Lethal alpha-thalassaemia created by gene targeting in mice and its genetic rescue.

Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.

The response of T cells to histocompatibility-2 antigens. Dose-response kinetics.

The DNA synthetic response of a wide variety of parental thymocyte doses was studied in the lymphoid tissues of lethally irradiated F(1) mice. The response curves of the thymocytes were strikingly similar in shape to those of T cells responding to antigens, such as sheep red cells, which are more labile than the histocompatibility antigens of the F(1) host. The response was characterized by a dose-dependent latent period of 1-3 days, followed by a sharp increase in activity and a significant subsequent shutoff. Larger thymocyte doses tended to shorten the latent period. A comparison of the responses of different cell doses to one another indicated that the response usually simulated a suppressed first-order reaction. However, occasional multiple order reactions were observed. We have interpreted these data to indicate that both positive and negative interactions occur between thymocytes, without the mediation of B cell products such as conventional antibodies.

Hemoglobin I mutation encoded at both alpha-globin loci on the same chromosome: concerted evolution in the human genome.

Genetic analysis of an individual expressing an unexpectedly high level of hemoglobin I, an alpha-globin structural mutant, reveals that the mutation is present at both the alpha 1- and the alpha 2-globin gene loci. Kindred analysis confirms that the two affected genes are located in cis. The most likely explanation for this finding is that a recent conversion event occurred within the human alpha-globin gene cluster.

Differentiation of the mRNA transcripts originating from the alpha 1- and alpha 2-globin loci in normals and alpha-thalassemics.

The alpha-globin polypeptide is encoded by two adjacent genes, alpha 1 and alpha 2. In the normal diploid state (alpha alpha/alpha alpha) all four alpha-globin genes are expressed. Loss or dysfunction of one or more of these genes leads to deficient alpha-globin production and results in alpha-thalassemia. We present a technique to differentially assess the steady-state levels of the alpha 1- and alpha-2-globin messenger RNA (mRNA) transcripts and thus delineate the relative level of expression of the two alpha-globin loci in a variety of alpha-thalassemia states. Only alpha 1 mRNA was produced in the alpha-thalassemia-2 haplotype (-alpha) (one of the two alpha-globin genes deleted from chromosome 16). This confirms previous gene mapping data which demonstrate deletion of the alpha 2 gene. The triple alpha-globin gene haplotype (alpha alpha alpha) is the reciprocal of the alpha-thalassemia-2 haplotype and thus contains an extra alpha 2-globin gene. RNA from this haplotype contained a greater than normal level of alpha 2-relative to alpha 1-globin mRNA. This data implies that the extra alpha 2 gene in the triple alpha-globin haplotype is functional. We detected a relative instability of the alpha 2-globin mRNA encoding the alpha-globin structural mutant Constant Spring. This instability may contribute to the low level of expression of the alpha-Constant Spring protein. In a Chinese patient with nondeletion hemoglobin-H disease (- -/alpha alpha T) (both alpha-globin genes are present but not fully functional) a normal ratio was maintained between the levels of alpha 1- and alpha 2-globin mRNA, implying that mRNA production from both alpha-globin genes is suppressed in a balanced manner. These observations extended previous findings concerning the structural rearrangements in the deletion types of alpha-thalassemia and the pathophysiology of two nondeletion variants.

Locus assignment of alpha-globin structural mutations by hybrid-selected translation.

The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants.

alpha-Thalassemia caused by an unstable alpha-globin mutant.

In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins.

Translational profiles of alpha 1-, alpha 2-, and beta-globin messenger ribonucleic acids in human reticulocytes.

In human reticulocytes, the critical balancing of alpha- and beta-globin synthesis may be controlled in part by differential translation of the three major adult globin messenger RNAs (mRNAs), alpha 1, alpha 2, and beta. In this study, we determined, as a parameter of translational efficiency, the relative ribosome loading of these three mRNAs. Using oligonucleotide probes specific for the alpha 1- and alpha 2-globin mRNAs, we find that these two mRNAs have identical translational profiles. Their distribution contrasts with that of beta-globin mRNA, which is present on heavier polyribosomes and is less prevalent in pre-80S messenger ribonucleoprotein fractions. The relative distribution of alpha- vs. beta-globin mRNA is consistent with more efficient beta-globin translation. In contrast, the parallel distributions of alpha 1- and alpha 2-globin mRNAs suggests they are translated with equal efficiencies. Considering the relative concentrations of the two alpha-globin mRNAs in normal reticulocytes, this result predicts a dominant role for the alpha 2-globin locus in human alpha-globin expression.

