◽

10.1182/blood.v78.10.2557.2557 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2557-2563 ◽

Cited By ~ 29

Author(s):

L Maouche ◽

C Tournamille ◽

C Hattab ◽

G Boffa ◽

JP Cartron ◽

...

Keyword(s):

Sequence Homology ◽

Transcription Initiation ◽

Stop Codon ◽

Fetal Liver ◽

Splice Junction ◽

Initiation Site ◽

Erythropoietin Receptor ◽

Cdna Libraries ◽

Coding Region ◽

Human Erythropoietin

Abstract The genomic and complementary DNAs of the human erythropoietin receptor (hEpo-R) have been isolated and characterized from a genomic placental library and from two cDNA libraries prepared from bone marrow and fetal liver. The five different partial cDNAs isolated were aberrant in the predicted reading frames as compared with the Epo-R protein sequence, because all retained insert sequences that may represent splicing intermediates (three clones), cloning artifact (one clone), or a new sequence at a splice junction (one clone) of the gene. The cDNAs were used to isolate several genomic clones encompassing the complete hEpo-R gene. This gene, which encodes a 508-amino acid polypeptide chain of predicted M(r) 55,000, is organized into eight exons spread over 6 kb of DNA and exhibited a high degree of sequence homology (81.6% in the coding region) and structural organization with its murine counterpart. Primer extension analysis indicated that the transcription initiation site is located 141 bp upstream of the initiation codon. Sequence homology 320 bp upstream of the cap site was significantly lower (60%) and diverged completely further upstream as compared with the murine gene. Similarly, the human and murine sequences were largely divergent downstream of the stop codon, indicating that a strong conservation during evolution was restricted to the coding sequence of the Epo-R protein. The 320-bp region upstream of the cap site does not contain the typical TATA or CAAT boxes present in many tissue-specific genes, but does include potential binding sites for the ubiquitous Sp1 and the erythroid-specific GATA-1 trans-activating factors. These boxes are well conserved in sequence and position relative to the cap site within the promoter region of the human and murine genes, but the CACCC boxes present in the murine gene are absent in the human gene.

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Cloning of the human erythropoietin receptor gene

Blood ◽

10.1182/blood.v78.10.2548.bloodjournal78102548 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2548-2556 ◽

Cited By ~ 1

Author(s):

CT Noguchi ◽

KS Bae ◽

K Chin ◽

Y Wada ◽

AN Schechter ◽

...

Keyword(s):

Binding Site ◽

Translational Control ◽

Transcription Initiation ◽

Repetitive Sequences ◽

Cdna Probe ◽

Gc Content ◽

Erythropoietin Receptor ◽

Coding Region ◽

Epo Receptor ◽

Human Erythropoietin

We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5′ region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5′ of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5′ flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5′ of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5′ to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

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Cloning of the human erythropoietin receptor gene

Blood ◽

10.1182/blood.v78.10.2548.2548 ◽

1991 ◽

Vol 78 (10) ◽

pp. 2548-2556 ◽

Cited By ~ 56

Author(s):

CT Noguchi ◽

KS Bae ◽

K Chin ◽

Y Wada ◽

AN Schechter ◽

...

Keyword(s):

Binding Site ◽

Translational Control ◽

Transcription Initiation ◽

Repetitive Sequences ◽

Cdna Probe ◽

Gc Content ◽

Erythropoietin Receptor ◽

Coding Region ◽

Epo Receptor ◽

Human Erythropoietin

Abstract We have isolated and characterized a genomic clone of the human erythropoietin (Epo) receptor from a placental genomic library using a cDNA probe for the murine Epo receptor. The coding region spans about 6.5 kb with seven intervening sequences ranging in size from 81 bp to 2.1 kb. A stretch of 123 purines is found in the 5′ region from -456 to -578 upstream from the first codon and flanking the Alu repetitive sequences located further upstream. The human Epo receptor contains a palindromic sequence 5′ of the translated region that consists of an almost perfect inverted repeat of 12 nucleotides (CAGCTGC(G/C)TCCG) centered about G at -92 from the first codon. An inverted SP1 binding site (CCGCCC) and an inverted GATA-1 binding site (TTATCT) are located at positions -151 and -179, respectively, and CACCC sequences are located at -585 and further upstream. No TATA or CAAT sequences are in this 5′ flanking region. However, this region as far as -275 has a 72% GC content compared with an overall GC content of 56%. A 1-kb BamHI fragment of the human Epo receptor containing 700 bp of sequences 5′ of the coding region was transcribed in an in vitro transcription assay; initiation of transcription appeared to be around 132 +/- 5 just downstream from the inverted SP1 site at -151. T1 analysis of human Epo receptor messenger RNA also maps the site of transcription initiation to this region. Within 180 nucleotides 5′ to the first exon are three regions with 70% or greater homology with the murine Epo receptor. The study of this gene, including its similarities with the murine Epo receptor, should help elucidate aspects of the transcriptional and possible translational control of the Epo receptor in human erythroid cells and thus its role in signal transduction and erythroid differentiation.

