Hemoglobin Messenger RNA Research Articles

Hypoxia Up-Regulates Expression of Hemoglobin in Alveolar Epithelial Cells

Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2α protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.

The identification of newborns using messenger RNA profiling analysis

The ability to determine the physical characteristics of an individual depositing a bloodstain at a crime scene would be an invaluable tool to investigators, akin to eyewitness information. One useful biometric that may be amenable to molecular genetic analysis is the biological age of an individual. In theory, it may be possible to determine patterns of gene expression that are age specific, thereby permitting the distinction among tissue samples originating from individuals of different ages (e.g., newborn, adolescent, middle-age, elderly). We have discovered two novel isoforms of gamma hemoglobin messenger RNA, designated HBG1n and HBG2n, which exhibit an extremely restricted pattern of gene expression, being confined to newborn individuals. Multiplex quantitative reverse transcription PCR (qRT-PCR) assays incorporating these novel mRNAs have been designed, tested, and evaluated for their potential forensic use. The results indicate that the assays provide the ability to determine whether a bloodstain originated from a newborn.

Estimating the total number of nucleotide substitutions since the common anchestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Estimating the total number of nucleotide substitutions since the common anchestor of a pair of homologous genes: Comparison of several methods and three beta hemoglobin messenger RNA's

Translation of globin messenger RNA by the mouse ovum.

It has been demonstrated that the Xenopus oocyte can translate rabbit haemoglobin messenger RNA (mRNA) following microinjection of the message into the cell. The Xenopus oocyte has since been shown to be capable of translating a variety of messenger RNAs from different species. This system has proved useful in un-erstanding the mechanism of message translation and has also provided information about the translation capability of the Xenopus oocyte. Several other cell types, including HeLa cells and fibroblasts, can also translate exogenous message injected into the cell. However, there have been no reports of injection of mRNA into oocytes or fertilised one-cell ova of mammalian species. Nevertheless, the latter system could be of considerable use in studying the processing of exogenous messages in a mammalian system undergoing development, as well as providing insight into the way the early embryo processes injected messages and the protein products of such messages. We report here the results of injecting message into the fertilised one-cell mouse ovum and show that both mouse and rabbit globin mRNA are translated in this system.

Nucleotide Sequence Analysis of Coding and Noncoding Regions of Human β-Globin mRNA

Publisher Summary This chapter explores that the analysis of human hemoglobin messenger RNA was undertaken to determine the exact nucleotide sequence of translated and untranslated regions. Knowledge of the translated sequences of globin mRNA has important implications for determination of specific codon usage by eitkaryotic cells and for measurements of the rate of mutational events when human and subhuman species are compared. The chapter reviews the sequence analysis studies that were carried out on human β-globin mRNA. Thalassemic, sickle-cell and Hb A mRNA served as substrates for the preparation of complementary DNA by RNA-dependent DNA polymcmse. cDNA was transcribed by E. coli RNA polymerase, and the resulting cRNA was analyzed. Additional sequence information was obtained by analysis of iodinated native a-thalassemic mRNA and by digestion of cDNA with restriction endonucleases. The chapter discusses that the data derived allow assignment of over 350 nucleotides to the coding region and 43 nucleotides to the noncoding region adjacent to the termination codon. There are extensive regions of homology between the translated regions of rabbit and human β-mRNA; a short homologous region can be demonstrated between the untranslated 3'-terminal sequence of human mRNA and other animal cell mRNAs.

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

Translational response of an E. coli “permeabilized” cell system (PCS) to hemoglobin messenger RNA

Gel Chromatographic Analysis of Nascent Globin Chains: EVIDENCE OF NONUNIFORM SIZE DISTRIBUTION

Abstract A gel chromatographic analytical procedure using Bio-Gel A-0.5m in 6 m guanidine HCl-0.1 m 2-mercaptoethanol has been developed and standardized. The procedure permits the molecular weight of peptides in the size range of 2 to 146 amino acids in length to be established from the distribution coefficient of the peptide on the gel column. This procedure has been applied to an analysis of the size distribution of nascent peptides from rabbit reticulocyte ribosomes. These studies indicate that the size distribution of nascent peptides is nonuniform and that translation of hemoglobin messenger RNA molecules does not proceed at a constant rate in the reticulocyte.

