Globin mRNA Sequences Research Articles

Fetal cell-free nucleic acids in the maternal circulation: new clinical applications.

Six years after the demonstration of the presence of cell-free fetal nucleic acids in maternal plasma, perinatal clinical applications continue to expand. The focus of this article is on advances that have occurred since the CNAPS II conference held in Hong Kong in 2001. Circulating fetal DNA levels (fDNA) are elevated in pregnancies complicated by fetal trisomies 13 and 21 but not 18. Measurement of fDNA levels improves the performance of the current standard maternal serum screen, by increasing the detection of Down syndrome cases by 5% with no increase in the false-positive rate. fDNA levels are elevated in women who have developed clinical symptoms of preeclampsia, but they are also elevated by the early second trimester in women who will eventually develop preeclampsia. fDNA and mRNA gamma globin measurement may have clinical utility as markers for fetomaternal hemorrhage in the late first trimester. Cell-free fetal DNA levels are quite high in the amniotic fluid, permitting fetal genomic isolation and analysis using comparative genomic hybridization techniques. Fetal DNA crosses the blood-brain barrier and is detectable in maternal cerebrospinal fluid in a subset of pregnant women. The biological implications of this are currently unknown. Review of the literature suggests that the placenta is the predominant source of the circulating fetal nucleic acids. However, detection of gamma globin mRNA sequences in the plasma of pregnant women suggests that fetal blood cells also contribute to the pool of nucleic acids. Widespread incorporation of fetal nucleic acid measurement into routine prenatal care depends on the identification of a readily accessible gender-independent fetal marker.

Identification of two critical base pairings in 5′ untranslated regions affecting translation efficiency of synthetic uncapped globin mRNAs

We observed a marked difference between the in vitro translation efficiency of two uncapped synthetic mRNAs, displaying the entire human alpha or beta globin mRNA sequences and some additional non-globin sequences in 5'. The comparison of the translation efficiencies of chimeric mRNAs indicated that the alpha 5' untranslated region (5' UTR) is responsible for a low translation efficiency that cannot be explained neither by primary sequence nor by the overall stability of 5' UTR secondary structures only. By point mutations in this alpha 5' UTR, we identified two base pairings at position -1 and -2 preceding the initiation codon which are associated with a negative effect on translation efficiency.

Displacement synthesis and cloning of symmetrical complementary DNA containing duck globin mRNA sequences

A displacement synthesis procedure was used to construct symmetrical recombinant cDNAs. A double-stranded cDNA containing a hairpin loop was extended by the addition of a homopolymer to the 3′ end. This was followed by displacement, or “third strand” synthesis that was primed by an oligonucleotide hybridized to the homopolymer. Ideally, the product should be a DNA containing an inverted repeat with twofold rotational symmetry about nonsymmetrical sequences representing the hairpin loop in the original double-stranded cDNA. Duck globin cDNAs were synthesized by the displacement mode of construction and cloned in pBR322. An α-globin recombinant, pDGPα-2, was isolated and sequenced. This recombinant was found to have two 3′ half regions (mRNA sequence sense) inverted about a nonsymmetrical 5′ half region and an adjacent oligo(dGṡdC) homopolymer. The sequence arrangement indicates that the cDNA folded back on itself, forming a large loop, to prime synthesis of a second strand. We propose that the internal oligo(dGṡ dC) arose through dynamic shifts in cDNA intrastrand structure during the course of synthesis.

Human β-globin promoter and coding sequences transcribed by RNA polymerase III

We have investigated low abundance RNAs transcribed in vitro and in vivo from the human β-globin gene. These RNAs contain globin mRNA sequences covalently linked to sequences transcribed from the 5′ flanking region between −235 and the mRNA cap site (+1). Their synthesis in vitro is sensitive to high (100 μg/ml) levels of α-amanitin but not to low (2 μg/ml) levels, and one region of the DNA template bordering their 5′ termini is similar to a small segment of Alu repetitive DNA and to the RNA polymerase III promoter consensus sequence. Therefore, these RNAs are transcribed by RNA polymerase III but extend into the mRNA-coding region that is usually transcribed by polymerise II. The polymerise III transcripts are polyadenylated and are probably spliced. Their presence in bone marrow cells and peripheral blood reticulocytes implies that they play some role in the erythroid cell.