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Drd4 gene polymorphisms are associated with personality variation in a passerine bird

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2007.0337 ◽

2007 ◽

Vol 274 (1619) ◽

pp. 1685-1691 ◽

Cited By ~ 129

Author(s):

Andrew E Fidler ◽

Kees van Oers ◽

Piet J Drent ◽

Sylvia Kuhn ◽

Jakob C Mueller ◽

...

Keyword(s):

Transcription Initiation ◽

Gene Polymorphisms ◽

Parus Major ◽

Passerine Bird ◽

Initiation Site ◽

Novelty Seeking ◽

Coding Region ◽

Free Living ◽

Single Nucleotide ◽

Genetic Epistasis

Polymorphisms in several neurotransmitter-associated genes have been associated with variation in human personality traits. Among the more promising of such associations is that between the human dopamine receptor D4 gene ( Drd4 ) variants and novelty-seeking behaviour. However, genetic epistasis, genotype–environment interactions and confounding environmental factors all act to obscure genotype–personality relationships. Such problems can be addressed by measuring personality under standardized conditions and by selection experiments, with both approaches only feasible with non-human animals. Looking for similar Drd4 genotype–personality associations in a free-living bird, the great tit ( Parus major ), we detected 73 polymorphisms (66 SNPs, 7 indels) in the P. major Drd4 orthologue. Two of the P. major Drd4 gene polymorphisms were investigated for evidence of association with novelty-seeking behaviour: a coding region synonymous single nucleotide polymorphism (SNP830) and a 15 bp indel (ID15) located 5′ to the putative transcription initiation site. Frequencies of the three Drd4 SNP830 genotypes, but not the ID15 genotypes, differed significantly between two P. major lines selected over four generations for divergent levels of ‘early exploratory behaviour’ (EEB). Strong corroborating evidence for the significance of this finding comes from the analysis of free-living, unselected birds where we found a significant association between SNP830 genotypes and differing mean EEB levels. These findings suggest that an association between Drd4 gene polymorphisms and animal personality variation predates the divergence of the avian and mammalian lineages. Furthermore, this work heralds the possibility of following microevolutionary changes in frequencies of behaviourally relevant Drd4 polymorphisms within populations where natural selection acts differentially on different personality types.

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Structure and tissue-specific expression of the Drosophila melanogaster organellar-type Ca2+-ATPase gene

Biochemical Journal ◽

10.1042/bj3100757 ◽

1995 ◽

Vol 310 (3) ◽

pp. 757-763 ◽

Cited By ~ 18

Author(s):

A Magyar ◽

E Bakos ◽

A Váradi

Keyword(s):

Drosophila Melanogaster ◽

Transcription Initiation ◽

Initiation Site ◽

Drosophila Genome ◽

Coding Region ◽

Specific Expression ◽

S1 Nuclease ◽

Protein Coding ◽

Atpase Gene ◽

High Level

A 14 kb genomic clone covering the organellar-type Ca(2+)-ATPase gene of Drosophila melanogaster has been isolated and characterized. The sequence of a 7132 bp region extending from 1.1 kb 5′ upstream of the initiation ATG codon over the polyadenylation signal at the 3′ end has been determined. The gene consists of nine exons including one with an exceptional size of 2172 bp representing 72% of the protein coding region. Introns are relatively small (< 100 bp) except for the 3′ intron which has a size of 2239 bp, an exceptionally large size among Drosophila introns. Five of the introns are in the same positions in Drosophila, Artemia and rabbit SERCA1 Ca(2+)-ATPase genes. There is only one organellar-type Ca(2+)-ATPase gene in the Drosophila genome, as was shown by Southern-blot analysis [Váradi, Gilmore-Hebert and Benz (1989) FEBS Lett. 258, 203-207] and by chromosomal localization [Magyar and Váradi (1990) Biochem. Biophys. Res. Commun. 173, 872-877]. Primer extension and S1-nuclease assays revealed a potential transcription initiation site 876 bp upstream of the translation initiation ATG with a TATA-box 23 bp upstream of this site. Analysis of the 5′ region of the Drosophila organellar-type Ca(2+)-ATPase gene suggests the presence of potential recognition sequences of various muscle-specific transcription factors and shows a region with remarkable similarity to that in the rabbit SERCA2 gene. The tissue distribution of expression of the organellar-type Ca(2+)-ATPase gene has been studied by in situ RNA-RNA hybridization on microscopic sections. A low mRNA abundance can be detected in each tissue of adult flies, suggesting a housekeeping function for the gene. On the other hand a pronounced tissue specificity of expression has also been found as the organellar-type Ca(2+)-ATPase is expressed at a very high level in cell bodies of the central nervous system and in various muscles.