Compensatory increase in alpha 1-globin gene expression in individuals heterozygous for the alpha-thalassemia-2 deletion.

alpha-Globin is encoded by the two adjacent genes, alpha 1 and alpha 2. Although it is clearly established that both alpha-globin genes are expressed, their relative contributions to alpha-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in alpha-globin gene activity secondarily to the loss of function of one or more of these genes (alpha-thalassemia [Thal]) have not been directly investigated. This study further defines the expression of the two human alpha-globin genes by determining the relative levels of alpha 1 and alpha 2 mRNA in the reticulocytes of normal individuals and in individuals heterozygous for the common 3.7-kilobase deletion within the alpha-globin gene cluster that removes the alpha 2-globin gene (the rightward type alpha-Thal-2 deletion). To quantitate accurately the ratio of the two alpha-globin mRNAs, we have modified a previously reported S1 nuclease assay to include the use of 32P end-labeled probes isolated from alpha 1- and alpha 2-globin complementary DNA recombinant plasmids. In individuals with a normal alpha-globin genotype (as determined by Southern blot analysis [alpha alpha/alpha alpha]), alpha 2-globin mRNA is present at an average 2.8-fold excess to alpha 1. In individuals heterozygous for the rightward type alpha-Thal-2 deletion (-alpha/alpha alpha) the alpha 2/alpha 1 mRNA ratio is 1:1. These results suggest that the loss of the alpha 2-globin gene in the alpha-Thal-2 deletion is associated with a 1.8-fold compensatory increase alpha 1-globin gene expression.

An initiation codon mutation (AUG----GUG) of the human alpha 1-globin gene. Structural characterization and evidence for a mild thalassemic phenotype.

alpha-globin is encoded by two adjacent genes, alpha 1 and alpha 2. Recent evidence suggests that these genes are not equally expressed and that the alpha 2-globin gene encodes the majority of alpha-globin. This finding would predict that a thalassemic mutation of the alpha 2-globin gene would result in a more severe loss of alpha-chain synthesis than a similar mutation in the alpha 1-globin gene. In a previous study we described a nondeletion alpha-thalassemia defect in the alpha 2-globin gene resulting from an AUG----ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG----GUG, present in the alpha 1-globin gene. The alpha 1- and alpha 2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the alpha 2 mutant results in a more severe loss of alpha-globin synthesis and a more severe clinical alpha-thalassemia phenotype than the corresponding alpha 1-globin gene mutation. This difference reflects the dominant role of alpha 2-globin gene in overall alpha-globin synthesis.

Characterization and histologic localization of human growth hormone-variant gene expression in the placenta.

The human growth hormone-variant (hGH-V) gene is one of five highly similar growth hormone-related genes clustered on the short arm of chromosome 17. Although the pattern of expression of the adjacent normal growth hormone (hGH-N) and chorionic somatomammotropin (hCS) genes in this cluster are well characterized, the expression of the hGH-V gene remains to be defined. In previous studies, we have demonstrated that the hGH-V gene is transcribed in the term placenta and expressed as two alternatively spliced mRNAs: one is predicted to encode a 22-kD hormone (hGH-V), the other retains intron 4 in its sequence resulting in the predicted synthesis of a novel 26-kD hGH-V-related protein (hGH-V2). In the present report, we document the expression of both of these hGH-V mRNA species in the villi of the term placenta, demonstrate an increase in their concentrations during gestation, and directly sublocalize hGH-V gene expression to the syncytiotrophoblastic epithelium of the term placenta by in situ cDNA-mRNA histohybridization. The demonstrated similarity in the developmental and tissue-specific expression of the hGH-V gene with that of the related hCS gene suggests that these two genes may share common regulatory elements.

Human growth hormone gene and the highly homologous growth hormone variant gene display different splicing patterns.