Globin gene expression in Xenopus laevis: Anemia induces precocious globin transition and appearance of adult erythroblasts during metamorphosis

The expression of Xenopus laevis larval and adult globin genes after phenylhydrazine-induced anemia has been investigated at the cellular and molecular levels by means of cloned cDNA probes specific for the four main larval and the four main adult globin mRNA species. In the circulating blood of anemic metamorphic larvae there are at least two distinct populations of erythroblasts containing either larval or adult globin mRNA sequences. The cells expressing adult sequences replace those expressing larval ones at the end of metamorphosis. Under the influence of anemia the qualitative pattern of transcribed RNA species is not changed, but the larval to adult transition takes place earlier during development. This would imply that this transition is not strongly correlated with the morphological changes of metamorphosis. The abundance of the different larval globin mRNA species is similar and, as compared to control animals, not affected by the phenylhydrazine treatment. In anemic adults no reactivation of larval gene expression has been observed. The different adult globin mRNA species are present in comparable abundance, but the phenylhydrazine treatment appears to enhance expression of the α A II and β A II genes, as compared to nonanemic individuals.

Human beta and delta globin messenger RNAs turn over at different rates

We have tested the hypothesis of Winslow & Ingram (1966) that the steady-state levels of human beta and delta globin proteins are determined, in part, by the stabilities of their respective messenger RNAs. Nucleated bone marrow cells were cultured under different RNA "chase" conditions, and samples were harvested at intervals thereafter. The quantities of beta and delta globin mRNA sequences in total RNA from these cells were measured by solution hybridization with specific 32P-labeled DNA probes. The average half-lives of beta and delta globin mRNAs are 16.5 and 4.5 hours, respectively. Both mRNAs are polyadenylated. The rapid turnover of delta globin mRNA accounts, at least in part, for the low level of delta globin mRNA in non-nucleated peripheral blood reticulocytes. The possibility that the rate of mRNA decay is determined by nucleotide sequence signals located in the 3' untranslated region is discussed.

In vitro transformation by avian erythroblastosis virus recruits cells of erythroid lineage with varying degree of differentiation

We have previously shown that AEV infection of leukosis-free chickens provokes an erythroleukemia in the infected birds, and that the target cell for virus transformation is most likely an early pro-erythroblast comparable to the BFU-E. The virus infection correlates with a block in differentiation which was studied at a specific cellular gene level, namely that of the globin genes. In live birds, the viral transformation has been found to affect the transcriptional activity to two out of three adult globin genes and the phenotypic expression of the third. Indeed, the alpha A gene that is transcribed is silent as well, since its transcription product is fully eliminated in the nucleus following an "abortive processing". Chick bone marrow cells can also be transformed in vitro by AEV in which case the virus recruits, as targets, cells of the erythroid lineage belonging to a wider spectrum and hence in varying degrees of differentiation. In contrast to the results obtained with in vivo transformed cells, molecular hybridization with globin cDNA probes showed that in the erythroblasts transformed in vitro under tissue culture conditions all the three adult globin genes are transcriptionally active. In fact, low but detectable amounts of globin mRNA sequences are present in both the nuclear and cytoplasmic compartment of the cell prior to exposure to any chemical inducers of red cell differentiation. Judging from our present results, we may propose that AEV transformation in vitro does not shut off out only modulates the already functioning erythroid cell differentiation program.

In vitro transformation by avian erythroblastosis virus recruits cells of erythroid lineage with varying degree of differentiation.