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The Genes Encoding Formamidopyrimidine and MutY DNA Glycosylases in Escherichia coli Are Transcribed as Part of Complex Operons

Journal of Bacteriology ◽

10.1128/jb.181.14.4223-4236.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4223-4236 ◽

Cited By ~ 12

Author(s):

Christine M. Gifford ◽

Susan S. Wallace

Keyword(s):

Escherichia Coli ◽

Gene Order ◽

Intergenic Region ◽

Transcription Initiation ◽

Excision Repair ◽

Initiation Site ◽

Dna Glycosylase ◽

Rnase E ◽

Coding Region ◽

Genes Encoding

ABSTRACT Escherichia coli formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine. The genes encoding these proteins, fpg and mutY, are both cotranscribed as part of complex operons. fpg is the terminal gene in an operon with the gene order radC,rpmB, rpmG, and fpg. This operon has transcription initiation sites upstream of radC, in theradC coding region, and immediately upstream offpg. There is a strong attenuator in therpmG-fpg intergenic region and three transcription termination sites downstream of fpg. There is an additional site, in the radC-rpmB intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site. mutY is the first gene in an operon with the gene order mutY, yggX, mltC, andnupG. This operon has transcription initiation sites upstream of mutY, in the mutY coding region, and immediately upstream of nupG. There also appear to be attenuators in the yggX-mltC and mltC-nupGintergenic regions. The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms.

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Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3244 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3244-3252 ◽

Cited By ~ 37

Author(s):

J M Huibregtse ◽

D R Engelke

Keyword(s):

Stationary Phase ◽

Transcription Initiation ◽

Initiation Site ◽

Dnase I ◽

Transcription Initiation Site ◽

Upstream Region ◽

Stable Complex ◽

Coding Region ◽

Flanking Region ◽

Genomic Footprinting

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

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Regulatory sequences for the amplification and replication of the ribosomal DNA minichromosome in Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7237 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7237-7247 ◽

Cited By ~ 21

Author(s):

P Blomberg ◽

C Randolph ◽

C H Yao ◽

M C Yao

Keyword(s):

Transcription Initiation ◽

Tetrahymena Thermophila ◽

Initiation Site ◽

Core Region ◽

Rrna Gene ◽

Regulatory Sequences ◽

Coding Region ◽

Cis Acting ◽

Rrna Transcription ◽

Stable Maintenance

We have analyzed the cis-acting sequences that regulate rRNA gene (rDNA) replication in Tetrahymena thermophila. The macronucleus of this ciliated protozoan contains 9,000 copies of a 21-kbp minichromosome in the form of a palindrome comprising two copies of the rDNA. These are derived from a single chromosomally integrated copy during conjugation through selective amplification and are maintained by replicating once per cell cycle during vegetative growth. We have developed a transformation vector and carried out a deletion analysis to determine the minimal sequences required for replication, amplification, and/or stable maintenance of the rDNA molecule. Using constructs containing progressively longer deletions, we show that only a small portion (approximately 900 bp) of the rDNA is needed for extrachromosomal replication and stable maintenance of this molecule. This core region is very near but does not include the rRNA transcription initiation site or its putative promoter, indicating that replication is not dependent on normal rRNA transcription. It includes two nearly identical nuclease-sensitive domains (D1 and D2), one of which (D1) corresponds to the physical origin of replication determined previously. Deletion of both domains abolishes replication, whereas deletion of either domain allows the molecules to replicate, indicating that only one domain is required. In addition to this core region, we have found several DNA segments, including a tandem array of a 21-nucleotide repeat (type II repeats) and sequences within the rRNA coding region, that play distinctive and important roles in maintaining the rDNA at a high copy number.