Stably transfected cell lines containing the normal human growth hormone (hGH-N) and human growth hormone-variant (hGH-V) genes have been established in order to study the expression of these two highly homologous genes. Each gene was inserted into a bovine papillomavirus shuttle vector under the transcriptional control of the mouse metallothionein gene promoter and the resultant recombinants were transfected into mouse C127 cells. The transfected cells containing the hGH-N gene secrete two hGH proteins, 91% migrating at 22 kD and 9% migrating at 20 kD, the same relative proportions synthesized in vivo by the human pituitary. S1 nuclease analysis of mRNA from these cells confirms that 20 kD hGH is encoded by an alternatively spliced product of the primary hGH-N gene transcript in which the normal exon 3 splice-acceptor site is bypassed for a secondary site 15 codons within exon 3. Although the hGH-V gene is identical to the hGH-N gene for at least 15 nucleotides on either side of the normal and alternative exon 3 AG splice-acceptor sites, hGH-V synthesizes only a 22-kD protein. Reciprocal exchange of exon 3 and its flanking intron sequences between the hGH-N gene and the hGH-V gene, eliminates the synthesis of the 20-kD protein in both resultant chimeric genes. These results directly demonstrate that both the major 22-kD and the minor 20-kD forms of pituitary hGH are encoded by the alternative splicing products of a single hGH-N gene transcript. This alternative splicing is neither species nor tissue-specific and appears to be regulated by at least two separate regions remote from the AG splice-acceptor site.

Molecular basis for nondeletion alpha-thalassemia in American blacks. Alpha 2(116GAG----UAG).

An American black woman was found to have the phenotype of moderately severe alpha-thalassemia normally associated with the loss of two to three alpha-globin genes despite an alpha-globin gene map that demonstrated the loss of only a single alpha-globin gene (-alpha/alpha alpha). Several individuals in her kindred with normal alpha-globin gene mapping studies (alpha alpha/alpha alpha) had mild alpha-thalassemia hematologic values consistent with the loss of one to two alpha-globin genes. These data suggested the presence of a nondeletion alpha-thalassemia defect in this family which segregates with the intact alpha alpha gene cluster. An abnormally migrating and highly unstable alpha-globin gene product was demonstrated by in vitro translation of the reticulocyte mRNA from the proposita and this mutant alpha-globin protein was mapped to the alpha 2-globin gene by hybrid-selected translation. The abnormal alpha 2-globin gene was cloned and sequenced. A single base mutation that results in a premature termination codon was identified at codon 116 (GAG----UAG). The defined alpha-globin genotype of the proposita (-alpha/alpha 116UAG alpha) and the positioning of this nonsense mutation at the alpha 2-globin gene locus are fully consistent with the observed alpha-thalassemia phenotype.

beta zero thalassemia in Sardinia is caused by a nonsense mutation.

We report the characterization of a molecular lesion of beta thalassemia in Sardinia. Beta thalassemia in this area is predominantly the beta zero type with low levels of beta-globin mRNA. Translation assay of this messenger RNA in a cell-free system showed beta-globin chain synthesis only with the addition of an amber (UAG) suppressor transfer RNA. Double-stranded complementary DNA prepared from reticulocyte mRNA from a Sardinian patient was cloned in a bacterial plasmid and a beta-globin complementary DNA containing clone was isolated and sequenced. At the position corresponding to amino acid number 39, a single nucleotide mutation converted a glutamine codon (CAG) to an amber termination codon (UAG). We previously reported an amber nonsense mutation at amino acid 17 as a cause of Chinese beta zero thalassemia. Thus, beta zero thalassemia in Sardinia represents the second example of a nonsense mutation, and we predict that other beta zero thalassemias with mutations at various points along the beta-globin chain will be found to form a discrete subgroup of beta zero thalassemia. These experiments further illustrate the heterogeneity of lesions that lead to defective globin chain synthesis in beta thalassemia.

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein.

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.

Erythroid cell-specific determinants of alpha-globin mRNA stability.

Although globin mRNAs are considered prototypes of highly stable messages, the mechanisms responsible for their longevity remain largely undefined. As an initial step in identifying potential cis-acting elements or structures which contribute to their stability, we analyzed the defect in expression of a naturally occurring alpha 2-globin mutant, alpha Constant Spring (CS). The CS mutation is a single-base change in the translation termination codon (UAA-->CAA) that allows the ribosome to read through into the 3' nontranslated region (NTR). The presence of CS mRNA in transcriptionally active erythroid precursors and its absence (relative to normal alpha-globin mRNA) in the more differentiated transcriptionally silent erythrocytes suggest that this mutation disrupts some feature of the alpha-globin mRNA required for its stability. Using a transient transfection system, we demonstrate that in murine erythroleukemia cells the CS mRNA is unstable compared with the normal alpha 2-globin mRNA. The analyses of several other naturally occurring and site-directed mutant alpha-globin genes in murine erythroleukemia cells indicate that entry of a translating ribosome into the 3' NTR targets the message for accelerated degradation in erythroid cells. In contrast, both the CS and alpha 2-globin mRNAs are stable in several nonerythroid cell lines. These results suggest that translational readthrough disrupts a determinant associated with the alpha 2-globin 3' NTR which is required for mRNA stability in erythroid cells.

Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Erythroid cell-specific mRNA stability elements in the alpha 2-globin 3' nontranslated region.

Very little is known about the mechanisms mediating longevities of mRNAs. As a means of identifying potential cis- and trans-acting elements which stabilize an individual mRNA, naturally occurring mutations that decreased stability of the normally long-lived globin mRNA were analyzed. Our previous studies demonstrated that a subset of mutations which allowed the translating ribosome to read through into the alpha 2-globin 3' nontranslated region (NTR) targeted the mutant mRNAs for accelerated turnover in erythroid cells but not in several nonerythroid cell lines (I. M. Weiss and S. A. Liebhaber, Mol. Cell. Biol. 14:8123-8132, 1994). These results suggested that translational readthrough interfered with some feature of the alpha 2-globin 3' NTR required for message stability in erythroid cells. To define the cis-acting sequences which comprise this erythroid cell-specific stability determinant, scanning mutagenesis was performed on the alpha 2-globin 3' NTR, and the stability of each mutant mRNA was examined during transient expression. Three cytidine-rich regions which are required for longevity of the alpha 2-globin mRNA were identified. However, in contrast to the readthrough mutations, base substitutions in these elements destabilize the message through a translation-independent mechanism. To account for these results, we propose that the cis-acting elements form a complex or determinant in the normal alpha 2-globin mRNA which protects the message from degradation in erythroid cells. Disruption of this determinant, by translational readthrough or because mutations in an element prevent or inhibit its formation, targets the message for accelerated turnover in these cells.

Developmental silencing of the embryonic zeta-globin gene: concerted action of the promoter and the 3'-flanking region combined with stage-specific silencing by the transcribed segment.

Globin gene switching is a well-described model of eucaryotic developmental control. In the case of the human alpha-globin gene cluster, migration of erythropoietic activity from the embryonic yolk sac to the fetal liver is parallaled by the zeta-globin gene silencing and enhanced expression of the alpha-globin genes. To map critical cis determinants of this switch, the human zeta-globin gene, the alpha-globin gene, and chimeric recombinants were introduced into the mouse genome. Consistent with previous studies, expression of the individual alpha- and zeta-globin transgenes was found to be developmentally appropriate. Contrary to current models, however, the alpha- and zeta-globin gene promoters were not sufficient to establish this control. Instead, full silencing of the zeta-globin gene required the combined activities of this promoter, transcribed region, and 3'-flanking sequences. Individually, the silencing activities of the zeta-globin gene promoter and 3'-flanking region were minimal but increased markedly when both regions were present. The zeta-globin transcribed region appeared to contribute to gene silencing by a mechanism specifically activated in definitive erythroblasts in the fetal liver. These data demonstrate that a complex set of controls, requiring at least three determinants and involving at least two independent mechanisms, is necessary for full developmental silencing of the human zeta-globin gene.

Sequence divergence in the 3' untranslated regions of human zeta- and alpha-globin mRNAs mediates a difference in their stabilities and contributes to efficient alpha-to-zeta gene development switching.

The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of zeta- and alpha-globin in the embryonic yolk sac to exclusive expression of alpha-globin during fetal and adult life. Recent studies with transgenic mice demonstrate that in addition to transcriptional control elements, full developmental silencing of the human zeta-globin gene requires elements encoded within the transcribed region. In the current work, we establish that these latter elements operate posttranscriptionally by reducing the relative stability of zeta-globin mRNA. Using a transgenic mouse model system, we demonstrate that human zeta-globin mRNA is unstable in adult erythroid cells relative to the highly stable human alpha-globin mRNA. A critical determinant of the difference between alpha- and zeta-globin mRNA stability is mapped by in vivo expression studies to their respective 3' untranslated regions (3'UTRs). In vitro messenger ribonucleoprotein (mRNP) assembly assays demonstrate that the alpha- and zeta-globin 3'UTRs assemble a previously described mRNP stability-determining complex, the alpha-complex, with distinctly different affinities. The diminished efficiency of alpha-complex assembly on the zeta 3'UTR results from a single C-->G nucleotide substitution in a crucial polypyrimidine tract contained by both the human alpha- and zeta-globin mRNA 3'UTRs. A potential pathway for accelerated zeta-globin mRNA decay is suggested by the observation that its 3'UTR encodes a shortened poly(A) tail. Based upon these data, we propose a model for zeta-globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of zeta-globin transcripts.