We have previously shown that AEV infection of leukosis-free chickens provokes an erythroleukemia in the infected birds, and that the target cell for virus transformation is most likely an early pro-erythroblast comparable to the BFU-E. The virus infection correlates with a block in differentiation which was studied at a specific cellular gene level, namely that of the globin genes. In live birds, the viral transformation has been found to affect the transcriptional activity to two out of three adult globin genes and the phenotypic expression of the third. Indeed, the alpha A gene that is transcribed is silent as well, since its transcription product is fully eliminated in the nucleus following an "abortive processing". Chick bone marrow cells can also be transformed in vitro by AEV in which case the virus recruits, as targets, cells of the erythroid lineage belonging to a wider spectrum and hence in varying degrees of differentiation. In contrast to the results obtained with in vivo transformed cells, molecular hybridization with globin cDNA probes showed that in the erythroblasts transformed in vitro under tissue culture conditions all the three adult globin genes are transcriptionally active. In fact, low but detectable amounts of globin mRNA sequences are present in both the nuclear and cytoplasmic compartment of the cell prior to exposure to any chemical inducers of red cell differentiation. Judging from our present results, we may propose that AEV transformation in vitro does not shut off out only modulates the already functioning erythroid cell differentiation program.

Differing Responses of Globin and Glycophorin Gene Expression to Hemin in the Human Leukemia Cell Line K562

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3-4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3-5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

Posttranscriptional defects in beta-globin messenger RNA metabolism in beta-thalassemia: abnormal accumulation of beta-messenger RNA precursor sequences.

The production of beta-globin messenger RNA (mRNA) in beta-thalassemic erythroblasts was studied during pulse-chase incubations with [3H]uridine. Globin [3H]mRNA was quantitated by molecular hybridization to recombinant DNA probes complementary to globin mRNA and mRNA precursor sequences. Each of six patients with beta +-thalassemia produced normal amounts of globin alpha and beta [3H]mRNA during a 20-min pulse incubation, but the beta/alpha [3H]mRNA ratio declined to steady-state levels during a chase incubation, suggesting posttranscriptional defects in beta-globin mRNA metabolism. beta-globin mRNA precursor production was estimated by measurement of [3H]RNA sequences hybridizing to a pure DNA probe containing only the large intervening sequence (intron) of the beta-mRNA precursor. Four of the patients exhibited abnormal accumulation of 3H-beta-intron sequences (2-10 times normal), indicating abnormal posttranscriptional processing. In the remaining two patients, one of whom is known to carry a mutation in the small intron of the beta-globin gene, accumulation of large 3H beta-intron RNA and beta-globin [3H]mRNA was normal in nuclei, but the ratio of beta/alpha [3H]mRNA in cytoplasm was reduced, suggesting a different posttranscriptional defect in beta-mRNA processing. These findings imply the existence of heterogeneous posttranscriptional abnormalities in beta-globin mRNA metabolism in different patients with beta-thalassemia. The initial rates of gamma- and delta-mRNA synthesis were low in all patients, suggesting that the low level of expression of these genes in adults is mediated at the transcriptional level.

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.

Transcriptional activity of globin genes in uninducible variants of Friend leukemic cells.

Transcriptional activity of globin genes in inducible and uninducible Friend leukemic cells was studied by two different approaches in an attempt to identify the biochemical basis for the loss of inducibility. (1) Nuclei were isolated, and transcripts synthesized endogenously were purified by the Hg-UTP/sulfhydryl-Sepharose method. Globin mRNA sequences were quantitated by hybridization with [32P]globin cDNA. The results showed that the transcription of globin genes was stimulated in DMSO-treated inducible cell nuclei, whereas there was no activation of globin gene transcription in nuclei of uninducible variants cultured with DMSO. (2) Nuclei were prepared from inducible and uninducible Friend cells cultured in the presence or absence of DMSO, and were then subjected to limited digestion with DNase I. DNA was isolated from the nuclei after about 10% of total DNA had become acid-soluble, and was hybridized with globin cDNA. Globin genes either in inducible or in uninducible Friend cells were specifically susceptible to DNase I irrespective of the addition of inducer. From these studies, it was concluded that globin genes of uninducible Friend cells retained the "active conformation" but were not actively transcribed even in the presence of inducers.