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Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3244-3252.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3244-3252

Author(s):

J M Huibregtse ◽

D R Engelke

Keyword(s):

Stationary Phase ◽

Transcription Initiation ◽

Initiation Site ◽

Dnase I ◽

Transcription Initiation Site ◽

Upstream Region ◽

Stable Complex ◽

Coding Region ◽

Flanking Region ◽

Genomic Footprinting

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Truncated Human Erythropoietin Receptor Causes Fetal Polycythemia and Is Associated with a Prolonged Primitive Erythropoiesis and Hyperactive Definitive Erythropoiesis.

Blood ◽

10.1182/blood.v110.11.3664.3664 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3664-3664

Author(s):

Donghoon Yoon ◽

Josef Prchal

Keyword(s):

Fetal Liver ◽

Perinatal Period ◽

Erythropoietin Receptor ◽

Erythroid Cell ◽

Fetal Life ◽

Human Erythropoietin ◽

Significant Difference ◽

Primitive Erythropoiesis ◽

Definitive Erythropoiesis ◽

Fetal Erythropoiesis

Abstract Gain-of-function mutations that cause truncations of the cytoplasmic domain of the human erythropoietin receptor (EpoR) result in the dominantly inherited disorder, primary familial congenital polycythemia. The EpoR truncation causes polycythemia, which is not always seen at birth, and in vitro hypersensitivity of erythroid precursors to erythropoietin (Epo). We have replaced the murine EpoR (MEpoR) gene with either a wild-type human EPOR (wthEPOR) gene or a mutant human EPOR gene (mthEPOR). This mutation, initially identified in a family with polycythemia, produces a truncated EpoR with a deletion of the cytoplasmic C-terminal domain after the first tyrosine residue. To investigate the effects of wthEPOR and mthEPOR genes on fetal erythropoiesis, we examined mouse fetuses homozygous for either wthEPOR or mthEPOR from E12.5 up to 5 weeks postnatally (PN). The mthEPOR fetuses were polycythemic and the wthEPOR fetuses were anemic during the early embryonic stage (E12.5∼E16.5) and at the adult stage (3 weeks and older). However, during the perinatal period (E18.5∼PN2 weeks), both of the phenotypes temporarily disappeared. To study the pathophysiology of the phenotypes, we analyzed erythropoiesis using differential expression of CD71 and TER119 surface antigens by FACS in the peripheral blood (PB) and fetal liver (FL). PB from wthEPOR and mthEPOR had a significantly higher population of earlier progenitors (proerythroblasts and early basophilic erythroblasts) from E12.5 to E16.5 compared to MEpoR. From E18.5 on, however, all genotypes showed a similar pattern. At E14.5, FL of wthEPOR and mthEPOR homozygotes were similar to MEpoR FLs at E12.5. Since a dynamic switch of primitive to definitive erythropoiesis occurs around E14.5, we evaluated nucleated erythroid cell populations in PB from E12.5∼E15.5 using the DNA dye DRAQ5 followed by FACS analysis. From E13.5 to E14.5, mthEPOR fetuses contained more nucleated cells than MEpoR fetuses (43 to 55% vs 37 to 29%; mthEPOR vs MEpoR), whereas wthEPOR fetuses had comparable numbers to MEpoR fetuses (36 to 25% vs 37 to 29%; wthEPOR vs MEpoR). Interestingly, all genotypes from E15.5 and thereafter contained over 90% enucleated cells with no significant difference in their PB. To understand the molecular mechanism of these features, we have analyzed Stat5 phosphorylation after Epo stimulation in FL from E14.5 and E18.5. At both stages, the mthEPOR FL had a markedly increased and sustained Epo-sensitive Stat5 phosphorylation compared to MEpoR FL. In contrast, in wthEPOR FL Stat5 phosphorylation was significantly lower even at the highest concentration of Epo. The membrane-localized EpoR levels were measured by fluorescence labeled anti-EpoR antibody. All genotypes showed comparable levels of EpoR on the membrane. We demonstrate here that with the exception of the perinatal period, mthEPOR causes polycythemia whereas wthEPOR causes anemia, even in fetal life. We propose that the polycythemia of mthEPOR during early embryogenesis (E12.5∼E14.5) is due to a delayed switch from primitive to fetal erythropoiesis, followed by hyperactive definitive erythropoiesis. These characteristics (the prolonged primitive erythropoiesis and hyperactive definitive erythropoiesis) could be explained, at least in part, by hyperactive and sustained Stat5 activation.

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