Polynucleotide sequences in eukaryotic DNA and RNA that form ribonuclease-resistant complexes with polyuridylic acid

When annealed with synthetic polynucleotides and treated with ribonuclease under appropriate conditions, poly(U) forms the ribonuclease-resistant complexes poly(rA) · poly(U) (1:1), poly(dA) · 2poly(U) (1:2) and poly · (dA)poly(dT) · poly(U) (1:1:1). This forms the basis of a quantitative assay of poly(rA), poly(dA) and poly(dA) · poly(dT) sequences in unlabelled nucleic acids. Using this assay, duck haemoglobin messenger RNA is shown to contain a poly(rA) sequence approximately 100 nucleotides long. Eukaryotic DNAs contain small amounts of sequences that react with poly(U). In the case of duck DNA, these sequences are considerably shorter than the mRNA-associated sequences and are interspersed widely with other sequences. It is concluded that if duck DNA does contain poly(dA) sequences corresponding to mRNA-associated poly(rA) sequences, there are fewer than 8000 of these per haploid genome.

The translation of rabbit hemoglobin messenger RNA in a cell-free extract from chick embryo brain

The translation of rabbit hemoglobin messenger RNA in an unfractionated cytoplasmic extract from chick embryo brain was studied. This translation was not dependent upon reticulocyte-specific factors. An analysis of the product synthesized in vitro with the embryo brain cell-free extract and rabbit hemoglobin messenger RNA by carboxymethyl cellulose chromatography showed that the system was capable of synthesizing both the α and β globin chains. Analysis of the tryptic peptides of the in vitro synthesized α chain by ion-exchange chromatography showed that the embryo brain extract with rabbit hemoglobin messenger RNA was capable of synthesizing the complete α chain of rabbit hemoglobin. The results suggest that no stringent tissue-specific controls exist for the translation of globin messenger RNA and were discussed in this context.

Evidence for template-specific sites in DNA polymerases

Using rabbit hemoglobin messenger RNA as template, E. coli polymerase I produces poly (dT), poly (dA)·(dT) and antimessenger DNA products. Mild heating of the enzyme causes a differential loss in activity as indicated by three rates of inactivation for the three types of synthesis. Heat inactivation studies have also been carried out with DNA polymerases from oncogenic RNA viruses and mammalian sources using various homopolymer-oligomer pairs as primertemplates. In general, for any given enzyme these synthetic primer-templates reveal different extents of inactivation of the polymerase. These findings may be interpreted to suggest a) that the binding of DNA polymerase to various primer-templates produces conformational changes in the enzyme which are dependent on the type of template bound, or b) that many, if not all, DNA polymerases have different subsites for different templates.

Reticulocyte RNA-dependent RNA polymerase.

A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes.

Hemoglobin messenger RNA from human bone marrow. Isolation and translation in homozygous and heterozygous beta-thalassemia.

A method for isolating human hemoglobin messenger RNA (mRNA) from bone marrow cells was developed to investigate the molecular basis for the defect in globin synthesis in beta thalassemia. Active mRNA was isolated from the bone marrow cells and peripheral reticulocytes of patients with homozygous beta thalassemia, heterozygous beta thalassemia, sickle cell trait, double heterozygosity for beta thalassemia and sickle cell trait, as well as from a patient with normal hemoglobin synthesis but with an elevated reticulocyte count secondary to hereditary spherocytosis. The mRNA was prepared for assay in an mRNA-dependent rabbit reticulocyte cell-free system and the amount of alpha and beta globin chains synthesized was determined by carboxymethylcellulose column chromatography. The relative synthesis of alpha to beta chains in response to normal hemoglobin mRNA was found to be a function of the amount of mRNA added to the assay system: increasing the amount of mRNA led to a decrease in the alpha-to-beta-chain synthetic ratio. Therefore, assays were carried out at limiting concentrations of mRNA. The molecular defect in homozygous beta thalassemia was shown to be carried in the mRNA of bone marrow cells as well as in the mRNA from peripheral reticulocytes, because much less beta than alpha globin was produced in the cell-free system in response to mRNA from either type of cell. In patients doubly heterozygous for beta thalassemia and sickle cell trait, little or no synthesis of beta(A) globin occurred in the bone marrow cells or the peripheral reticulocytes. The alpha to beta(S) synthetic ratio of the intact bone marrow cells was approximately 1, while the same ratio in the peripheral reticulocytes was between 1.5 and 2. The virtual absence of translatable beta globin mRNA in the mRNA prepared from the cells of these doubly heterozygous patients further demonstrates that the molecular defect produced by the beta thalassemia gene is in the beta globin mRNA.