Restriction mapping of cDNA recombinants including the adult chicken and duck globin messenger sequences: a comparative study

A comparison of the organization of six avian adult globin messenger sequences is based on previously reported recombinant duck adult globin cDNA plasmids (Therwath et al., 1980) and the actual construction and characterization of pBR322 recombinant plasmids including the β and the normal α A and α D chicken adult globin mRNA sequences. Identification of the cloned DNA was performed using hybridization-selection under conditions permitting complete purification in one step of the three globin mRNAs, and translation of the corresponding mRNA. Orientation of the globin insert in the vector was determined, taking into account the computer prediction of the restriction sites based on the known amino acid sequences of the three globin chains (Roizès and Pelaquier, 1980) and those actually observed, and by identification of restriction fragments using 3'-specific probes. Identification, orientation and restriction mapping of these cloned DNAs reveals extensive homologies in organisation of β sequences between duck and chicken, as well as among the α sequences in every two possible combinations.

Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts.

A mixture of two recombinant plasmids was microinjected into mouse thymidine kinase-negative fibroblasts (L cells). One plasmid contained the thymidine kinase gene of herpes simplex virus type I and the other contained the human beta globin gene. Seven fibroblast colonies arising from injected cells incubated in hypoxanthine/aminopterin/thymidine medium were analyzed. These microinjected cells were shown to: (i) produce functionally active herpes simplex type I thymidine kinase enzyme, (ii) replicate the human beta globin gene, and (iii) produce human beta globin mRNA sequences at low levels. Thus, the genetic defect (lack of thymidine kinase activity) was corrected by the microinjected thymidine kinase gene, and a coinjected human beta globin gene was replicated and weakly expressed.

Partial sequence analysis of Xenopus α- and β-globin mRNA as determined from recombinant DNA plasmids

Recombinant plasmids containing Xenopus globin mRNA sequences have been constructed using the mRNA:cDNA hybrid conditions of Zain et al. (1979, Cell 16, 851–861). The partial nucleotide sequence of two of these recombinants has been determined. They have been identified as containing α- and β-globin-like sequences by homology to other amphibian globin proteins. The nucleotide sequence of these recombinants permits the comparison of conserved regions in both the coding and 3′ nontranslated regions of Xenopus globin mRNAs with the known sequences of other eukaryotic globin proteins and mRNAs. Among the features which have been conserved though evolution is the sequence AAUAAA close to the 3′ terminus of the nontranslated region. Extensive regions of homology occur between the 3′ nontranslated regions of Xenopus α- and β-globin mRNA.

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.

Specific inducibility of globin mRNA in Friend leukemia cells in low serum concentration.

Friend leukemia cells were adapted to grow in a medium containing 0.5% serum by gradually decreasing concentration of the serum from 15%. Upon treatment of the cells with dimethyl sulfoxide, appearance of benzidine-positive cells was suppressed in the cells cultivated in a 0.5% serum medium, whereas globin mRNA sequences, as determined by hybridization of cytoplasmic RNA with globin cDNA, increased to the same order as in the cells grown in a medium containing 15% serum. A small but significant amount of globin was synthesized in cells treated with dimethyl sulfoxide in 0.5% serum medium, as determined by dodecyl sulfate-polyacrylamide gel electrophoresis of 3H-labeled cellular proteins.

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. Direct measurements of heme synthesis indicate that Fw cells may be significantly deficient in heme metabolism.

Globin mRNA Precursor

The globin mRNA sequences present in duck erythroblast nuclei appear under non-denaturing conditions to be associated with heterogeneous nuclear RNA (hnRNA) molecules of various sizes. Under denaturing conditions, however, the bulk of the globin mRNA sequences associated with hnRNA are released as molecules of size close to that of the active globin mRNA. To find out whether hydrogen-bonded structures occur in situ or arise after RNA extraction, nuclei were treated with aminomethyltrioxalen and exposed to ultraviolet light. This treatment generates covalent links between opposite strands of double-stranded nuclei acids, which were visualised by electron microscopy. It appears that, after cross-linking, a fraction of the globin mRNA sequences present in nuclei is associated with high-molecular-weight hnRNA molecules by a link found associated with a band of 0.9 x 10(6) molecular weight approximately. It is suggested that within the erythroblast nucleus, globin mRNA sequences are associated by hydrogen bonds with RNA of high molecular weight. These structures may represent intermediate steps in globin mRNA processing.