Haemoglobin messenger RNA in spleen of anaemic rabbits

Haemoglobin meseenger RNA was isolated from spleen erythroïd cells of anaemic rabbits. The mRNA fraction looks quite homogeneous when analyzed by sucrose gradient centrifugation; it sediments in the 9S region. When added to an ascites cell free system, spleen 9S RNA stimulates the incorporation of radioactive leucine into protein. The synthesized product has been characterized and identified as α and β chains of rabbit globin.

Synthesis of rabbit globin by reticulocyte postribosomal supernatant and heterologous ribosomes.

Cell‐free protein synthesis has been studied in heterologous systems using rabbit reticulocyte postribosomal supernatant and ribosomes obtained from HeLa or mouse myeloma cells. These cell‐free systems are quite active and incorporate labelled amino acids linearly for more than 30 min. The proteins synthesized have been analyzed by acrylamide gel electrophoresis, column chromatography and paper electrophoresis of the tryptic peptides. More than 70% of the protein synthesized with either type of ribosomes is α chain of rabbit hemoglobin; a small amount of β chain is also synthesized. This result can only be explained by the presence in the postribosomal supernatant of reticulocytes of messenger RNA for hemoglobin. This mRNA is translated more efficiently than the endogenous mRNA present on HeLa or myeloma ribosomes; previous measurements of the mRNA of reticulocyte postribosomal supernatant indicate that it is less on a weight basis than 1/20 of the mRNA associated with the corresponding amount of ribosomes used in an incubation. The preferential translation of mRNA present in the supernatant, presumably in a ribonucleoprotein particle, is discussed.

Microinjection of rabbit hemoglobin messenger RNA into amphibian oocytes and embryos.

After microinjection of hemoglobin mRNA from rabbit reticulocytes (9S mRNA) into Xenopus laevis oocytes, the cytological structure remains normal. The peroxidase reaction does not show an accumulation of synthesized hemoglobin in the germinal vesicle. Fertilized Pleurodeles Waltlii eggs synthesize rabbit hemoglobin after injection of 9S mRNA; hemoglobin synthesis decreases as development progresses. Micromeres and macromeres synthesize equal amounts of hemoglobin, despite the fact that the micromeres contain many more ribosomes. Descendants of injected blastomeres divide more slowly and do not differentiate; they ultimately undergo cytolysis.

Presence of haemoglobin messenger RNA in the postribosomal supernatant of rabbit reticulocytes and conditions necessary for its translation

FEBS LettersVolume 26, Issue 1-2 p. 77-82 Full-length articleFree Access Presence of haemoglobin messenger RNA in the postribosomal supernatant of rabbit reticulocytes and conditions necessary for its translation S.A. Bonanou-Tzedaki, S.A. Bonanou-Tzedaki Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this authorI.B. Pragnell, I.B. Pragnell Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this authorH.R.V. Arnstein, H.R.V. Arnstein Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this author S.A. Bonanou-Tzedaki, S.A. Bonanou-Tzedaki Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this authorI.B. Pragnell, I.B. Pragnell Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this authorH.R.V. Arnstein, H.R.V. Arnstein Department of Biochemistry, University of London King's College, Strand, London WC2R 2LS, EnglandSearch for more papers by this author First published: October 01, 1972 https://doi.org/10.1016/0014-5793(72)80546-5Citations: 28AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 S.A. Bonanou-Tzedaki, H.R.V. Arnstein, European J. Biochem., 24, (1971), 156– 2 G. Shapira, P. Padieu, N. Maleknia, J. Kruh, J.C. Dreyfus, J. Mol. Biol., 20, (1966), 427– 3 L. Grossbard, J. Banks, P.A. Marks, Arch. Biochem. Biophys., 125, (1968), 580– 4 P.M. Prichard, J.M. Gilbert, D.A. Shafritz, W.F. Anderson, Nature, 226, (1970), 511– 5 G. Blobel, D. Sabatini, Proc. Natl. Acad. Sci. U.S., 68, (1971), 390– 6 A.K. Falvey, T. Staehelin, J. Mol. Biol., 53, (1970), 1– 7 H.M. Dintzis, Proc. Natl. Acad. Sci. U.S., 47, (1961), 247– 8 S.N. Seal, J.D. Bewley, A. Marcus, J. Biol. Chem., 247, (1971), 2592– 9 M. Zasloff, S. Ochoa, Proc. Natl. Acad. Sci. U.S., 68, (1971), 3059– 10 S. Sabol, S. Ochoa, Nature New Biology, 234, (1971), 233– 11 M.B. Mathews, I.B. Pragnell, M. Osborn and H.R.V. Arnstein, manuscript submitted. 12 I.B. Pragnell, S.A. Bonanou-Tzedaki and H.R.V. Arnstein, in preparation. 13 M. Jabos-Lorena, C. Baglioni, Proc. Natl. Acad. Sci. U.S., 69, (1972), 1425– Citing Literature Volume26, Issue1-2October 01, 1972Pages 77-82 ReferencesRelatedInformation

Identification of a developmental program using bromodeoxyuridine

Incorporation of bromodeoxyuridine into the DNA of precursor erythroblasts (hematocytoblasts) prevents the appearance of hemoglobin in their progeny. Erythroblasts already synthesizing hemoglobin are unaffected by incorporation of the analogue. This resistance extends over as many as 3 to 4 continuous divisions, at substitutions for thymidine threefold higher than that needed to prevent the initial appearance of hemoglobin. No alterations were observed in the development of these erythroblasts once hemoglobin synthesis was begun. To the limit of resolution of our techniques, this insensitivity is unlikely to be based upon (a) a stable hemoglobin messenger RNA, (b) a species of DNA that is not replicated in the presence of the analogue during the resistant period, or (c) a differential flux of BrdUrd into restricted segments of the genome. It is hypothesized that BrdUrd prevents the initiation of new modes of synthetic activity.

The effect of reticulocyte ribosome “factors” on the translation of haemoglobin messenger RNA in living frog oocytes

1. The effect of ribosome “factors” (materials released from polysomes by 0.5 or 1.0 M KCl) on the translation of messenger RNA has been tested by injecting factors and mRNA into living frog oocytes. Factors were prepared from mammalian reticulocytes as well as from frog oocytes and were tested with Hb mRNA from mammalian reticulocytes. Injected oocytes were labelled with amino acids during incubation, and the effect of factors was judged from the ratio of labelled haemoglobin to labelled oocyte proteins synthesised by injected oocytes. 2. The factors released from reticulocyte ribosomes by KCl may contain up to 30 % of the total haemoglobin message activity of reticulocyte polysomes. When Hb mRNA was supplied at a low concentration so that its supply limited the rate of haemoglobin synthesis, reticulocyte factors had no effect on its translation. When Hb mRNA was supplied at a high enough concentration for some other component to limit the rate of haemoglobin synthesis, reticulocyte and oocyte factors altered the pattern of protein synthesis only slightly in favour of haemoglobin and oocyte proteins, respectively.

Initiation factor IF-3-dependent binding of Escherichia coli ribosomes and N-formylmethionine transfer-RNA to rabbit globin messenger.

An initiation complex has been formed in high yields from E. coli ribosomes, 9S messenger RNA for rabbit hemoglobin, and N-formylmethionine-tRNA. Initiation factor IF-3 is required for the binding and puromycin is required for the release of fMet. Valyl-tRNA fails to bind to the second codon, whereas a mixture of 15 aminoacyl-tRNAs promotes incorporation. Together with quantitative data, the findings suggest that IF-3 directs the ribosomes to an AUG codon on one of the two globin messengers, at a site that is different from the normal starting point for globin synthesis.

Arrest of cell division and hemoglobin synthesis in differentiating yolk sac erythroid cells

Yolk sac derived erythroid cells of C57B1/Cas fetal mice which are X irradiated with 120 rads at the 10th day of fetal development perform three normal cell cycles and synthesize hemoglobin in a normal fashion until day 12. Between day 12 and 13 normal cells perform one final cell cycle and reduce to half the rate of hemoglobin synthesis per cell; conversely, irradiated erythroid cells do not divide and maintain the full rate of hemoglobin formation. At day 13 irradiated non-divided erythroid cells contain polyribosomes in the same amount and with the same size distribution as normally divided cells and yet synthesize hemoglobin at a two-fold faster rate. This difference of hemoglobin synthesizing capacity is not due to new formation of messenger RNA for hemoglobin, neither it is due to a faster rate of translation, but rather because at day 13 non-divided erythroid cells have maintained the full complement of messenger RNA-ribosomal complexes active for hemoglobin synthesis, while in normally divided erythroid cells the number of messenger RNA-ribosomal complexes active for hemoglobin synthesis is cleaved in the course of cell division. In the irradiated erythroid population the total amount of each globin chain produced in the course of differentiation is not decreased in spite of the arrest of cell division. These data suggest that the total amount of hemoglobin produced during the development of the progeny of a single erythroid cell is programmed in early stages of erythroid differentiation.